advanced concepts of energy transformation in metabolism biochemistry

Table of Contents

Chapter 1 - Oxidative Phosphorylation

Chapter 2 - Chemiosmosis

Chapter 3 - Mitochondrion

Chapter 4 - Microbial Metabolism

Chapter 5 - Nitrogen Cycle

Chapter 6 - Phototroph and Photophosphorylation

Chapter 7 - Chloroplast

Chapter- 1

Oxidative Phosphorylation

The electron transport chain in the mitochondrion is the site of oxidative phosphorylation in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized, releasing energy to power the ATP synthase.

Oxidative phosphorylation is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP). Although the many

forms of life on earth use a range of different nutrients, almost all carry out oxidative phosphorylation to produce ATP, the molecule that supplies energy to metabolism. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis.

During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within mitochondria, whereas, in prokaryotes, these proteins are located in the cells' inner membranes. These linked sets of proteins are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors.

The energy released by electrons flowing through this electron transport chain is used to transport protons across the inner mitochondrial membrane, in a process called chemiosmosis. This generates potential energy in the form of a pH gradient and an electrical potential across this membrane. This store of energy is tapped by allowing protons to flow back across the membrane and down this gradient, through a large enzyme called ATP synthase. This enzyme uses this energy to generate ATP from adenosine diphosphate (ADP), in a phosphorylation reaction. This reaction is driven by the proton flow, which forces the rotation of a part of the enzyme; the ATP synthase is a rotary mechanical motor.

Although oxidative phosphorylation is a vital part of metabolism, it produces reactive oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of free radicals, damaging cells and contributing to disease and, possibly, aging (senescence). The enzymes carrying out this metabolic pathway are also the target of many drugs and poisons that inhibit their activities.

Overview of energy transfer by chemiosmosis Oxidative phosphorylation works by using energy-releasing chemical reactions to drive energy-requiring reactions: The two sets of reactions are said to be coupled. This means one cannot occur without the other. The flow of electrons through the electron transport chain, from electron donors such as NADH to electron acceptors such as oxygen, is an exergonic process – it releases energy, whereas the synthesis of ATP is an endergonic process, which requires an input of energy. Both the electron transport chain and the ATP synthase are embedded in a membrane, and energy is transferred from electron transport chain to the ATP synthase by movements of protons across this membrane, in a process called chemiosmosis. In practice, this is like a simple electric circuit, with a current of protons being driven from the negative N-side of the membrane to the positive P-side by the proton-pumping enzymes of the electron transport chain. These enzymes are like a battery, as they perform work to drive current through the circuit. The movement of protons creates an electrochemical gradient across the membrane, which is often called the proton-motive force. This gradient has two components: a difference in proton

concentration (a H+ gradient) and a difference in electric potential, with the N-side having a negative charge. The energy is stored largely as the difference of electric potentials in mitochondria, but also as a pH gradient in chloroplasts.

ATP synthase releases this stored energy by completing the circuit and allowing protons to flow down the electrochemical gradient, back to the N-side of the membrane. This enzyme is like an electric motor as it uses the proton-motive force to drive the rotation of part of its structure and couples this motion to the synthesis of ATP.

The amount of energy released by oxidative phosphorylation is high, compared with the amount produced by anaerobic fermentation. Glycolysis produces only 2 ATP molecules, but somewhere between 30 and 36 ATPs are produced by the oxidative phosphorylation of the 10 NADH and 2 succinate molecules made by converting one molecule of glucose to carbon dioxide and water. This ATP yield is the theoretical maximum value; in practice, some protons leak across the membrane, lowering the yield of ATP.

Electron and proton transfer molecules

Reduction of coenzyme Q from its ubiquinone form (Q) to the reduced ubiquinol form (QH2)

The electron transport chain carries both protons and electrons, passing electrons from donors to acceptors, and transporting protons across a membrane. These processes use both soluble and protein-bound transfer molecules. In mitochondria, electrons are transferred within the intermembrane space by the water-soluble electron transfer protein cytochrome c. This carries only electrons, and these are transferred by the reduction and oxidation of an iron atom that the protein holds within a heme group in its structure.

Cytochrome c is also found in some bacteria, where it is located within the periplasmic space.

Within the inner mitochondrial membrane, the lipid-soluble electron carrier coenzyme Q10 (Q) carries both electrons and protons by a redox cycle. This small benzoquinone molecule is very hydrophobic, so it diffuses freely within the membrane. When Q accepts two electrons and two protons, it becomes reduced to the ubiquinol form (QH2); when QH2 releases two electrons and two protons, it becomes oxidized back to the ubiquinone (Q) form. As a result, if two enzymes are arranged so that Q is reduced on one side of the membrane and QH2 oxidized on the other, ubiquinone will couple these reactions and shuttle protons across the membrane. Some bacterial electron transport chains use different quinones, such as menaquinone, in addition to ubiquinone.

Within proteins, electrons are transferred between flavin cofactors, iron–sulfur clusters, and cytochromes. There are several types of iron–sulfur cluster. The simplest kind found in the electron transfer chain consists of two iron atoms joined by two atoms of inorganic sulfur; these are called [2Fe–2S] clusters. The second kind, called [4Fe–4S], contains a cube of four iron atoms and four sulfur atoms. Each iron atom in these clusters is coordinated by an additional amino acid, usually by the sulfur atom of cysteine. Metal ion cofactors undergo redox reactions without binding or releasing protons, so in the electron transport chain they serve solely to transport electrons through proteins. Electrons move quite long distances through proteins by hopping along chains of these cofactors. This occurs by quantum tunnelling, which is rapid over distances of less than 1.4×10−9 m.

Eukaryotic electron transport chains Many catabolic biochemical processes, such as glycolysis, the citric acid cycle, and beta oxidation, produce the reduced coenzyme NADH. This coenzyme contains electrons that have a high transfer potential; in other words, they will release a large amount of energy upon oxidation. However, the cell does not release this energy all at once, as this would be an uncontrollable reaction. Instead, the electrons are removed from NADH and passed to oxygen through a series of enzymes that each release a small amount of the energy. This set of enzymes, consisting of complexes I through IV, is called the electron transport chain and is found in the inner membrane of the mitochondrion. Succinate is also oxidized by the electron transport chain, but feeds into the pathway at a different point.

In eukaryotes, the enzymes in this electron transport system use the energy released from the oxidation of NADH to pump protons across the inner membrane of the mitochondrion. This causes protons to build up in the intermembrane space, and generates an electrochemical gradient across the membrane. The energy stored in this potential is then used by ATP synthase to produce ATP. Oxidative phosphorylation in the eukaryotic mitochondrion is the best-understood example of this process. The mitochondrion is present in almost all eukaryotes, with the exception of anaerobic protozoa such as Trichomonas vaginalis that instead reduce protons to hydrogen in a remnant mitochondrion called a hydrogenosome.

Typical respiratory enzymes and substrates in eukaryotes.

Respiratory enzyme Redox pair Midpoint potential

(Volts) NADH dehydrogenase NAD+ / NADH −0.32

Succinate dehydrogenase FMN or FAD / FMNH2 or FADH2 −0.20 Cytochrome bc1 complex Coenzyme Q10ox / Coenzyme Q10red +0.06 Cytochrome bc1 complex Cytochrome box / Cytochrome bred +0.12 Complex IV Cytochrome cox / Cytochrome cred +0.22 Complex IV Cytochrome aox / Cytochrome ared +0.29 Complex IV O2 / HO− +0.82 Conditions: pH = 7

NADH-coenzyme Q oxidoreductase (complex I)

Complex I or NADH-Q oxidoreductase. The abbreviations are discussed in the text. In all diagrams of respiratory complexes here, the matrix is at the bottom, with the intermembrane space above.

NADH dehydrogenase

Structure of NADH dehydrogenase

Electron transport chain. NADH dehydrogenase is "I", at the left

NAD+ to NADH

FMN to FMNH2

CoQ to CoQH2

The structure of the peripheral domain of a NADH dehydrogenase related protein; bacterial FMN dehydrogenase PDB 2FUG. This structure omits a large transmembrane domain, which lies to the bottom of the image and extends to the right. This section of the complex lies in the mitochondrial matrix.

The electron carriers of the NADH dehydrogenease complex. Seven primary iron sulphur centers lie in a line down the peripheral arm of the complex to carry electrons from the site of NADH dehydration to ubiquinone. The iron sulphur group on the right is not found in the eukaryotic complex. Note: This image includes two errors. At the top, it should indicate NADH → NAD+ via a FMN electron carrier/cofactor. At the bottom, it should indicate Ubiquinone → Ubiquinol.

NADH dehydrogenase (EC 1.6.5.3) (also referred to as "NADH:quinone reductase" or "Complex I") is an enzyme located in the inner mitochondrial membrane that catalyzes the transfer of electrons from NADH to coenzyme Q (CoQ). It is the "entry enzyme" of oxidative phosphorylation in the mitochondria.

Function NADH Dehydrogenase is the first enzyme (Complex I) of the mitochondrial electron transport chain. There are three energy-transducing enzymes in the electron transport chain - NADH dehydrogenase (Complex I), Coenzyme Q – cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV). NADH dehydrogenase is the largest and most complicated enzyme of the electron transport chain.

The reaction of NADH dehydrogenase is:

NADH + H+ + CoQ + 4H+in → NAD+ + CoQH2 + 4H+

out

In this process, the complex translocates four protons across the inner membrane per molecule of oxidized NADH, helping to build the electrochemical potential used to produce ATP.

The reaction can be reversed - referred to as aerobic succinate-supported NAD+ reduction - in the presence of a high membrane potential, but the exact catalytic mechanism remains unknown.

Complex I may have a role in triggering apoptosis. In fact, there has been shown to be a correlation between mitochondrial activities and programmed cell death (PCD) during somatic embryo development.

Mechanism All redox reactions take place in the extramembranous portion of NADH dehydrogenase. NADH initially binds to NADH dehydrogenase, and transfers two electrons to the flavin mononucleotide (FMN) prosthetic group of complex I, creating FMNH2. The electron acceptor - the isoalloxazine ring - of FMN is identical to that of FAD. The electrons are then transferred through the second prosthetic group of NADH dehydrogenase via a series of iron-sulfur (Fe-S) clusters, and finally to coenzyme Q (ubiquinone). This electron flow causes four hydrogen ions to be pumped out of the mitochondrial matrix. Ubiquinone (CoQ) accepts two electrons to be reduced to ubiquionol (CoQH2).

Composition and structure NADH Dehydrogenase is the largest of the respiratory complexes. In mammals, the enzyme contains 45 separate polypeptide chains. Of particular functional importance are the flavin prosthetic group (FMN) and eight iron-sulfur clusters (FeS). Of the 45 subunits, seven are encoded by the mitochondrial genome.

The structure is an "L" shape with a long membrane domain (with around 60 trans-membrane helices) and a hydrophilic peripheral domain, which includes all the known redox centres and the NADH binding site. Whereas the structure of the eukaryotic

complex is not well characterised, the peripheral/hydrophilic domain of the complex from a bacterium (Thermus thermophilus) has been crystallised (PDB 2FUG).

A recent study by Roessler et al. (2010) used electron paramagnetic resonance (EPR) spectra and double electron-electron resonance (DEER) to determine the path of electron transfer through the iron-sulfur complexes, which are located in the hydrophilic domain. Seven of these clusters form a chain from the flavin to the quinone binding sites; the eighth cluster is located on the other side of the flavin, and its function is unknown. The EPR and DEER results suggest an alternating or “roller-coaster” potential energy profile for the electron transfer between the active sites and along the iron-sulfur clusters, which can optimize the rate of electron travel and allow efficient energy conversion in Complex I.

A simulational study by Hayashi et. al. further identified the electron tunneling pathways in atomic resolution based on the tunneling current theory. The distinct pathways between neighboring Fe/S clusters primarily consist of two cysteine ligands and one additional key residue, which was supported by sensitivity of simulated electron transfer rates to their mutations and their conservation among various complex I homologues. Internal water between protein subunits was identified as an essential mediator enhancing the overall electron transfer rate to achieve physiologically significant value.

Inhibitors The best-known inhibitor of Complex I is rotenone (commonly used as an organic pesticide). Rotenone and rotenoids are isoflavonoids occurring in several genera of tropical plants such as Antonia (Loganiaceae), Derris and Lonchocarpus (Faboideae, Fabaceae). There have been reports of Indians using rotenone-containing plants to fish - due to its ichthyotoxic effect - as early as the 17th century. Rotenone binds to the ubiquinone binding site of Complex I as well as piericidin A, another potent inhibitor with a close structural homologue to ubiquinone.

Despite more than 50 years of study of NADH dehydrogenase, no inhibitors blocking the electron flow inside the enzyme have been found. Hydrophobic inhibitors like rotenone or piericidin most likely disrupt the electron transfer between the terminal FeS cluster N2 and ubiquinone. It has been shown that long-term systemic inhibition of Complex I by rotenone can induce selective degeneration of dopaminergic neurons.

NADH dehydrogenase is also blocked by adenosine diphosphate ribose - a reversible competitive inhibitor of NADH oxidation by binding to the enzyme at the nucleotide binding site. Both hydrophylic NADH and hydrophobic ubiquinone analogs act at the beginning and the end of the internal electron-transport pathway, respectively.

The acetogenin family are the most potent Complex I inhibitors. They have been shown to crosslink to the ND2 subunit, which suggests that ND2 is essential for quinone-binding. Interestingly, Rolliniastatin-2, an acetogenin, is the first Complex I inhibitor found that does not share the same binding site as rotenone.

Active/de-active transition The catalytic properties of eukaryotic Complex I are not simple. Two catalytically and structurally distinct forms exist in any given preparation of the enzyme: one is the fully competent, so-called “active” A-form and the other is the catalytically silent, dormant, “de-activated”, D-form. After exposure of idle enzyme to elevated, but physiological temperatures (>30°C) in the absence of substrate, the enzyme converts to the D-form. This form is catalytically incompetent but can be activated by the slow reaction (k~4 min-

1) of NADH oxidation with subsequent ubiquinone reduction. After one or several turnovers the enzyme becomes active and can catalyse physiological NADH:ubiquinone reaction at a much higher rate (k~104 min-1). In the presence of divalent cations (Mg2+, Ca2+), or at alkaline pH the activation takes much longer.

The high activation energy (270 kJ/mol) of the deactivation process indicates the occurrence of major conformational changes in the organisation of the Complex I. However, until now, the only conformational difference observed between these two forms is the number of cysteine residues exposed at the surface of the enzyme. Treatment of the D-form of complex I with the sulfhydryl reagents N-Ethylmaleimide or DTNB irreversibly blocks critical cysteine residue(s), abolishing the ability of the enzyme to respond to activation, thus inactivating it irreversibly. The A-form of complex I is insensitive to sulfhydryl reagents.

It was found that these conformational changes may have a very important physiological significance. The de-active, but not the active form of Complex I was susceptible to inhibition by nitrosothiols and peroxynitrite. It is likely that transition from the active to the deactive form of complex I takes place during pathological conditions when the turnover of the enzyme is limited at physiological temperatures, such as during hypoxia, or when the tissue nitric oxide:oxygen ratio increases (i.e. metabolic hypoxia).

Production of superoxide Recent investigations suggest that Complex I is a potent source of reactive oxygen species. Complex I can produce superoxide (as well as hydrogen peroxide), through at least two different pathways. During forward electron transfer, only very small amounts of superoxide are produced (probably less than 0.1% of the overall electron flow).

During reverse electron transfer, Complex I might be the most important site of superoxide production within mitochondria, with up to 5% of electrons being diverted to superoxide formation. Reverse electron transfer, the process by which electrons from the reduced ubiquinol pool (supplied by succinate dehydrogenase, glycerol-3-phosphate dehydrogenase, or dihydro-oorotate dehydrogenase in mammalian mitochondria) pass through Complex I to reduce NAD+ to NADH, driven by the inner mitochondrial membrane potential electric potential. Although it is not precisely known under what pathological conditions reverse-electron transfer would occur in vivo, in vitro

experiments indicate that it can be a very potent source of superoxide when succinate concentrations are high and oxaloacetate or malate concentrations are low.

Superoxide is a reactive oxygen species that contributes to cellular oxidative stress and is linked to neuromuscular diseases and aging. NADH dehdyrogenase produces superoxide by transferring one electron from FMNH2 to oxygen (O2). The radical flavin leftover is unstable, and transfers the remaining electron to the iron-sulfur centers. Interestingly, it is the ratio of NADH to NAD+ that determines the rate of superoxide formation.

Pathology Mutations in the subunits of Complex I can cause mitochondrial diseases, including Leigh syndrome. Point mutations in various Complex I subunits derived from mitochondrial DNA (mtDNA) can also result in Leber's Hereditary Optic Neuropathy. There is some evidence that Complex I defects may play a role in the etiology of Parkinson's disease, perhaps because of reactive oxygen species (Complex I can, like Complex III, leak electrons to oxygen, forming highly toxic superoxide).

Although the exact etiology of Parkinson’s disease is unclear, it is likely that mitochondrial dysfunction, along with proteasome inhibition and environmental toxins, may play a large role. In fact, the inhibition of Complex I has been shown to cause the production of peroxides and a decrease in proteasome activity, which may lead to Parkinson’s disease. Additionally, Esteves et al. (2010) found that cell lines with Parkinson’s disease show increased proton leakage in Complex I, which causes decreased maximum respiratory capacity.

Recent studies have examined other roles of NADH dehydrogenase activity in the brain. Andreazza et al. (2010) found that the level of Complex I activity was significantly decreased in patients with bipolar disorder, but not in patients with depression or schizophrenia. They found that patients with bipolar disorder showed increased protein oxidation and nitration in their prefrontal cortex. These results suggest that future studies should target Complex I for potential therapeutic studies for bipolar disorder. Similarly, Moran et al. (2010) found that patients with severe Complex I deficiency showed decreased oxygen consumption rates and slower growth rates. However, they found that mutations in different genes in Complex I lead to different phenotypes, thereby explaining the variations of pathophysiological manifestations of Complex I deficiency.

Exposure to pesticides can also inhibit Complex I and cause disease symptoms. For example, chronic exposure to low levels of dichlorvos, an organophosphate used as a pesticide, has been shown to cause liver dysfunction. This occurs because dichlorvos alters Complex I and II activity levels, which leads to decreased mitochondrial electron transfer activities and decreased ATP synthesis.

Genes The following is a list of humans genes that encode components of the NADH dehydrogenase (ubiquinone) complex:

• NADH dehydrogenase (ubiquinone) 1 alpha subcomplex o NDUFA1 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1,

7.5kDa o NDUFA2 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2,

8kDa o NDUFA3 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3,


9kDa o NDUFA4L – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-

like o NDUFA4L2 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,

4-like 2 o NDUFA5 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5,



14.5kDa o NDUFA8 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8,




14.7kDa o NDUFA12 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 o NDUFA13 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 o NDUFAB1 – NADH dehydrogenase (ubiquinone) 1, alpha/beta

subcomplex, 1, 8kDa o NDUFAF1 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,

assembly factor 1 o NDUFAF2 – NADH dehydrogenase (ubiquinone) 1 alpha subcomplex,



assembly factor 4 o NADH dehydrogenase (ubiquinone) 1 beta subcomplex

o NDUFB1 – NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7kDa










o NDUFB11 – NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17.3kDa

• NADH dehydrogenase (ubiquinone) 1, subcomplex unknown o NDUFC1 – NADH dehydrogenase (ubiquinone) 1, subcomplex unknown,

1, 6kDa o NDUFC2 – NADH dehydrogenase (ubiquinone) 1, subcomplex unknown,

2, 14.5kDa • NADH dehydrogenase (ubiquinone) Fe-S protein

o NDUFS1 – NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADH-coenzyme Q reductase)








• NADH dehydrogenase (ubiquinone) flavoprotein 1 o NDUFV1 – NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa

o NDUFV2 – NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa o NDUFV3 – NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa

• mitochondrially encoded NADH dehydrogenase subunit o MT-ND1 - mitochondrially encoded NADH dehydrogenase subunit 1 o MT-ND2 - mitochondrially encoded NADH dehydrogenase subunit 2 o MT-ND3 - mitochondrially encoded NADH dehydrogenase subunit 3 o MT-ND4 - mitochondrially encoded NADH dehydrogenase subunit 4 o MT-ND4L - mitochondrially encoded NADH dehydrogenase subunit 4L o MT-ND5 - mitochondrially encoded NADH dehydrogenase subunit 5 o MT-ND6 - mitochondrially encoded NADH dehydrogenase subunit 6

Succinate-Q oxidoreductase (complex II)

Complex II: Succinate-Q oxidoreductase

Succinate-Q oxidoreductase, also known as complex II or succinate dehydrogenase, is a second entry point to the electron transport chain. It is unusual because it is the only enzyme that is part of both the citric acid cycle and the electron transport chain. Complex II consists of four protein subunits and contains a bound flavin adenine dinucleotide (FAD) cofactor, iron–sulfur clusters, and a heme group that does not participate in electron transfer to coenzyme Q, but is believed to be important in decreasing production of reactive oxygen species. It oxidizes succinate to fumarate and reduces ubiquinone. As this reaction releases less energy than the oxidation of NADH, complex II does not transport protons across the membrane and does not contribute to the proton gradient.

In some eukaryotes, such as the parasitic worm Ascaris suum, an enzyme similar to complex II, fumarate reductase (menaquinol:fumarate oxidoreductase, or QFR), operates in reverse to oxidize ubiquinol and reduce fumarate. This allows the worm to survive in the anaerobic environment of the large intestine, carrying out anaerobic oxidative phosphorylation with fumarate as the electron acceptor. Another unconventional function of complex II is seen in the malaria parasite Plasmodium falciparum. Here, the reversed action of complex II as an oxidase is important in regenerating ubiquinol, which the parasite uses in an unusual form of pyrimidine biosynthesis.

Electron transfer flavoprotein-Q oxidoreductase

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-Q oxidoreductase), also known as electron transferring-flavoprotein dehydrogenase, is a third entry point to the electron transport chain. It is an enzyme that accepts electrons from electron-transferring flavoprotein in the mitochondrial matrix, and uses these electrons to reduce ubiquinone. This enzyme contains a flavin and a [4Fe–4S] cluster, but, unlike the other respiratory complexes, it attaches to the surface of the membrane and does not cross the lipid bilayer.

In mammals, this metabolic pathway is important in beta oxidation of fatty acids and catabolism of amino acids and choline, as it accepts electrons from multiple acetyl-CoA dehydrogenases. In plants, ETF-Q oxidoreductase is also important in the metabolic responses that allow survival in extended periods of darkness.

Q-cytochrome c oxidoreductase (complex III)

Shematic illustration of complex III reactions

The coenzyme Q: cytochrome c — oxidoreductase, sometimes called the cytochrome bc1 complex, and at other times complex III, is the third complex in the electron transport chain (EC 1.10.2.2), playing a critical role in biochemical generation of ATP (oxidative phosphorylation). Complex III is a multisubunit transmembrane lipoprotein encoded by both the mitochondrial (cytochrome b) and the nuclear genomes (all other subunits). Complex III is present in the mitochondria of all animals and all aerobic eukaryotes and the inner membranes of most eubacteria. Mutations in Complex III cause exercise intolerance as well as multisystem disorders.

Structure

Structure of complex III

Compared to the other major proton-pumping subunits of the electron transport chain, the number of subunits found can be small, as small as three polypeptide chains. This number does increase, and eleven subunits are found in higher animals. Three subunits have prosthetic groups. The cytochrome b subunit has two b-type hemes (bL and bH), the cytochrome c subunit has one c-type heme (c1), and the Rieske Iron Sulfur Protein subunit (ISP) has a two iron, two sulfur iron-sulfur cluster (2Fe•2S).

Structures of complex III: PDB 1KYO, PDB 1L0L

Reaction It catalyzes the reduction of cytochrome c by oxidation of coenzyme Q (CoQ) and the concomitant pumping of 4 protons from the mitochondrial matrix to the intermembrane space:

QH2 + 2 cytochrome c (FeIII) + 2 H+in → Q + 2 cytochrome c (FeII) + 4 H+

out

In the process called Q cycle, two protons are consumed from the matrix (M), four protons are released into the inter membrane space (IM) and two electrons are passed to cytochrome c.

Reaction Mechanism

The Q cycle

The reaction mechanism for complex III (Cytochrome bc1, Coenzyme Q: Cytochrome C Oxidoreductase) is named the Q cycle or the ubiquinone cycle as mentioned above. In

this cycle four protons get released into the P or Positive side (inter membrane space) but only two protons get taken up from the N or Negative side (matrix). As a result a proton gradient is formed across the membrane. Also, two ubiquinols get oxidized to ubiquinones and one ubiquinone gets reduced to ubiquinol! All this is accomplished by the transfer of two electrons from two ubiquinols to two cytochrome c's as well as two electrons from the same two ubiquinols to a ubiquinone. The reaction goes as follows.

1. Ubiquinol binds to cytochrome b. 2. The 2Fe/2S center and BL Heme each pull an electron off the bound ubiquinone, and two hydrogens are released into the intermembrane space. 3. The 2Fe/2S center transfers its electron to cytochrome c1 and the BL Heme transfers its electron to the BH Heme. 4. Cytocrome c1 transfers its electron to a water-soluble cytochrome c and the BH Heme transfers its electron to a nearby ubiquinone, turning the ubiquinone into a ubisemiquinone. 5. Cytochrome c diffuses and the fully oxidized ubiquinone is released. 6. Another ubiquinol binds to cytochrome b. 7. The 2Fe/2S center and BL Heme each pull an electron off the bound ubiquinone, and two hydrogens are released into the intermembrane space. 8. The 2Fe/2S center transfers its electron to cytochrome c1, and the BL Heme transfers its electron to the BH Heme. 9. Cytocrome c1 then transfers its electron to a water-soluble cytochrome c, and the BH Heme transfers its electron as well as two hydrogens from the matrix to the nearby ubisemiquinone, turning the ubisemiquinone into a ubiquinol. 10.The fully oxidized ubiquinone and ubiquinol are released.

Inhibitors of complex III There are three distinct groups of Complex III inhibitors.

• Antimycin A binds to the Qi site and inhibits the transfer of electrons in Complex III from heme bH to oxidized Q (Qi site inhibitor).

• Myxothiazol and stigmatellin binds to the Qo site and inhibits the transfer of electrons from reduced QH2 to the Rieske Iron sulfur protein. Myxothiazol and stigmatellin bind to distinct pockets within the Qo site.

o Myxothiazol binds very close to cytochrome bL (hence termed a "proximal" inhibitor).

o Stigmatellin binds near the Rieske Iron sulfur protein, with which it strongly interacts.

Some have been commercialized as fungicides (the strobilurin derivatives, best known of which is azoxystrobin; QoI inhibitors) and as anti-malaria agents (atovaquone).

Oxygen free radicals A small fraction of electrons leave the electron transport chain before reaching complex IV. Premature electron leakage to oxygen results in the formation of superoxide. The relevance of this otherwise minor side reaction is that superoxide and other reactive oxygen species are highly toxic and are thought to play a role in several pathologies, as well as aging (the free radical theory of aging). Electron leakage occurs mainly at the Qo site and is stimulated by antimycin A. Antimycin A locks the b hemes in the reduced state by preventing their re-oxidation at the Qi site, which, in turn, causes the steady-state concentrations of the Qo semiquinone to rise, the latter species reacting with oxygen to form superoxide. The effect of high membrane potential is thought to have a similar effect. Superoxide produced at the Qo site can be released both into the mitochondrial matrix and into the intermembrane space (from where it can reach the cytosol). This could be explained by the fact that Complex III might produce superoxide as membrane permeable HO2 rather than as membrane impermeable O2-.

Mutations in Complex III genes in human disease Mutations in Complex III-related genes typically manifest as exercise intolerance. Other mutations have been reported to cause septo-optic displaisa and mutlisystem disorders. However, mutations in BCS1L, a gene responsible for proper maturation of Complex III, can result in Björnstad syndrome and the GRACILE syndrome, which in neonates are lethal conditions that have multisystem and neurologic manifestations typifying severe mitochondrial disorders. The pathogenicity of several mutations has been verified in model systems such as yeast.

The extent to which these various pathologies are due to bioenergetic deficits or overeproduction of superoxide is presently unknown.

Cytochrome c oxidase (complex IV)

Complex IV: cytochrome c oxidase

Cytochrome c oxidase, also known as complex IV, is the final protein complex in the electron transport chain. The mammalian enzyme has an extremely complicated structure and contains 13 subunits, two heme groups, as well as multiple metal ion cofactors – in all three atoms of copper, one of magnesium and one of zinc.

This enzyme mediates the final reaction in the electron transport chain and transfers electrons to oxygen, while pumping protons across the membrane. The final electron acceptor oxygen, which is also called the terminal electron acceptor, is reduced to water in this step. Both the direct pumping of protons and the consumption of matrix protons in

the reduction of oxygen contribute to the proton gradient. The reaction catalyzed is the oxidation of cytochrome c and the reduction of oxygen:

Alternative reductases and oxidases

Many eukaryotic organisms have electron transport chains that differ from the much-studied mammalian enzymes described above. For example, plants have alternative NADH oxidases, which oxidize NADH in the cytosol rather than in the mitochondrial matrix, and pass these electrons to the ubiquinone pool. These enzymes do not transport protons, and, therefore, reduce ubiquinone without altering the electrochemical gradient across the inner membrane.

Another example of a divergent electron transport chain is the alternative oxidase, which is found in plants, as well as some fungi, protists, and possibly some animals. This enzyme transfers electrons directly from ubiquinol to oxygen.

The electron transport pathways produced by these alternative NADH and ubiquinone oxidases have lower ATP yields than the full pathway. The advantages produced by a shortened pathway are not entirely clear. However, the alternative oxidase is produced in response to stresses such as cold, reactive oxygen species, and infection by pathogens, as well as other factors that inhibit the full electron transport chain. Alternative pathways might, therefore, enhance an organisms' resistance to injury, by reducing oxidative stress.

Organization of complexes

The original model for how the respiratory chain complexes are organized was that they diffuse freely and independently in the mitochondrial membrane. However, recent data suggest that the complexes might form higher-order structures called supercomplexes or "respirasomes." In this model, the various complexes exist as organized sets of interacting enzymes. These associations might allow channeling of substrates between the various enzyme complexes, increasing the rate and efficiency of electron transfer. Within such mammalian supercomplexes, some components would be present in higher amounts than others, with some data suggesting a ratio between complexes I/II/III/IV and the ATP synthase of approximately 1:1:3:7:4. However, the debate over this supercomplex hypothesis is not completely resolved, as some data do not appear to fit with this model.

Prokaryotic electron transport chains In contrast to the general similarity in structure and function of the electron transport chains in eukaryotes, bacteria and archaea possess a large variety of electron-transfer enzymes. These use an equally wide set of chemicals as substrates. In common with eukaryotes, prokaryotic electron transport uses the energy released from the oxidation of

a substrate to pump ions across a membrane and generate an electrochemical gradient. In the bacteria, oxidative phosphorylation in Escherichia coli is understood in most detail, while archaeal systems are at present poorly understood.

The main difference between eukaryotic and prokaryotic oxidative phosphorylation is that bacteria and archaea use many different substances to donate or accept electrons. This allows prokaryotes to grow under a wide variety of environmental conditions. In E. coli, for example, oxidative phosphorylation can be driven by a large number of pairs of reducing agents and oxidizing agents, which are listed below. The midpoint potential of a chemical measures how much energy is released when it is oxidized or reduced, with reducing agents having negative potentials and oxidizing agents positive potentials.

Respiratory enzymes and substrates in E. coli.

Respiratory enzyme Redox pair

Midpoint potential

(Volts) Formate dehydrogenase Bicarbonate / Formate −0.43 Hydrogenase Proton / Hydrogen −0.42 NADH dehydrogenase NAD+ / NADH −0.32 Glycerol-3-phosphate dehydrogenase DHAP / Gly-3-P −0.19

Pyruvate oxidase Acetate + Carbon dioxide / Pyruvate ?

Lactate dehydrogenase Pyruvate / Lactate −0.19

D-amino acid dehydrogenase 2-oxoacid + ammonia / D-amino acid ?

Glucose dehydrogenase Gluconate / Glucose −0.14 Succinate dehydrogenase Fumarate / Succinate +0.03 Ubiquinol oxidase Oxygen / Water +0.82 Nitrate reductase Nitrate / Nitrite +0.42 Nitrite reductase Nitrite / Ammonia +0.36 Dimethyl sulfoxide reductase DMSO / DMS +0.16 Trimethylamine N-oxide reductase TMAO / TMA +0.13 Fumarate reductase Fumarate / Succinate +0.03

As shown above, E. coli can grow with reducing agents such as formate, hydrogen, or lactate as electron donors, and nitrate, DMSO, or oxygen as acceptors. The larger the difference in midpoint potential between an oxidizing and reducing agent, the more energy is released when they react. Out of these compounds, the succinate/fumarate pair is unusual, as its midpoint potential is close to zero. Succinate can therefore be oxidized to fumarate if a strong oxidizing agent such as oxygen is available, or fumarate can be

reduced to succinate using a strong reducing agent such as formate. These alternative reactions are catalyzed by succinate dehydrogenase and fumarate reductase, respectively.

Some prokaryotes use redox pairs that have only a small difference in midpoint potential. For example, nitrifying bacteria such as Nitrobacter oxidize nitrite to nitrate, donating the electrons to oxygen. The small amount of energy released in this reaction is enough to pump protons and generate ATP, but not enough to produce NADH or NADPH directly for use in anabolism. This problem is solved by using a nitrite oxidoreductase to produce enough proton-motive force to run part of the electron transport chain in reverse, causing complex I to generate NADH.

Prokaryotes control their use of these electron donors and acceptors by varying which enzymes are produced, in response to environmental conditions. This flexibility is possible because different oxidases and reductases use the same ubiquinone pool. This allows many combinations of enzymes to function together, linked by the common ubiquinol intermediate. These respiratory chains therefore have a modular design, with easily interchangeable sets of enzyme systems.

In addition to this metabolic diversity, prokaryotes also possess a range of isozymes – different enzymes that catalyze the same reaction. For example, in E. coli, there are two different types of ubiquinol oxidase using oxygen as an electron acceptor. Under highly aerobic conditions, the cell uses an oxidase with a low affinity for oxygen that can transport two protons per electron. However, if levels of oxygen fall, they switch to an oxidase that transfers only one proton per electron, but has a high affinity for oxygen.

ATP synthase (complex V)

ATP synthase. The FO proton channel and stalk are shown in blue, the F1 synthase domain in red and the membrane in gray.

ATP synthase, also called complex V, is the final enzyme in the oxidative phosphorylation pathway. This enzyme is found in all forms of life and functions in the same way in both prokaryotes and eukaryotes. The enzyme uses the energy stored in a proton gradient across a membrane to drive the synthesis of ATP from ADP and phosphate (Pi). Estimates of the number of protons required to synthesize one ATP have ranged from three to four, with some suggesting cells can vary this ratio, to suit different conditions.

This phosphorylation reaction is an equilibrium, which can be shifted by altering the proton-motive force. In the absence of a proton-motive force, the ATP synthase reaction will run from right to left, hydrolyzing ATP and pumping protons out of the matrix across the membrane. However, when the proton-motive force is high, the reaction is forced to run in the opposite direction; it proceeds from left to right, allowing protons to flow down their concentration gradient and turning ADP into ATP. Indeed, in the closely related vacuolar type H+-ATPases, the same reaction is used to acidify cellular compartments, by pumping protons and hydrolysing ATP.

ATP synthase is a massive protein complex with a mushroom-like shape. The mammalian enzyme complex contains 16 subunits and has a mass of approximately 600 kilodaltons. The portion embedded within the membrane is called FO and contains a ring of c subunits and the proton channel. The stalk and the ball-shaped headpiece is called F1 and is the site of ATP synthesis. The ball-shaped complex at the end of the F1 portion contains six proteins of two different kinds (three α subunits and three β subunits), whereas the "stalk" consists of one protein: the γ subunit, with the tip of the stalk extending into the ball of α and β subunits. Both the α and β subunits bind nucleotides, but only the β subunits catalyze the ATP synthesis reaction. Reaching along the side of the F1 portion and back into the membrane is a long rod-like subunit that anchors the α and β subunits into the base of the enzyme.

As protons cross the membrane through the channel in the base of ATP synthase, the FO proton-driven motor rotates. Rotation might be caused by changes in the ionization of amino acids in the ring of c subunits causing electrostatic interactions that propel the ring of c subunits past the proton channel. This rotating ring in turn drives the rotation of the central axle (the γ subunit stalk) within the α and β subunits. The α and β subunits are prevented from rotating themselves by the side-arm, which acts as a stator. This movement of the tip of the γ subunit within the ball of α and β subunits provides the energy for the active sites in the β subunits to undergo a cycle of movements that produces and then releases ATP.

Mechanism of ATP synthase. ATP is shown in red, ADP and phosphate in pink and the rotating γ subunit in black.

This ATP synthesis reaction is called the binding change mechanism and involves the active site of a β subunit cycling between three states. In the "open" state, ADP and phosphate enter the active site (shown in brown in the diagram). The protein then closes up around the molecules and binds them loosely – the "loose" state (shown in red). The enzyme then changes shape again and forces these molecules together, with the active site in the resulting "tight" state (shown in pink) binding the newly produced ATP molecule with very high affinity. Finally, the active site cycles back to the open state, releasing ATP and binding more ADP and phosphate, ready for the next cycle.

In some bacteria and archaea, ATP synthesis is driven by the movement of sodium ions through the cell membrane, rather than the movement of protons. Archaea such as Methanococcus also contain the A1Ao synthase, a form of the enzyme that contains additional proteins with little similarity in sequence to other bacterial and eukaryotic ATP synthase subunits. It is possible that, in some species, the A1Ao form of the enzyme is a specialized sodium-driven ATP synthase, but this might not be true in all cases.

Reactive oxygen species Molecular oxygen is an ideal terminal electron acceptor because it is a strong oxidizing agent. The reduction of oxygen does involve potentially harmful intermediates. Although the transfer of four electrons and four protons reduces oxygen to water, which is harmless, transfer of one or two electrons produces superoxide or peroxide anions, which are dangerously reactive.

These reactive oxygen species and their reaction products, such as the hydroxyl radical, are very harmful to cells, as they oxidize proteins and cause mutations in DNA. This cellular damage might contribute to disease and is proposed as one cause of aging.

The cytochrome c oxidase complex is highly efficient at reducing oxygen to water, and it releases very few partly reduced intermediates; however small amounts of superoxide anion and peroxide are produced by the electron transport chain. Particularly important is the reduction of coenzyme Q in complex III, as a highly reactive ubisemiquinone free radical is formed as an intermediate in the Q cycle. This unstable species can lead to electron "leakage" when electrons transfer directly to oxygen, forming superoxide. As the production of reactive oxygen species by these proton-pumping complexes is greatest at high membrane potentials, it has been proposed that mitochondria regulate their activity to maintain the membrane potential within a narrow range that balances ATP production against oxidant generation. For instance, oxidants can activate uncoupling proteins that reduce membrane potential.

To counteract these reactive oxygen species, cells contain numerous antioxidant systems, including antioxidant vitamins such as vitamin C and vitamin E, and antioxidant enzymes such as superoxide dismutase, catalase, and peroxidases, which detoxify the reactive species, limiting damage to the cell.

Inhibitors There are several well-known drugs and toxins that inhibit oxidative phosphorylation. Although any one of these toxins inhibits only one enzyme in the electron transport chain, inhibition of any step in this process will halt the rest of the process. For example, if oligomycin inhibits ATP synthase, protons cannot pass back into the mitochondrion. As a result, the proton pumps are unable to operate, as the gradient becomes too strong for them to overcome. NADH is then no longer oxidized and the citric acid cycle ceases to operate because the concentration of NAD+ falls below the concentration that these enzymes can use.

Compounds Use Effect on oxidative phosphorylation Cyanide Carbon monoxide Azide

Poisons Inhibit the electron transport chain by binding more strongly than oxygen to the Fe–Cu center in cytochrome c oxidase, preventing the reduction of oxygen.

Oligomycin Antibiotic Inhibits ATP synthase by blocking the flow of protons through the Fo subunit.

CCCP 2,4-Dinitrophenol Poisons

Ionophores that disrupt the proton gradient by carrying protons across a membrane. This ionophore uncouples proton pumping from ATP synthesis because it carries protons across the inner mitochondrial membrane.

Rotenone Pesticide Prevents the transfer of electrons from complex I to ubiquinone by blocking to the ubiquinone-binding site.

Malonate and oxaloacetate Competitive inhibitors of succinate dehydrogenase

(complex II).

Not all inhibitors of oxidative phosphorylation are toxins. In brown adipose tissue, regulated proton channels called uncoupling proteins can uncouple respiration from ATP synthesis. This rapid respiration produces heat, and is particularly important as a way of maintaining body temperature for hibernating animals, although these proteins may also have a more general function in cells' responses to stress.

History The field of oxidative phosphorylation began with the report in 1906 by Arthur Harden of a vital role for phosphate in cellular fermentation, but initially only sugar phosphates were known to be involved. However, in the early 1940s, the link between the oxidation of sugars and the generation of ATP was firmly established by Herman Kalckar, confirming the central role of ATP in energy transfer that had been proposed by Fritz

Albert Lipmann in 1941. Later, in 1949, Morris Friedkin and Albert L. Lehninger proved that the coenzyme NADH linked metabolic pathways such as the citric acid cycle and the synthesis of ATP.

For another twenty years, the mechanism by which ATP is generated remained mysterious, with scientists searching for an elusive "high-energy intermediate" that would link oxidation and phosphorylation reactions. This puzzle was solved by Peter D. Mitchell with the publication of the chemiosmotic theory in 1961. At first, this proposal was highly controversial, but it was slowly accepted and Mitchell was awarded a Nobel prize in 1978. Subsequent research concentrated on purifying and characterizing the enzymes involved, with major contributions being made by David E. Green on the complexes of the electron-transport chain, as well as Efraim Racker on the ATP synthase. A critical step towards solving the mechanism of the ATP synthase was provided by Paul D. Boyer, by his development in 1973 of the "binding change" mechanism, followed by his radical proposal of rotational catalysis in 1982. More recent work has included structural studies on the enzymes involved in oxidative phosphorylation by John E. Walker, with Walker and Boyer being awarded a Nobel Prize in 1997.

Chapter- 2

Chemiosmosis

Chemiosmosis is the movement of ions across a selectively-permeable membrane, down their electrochemical gradient. More specifically, it relates to the generation of ATP by the movement of hydrogen ions across a membrane during cellular respiration.

An Ion gradient has potential energy and can be used to power chemical reactions when the ions pass through a channel (red).

Hydrogen ions (protons) will diffuse from an area of high proton concentration to an area of lower proton concentration. Peter Mitchell proposed that an electrochemical concentration gradient of protons across a membrane could be harnessed to make ATP. He linked this process to osmosis, the diffusion of water across a membrane, which is why it is called chemiosmosis.

ATP synthase is the enzyme that makes ATP by chemiosmosis. It allows protons to pass through the membrane using the kinetic energy to phosphorylate ADP making ATP. The generation of ATP by chemiosmosis occurs in chloroplasts and mitochondria as well as in some bacteria.

The Chemiosmotic Theory Peter D. Mitchell proposed the chemiosmotic hypothesis in 1961. The theory suggests essentially that most ATP synthesis in respiring cells come from the electrochemical gradient across the inner membranes of mitochondria by using the energy of NADH and FADH2 formed from the breaking down of energy rich molecules such as glucose.

Chemiosmosis in a mitochondrion

Molecules such as glucose are metabolized to produce acetyl CoA as an energy-rich intermediate. The oxidation of acetyl CoA in the mitochondrial matrix is coupled to the reduction of a carrier molecule such as NAD and FAD. The carriers pass electrons to the electron transport chain (ETC) in the inner mitochondrial membrane, which in turn pass them to other proteins in the ETC. The energy available in the electrons is used to pump protons from the matrix across the inner mitochondrial membrane, storing energy in the

form of a transmembrane electrochemical gradient. The protons move back across the inner membrane through the enzyme ATP synthase. The flow of protons back into the matrix of the mitochondrion via ATP synthase provides enough energy for ADP to combine with inorganic phosphate to form ATP. The electrons and protons at the last pump in the ETC are taken up by oxygen to form water.

This was a radical proposal at the time, and was not well accepted. The prevailing view was that the energy of electron transfer was stored as a stable high potential intermediate, a chemically more conservative concept.

The problem with the older paradigm is that no high energy intermediate was ever found, and the evidence for proton pumping by the complexes of the electron transfer chain grew too great to be ignored. Eventually the weight of evidence began to favor the chemiosmotic hypothesis, and in 1978, Peter Mitchell was awarded the Nobel Prize in Chemistry.

Chemiosmotic coupling is important for ATP production in chloroplasts and many bacteria.

The proton-motive force In all mitochondria, chloroplasts and in many bacteria, chemiosmosis involves the proton-motive force (PMF) in some step. This can be described as the storing of energy as a combination of proton and voltage gradients across a membrane. The chemical potential energy refers to the difference in concentration of the protons on each side of the membrane and the electrical potential energy as a consequence of the charge separation across the membrane (when the protons move without a counter-ion, such as an electron).

In most cases the proton motive force is generated by an electron transport chain which acts as a proton pump, using the energy in electrons from an electron carrier to pump protons (hydrogen ions) out across the membrane, creating a separation of charge across the membrane. In mitochondria, free energy released from electrons by the electron transport chain is used to move protons from the mitochondrial matrix to the intermembrane space of the mitochondrion. Moving the protons out of the mitochondrion creates a lower concentration of positively charged protons inside it, resulting in a slight negative on the inside of the membrane: The electrical potential gradient is about -200 mV, inside negative. This charge difference and the proton concentration difference create a combined electrochemical gradient across the membrane. This electrochemical gradient for protons is both a concentration and charge difference and is often called the proton motive force (PMF). In mitochondria, the PMF is almost entirely made up of the electrical component but in chloroplasts the PMF is made up mostly of the pH gradient. In either case, the PMF needs to be about 50 kJ/mol for the ADP synthase to be able to make ATP.

In mitochondria

A diagram of chemiosmotic phosphorylation

Chemiosmotic phosphorylation is the third pathway that produces ATP from inorganic phosphate and an ADP molecule. This process is part of oxidative phosphorylation.

The complete breakdown of glucose in the presence of oxygen is called cellular respiration. The last steps of this process occur in mitochondria. The reduced molecules NADH and FADH2 are generated by the Krebs cycle and glycolysis. These molecules pass electrons to an electron transport chain, which uses the energy released to create a proton gradient across the inner mitochondrial membrane. ATP synthase then uses the energy stored in this gradient to make ATP. This process is called oxidative phosphorylation because oxygen is the final electron acceptor and the energy released by reducing oxygen to water is used to phosphorylate ADP and generate ATP.

In plants The light reactions of photosynthesis generate energy by chemiosmosis. Light energy (photons) are received by the antenna complex of Photosystem 2, which excites a pair of electrons to a higher energy level. These electrons travel down an electron transport chain, causing H+ to diffuse across the thylakoid membrane into the inter-thylakoid space. These H+ are then transported down their concentration gradient through an enzyme called ATP-synthase, creating ATP by phosphorylation of ADP to ATP. The electrons from the initial light reaction reach Photosystem 1, then are raised to a higher energy level by light energy and then received by an electron receptor and reduce NADP+ to NADPH+H. The electrons from Photosystem 2 get replaced by the splitting of water, called "photolysis." Two water molecules must be split in order to gain 2 electrons (as well as O2, the oxygen eudicots require for survival).

In prokaryotes Bacteria and archaea also can use chemiosmosis to generate ATP. Cyanobacteria, green sulfur bacteria, and purple bacteria create energy by a process called photophosphorylation. These bacteria use the energy of light to create a proton gradient using a photosynthetic electron transport chain. Non-photosynthetic bacteria such as E. coli also contain ATP synthase.

In fact, mitochondria and chloroplasts are believed to have been formed when early eukaryotic cells ingested bacteria that could create energy using chemiosmosis. This is called the endosymbiotic theory.

Chapter- 3

Mitochondrion

Two mitochondria from mammalian lung tissue displaying their matrix and membranes as shown by electron microscopy

Schematic of typical animal cell, showing subcellular components. Organelles: (1) nucleolus (2) nuclear membrane (3) Ribosomes (4) Vesicle (5) Rough endoplasmic reticulum (ER) (6) Golgi body (7) Cytoskeleton (8) Smooth ER (9) Mitochondria (13) Centrioles within centrosome

In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 10 micrometers (μm) in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria have been implicated in several human diseases, including mitochondrial disorders and cardiac dysfunction, and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος or mitos, thread + χονδρίον or chondrion, granule.

Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria. The organelle is composed of

compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria, whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported. The mitochondrial proteome is thought to be dynamically regulated. Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.

Structure

A mitochondrion contains outer and inner membranes composed of phospholipid bilayers and proteins. The two membranes, however, have different properties. Because of this double-membraned organization, there are five distinct compartments within the mitochondrion. There is the outer mitochondrial membrane, the intermembrane space (the space between the outer and inner membranes), the inner mitochondrial membrane, the cristae space (formed by infoldings of the inner membrane), and the matrix (space within the inner membrane).

Outer membrane

The outer mitochondrial membrane, which encloses the entire organelle, has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about 1:1 by weight). It contains large numbers of integral proteins called porins. These porins form

channels that allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling sequence at their N-terminus binds to a large multisubunit protein called translocase of the outer membrane, which then actively moves them across the membrane. Disruption of the outer membrane permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell death. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER) membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in ER-mitochondria calcium signaling and involved in the transfer of lipids between the ER and mitochondria.

Intermembrane space

The intermembrane space is the space between the outer membrane and the inner membrane. Because the outer membrane is freely permeable to small molecules, the concentrations of small molecules such as ions and sugars in the intermembrane space is the same as the cytosol. However, large proteins must have a specific signaling sequence to be transported across the outer membrane, so the protein composition of this space is different from the protein composition of the cytosol. One protein that is localized to the intermembrane space in this way is cytochrome c.

Inner membrane

The inner mitochondrial membrane contains proteins with five types of functions:

1. Those that perform the redox reactions of oxidative phosphorylation 2. ATP synthase, which generates ATP in the matrix 3. Specific transport proteins that regulate metabolite passage into and out of the

matrix 4. Protein import machinery. 5. Mitochondria fusion and fission protein

It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio (more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in 1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin contains four fatty acids rather than two and may help to make the inner membrane impermeable. Unlike the outer membrane, the inner membrane doesn't contain porins and is highly impermeable to all molecules. Almost all ions and molecules require special membrane transporters to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner membrane formed by the action of the enzymes of the electron transport chain.

Cristae

Cross-sectional image of cristae in rat liver mitochondrion to demonstrate the likely 3D structure and relationship to the inner membrane.

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical liver mitochondria the area of the inner membrane is about five times greater than the outer membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP, such as muscle cells, contain even more cristae. These folds are studded with small round bodies known as F1 particles or oxysomes. These are not simple random folds but rather invaginations of the inner membrane, which can affect overall chemiosmotic function.

Recent work has suggested that, within the recently identified longer mitochondria, their many cristae also act as weak mirrors at approximately half-wavelength intervals, (which collectively amounts to efficient mirrors which could offer the right conditions for laser activity). Hence it is proposed that this allows the production of directional ultraviolet or visible-light photon-signals, possibly related to mitosis control.

Matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase contained in the inner membrane. The matrix contains a highly-

concentrated mixture of hundreds of enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and the citric acid cycle.

Mitochondria have their own genetic material, and the machinery to manufacture their own RNAs and proteins. A published human mitochondrial DNA sequence revealed 16,569 base pairs encoding 37 total genes: 22 tRNA, 2 rRNA, and 13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner mitochondrial membrane, along with proteins encoded by genes that reside in the host cell's nucleus.

Organization and distribution Mitochondria are found in nearly all eukaryotes. They vary in number and location according to cell type. A single mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found in human liver cells, with about 1000–2000 mitochondria per cell making up 1/5th of the cell volume. The mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. Often they form a complex 3D branching network inside the cell with the cytoskeleton. The association with the cytoskeleton determines mitochondrial shape, which can affect the function as well. Recent evidence suggests vimentin, one of the components of the cytoskeleton, is critical to the association with the cytoskeleton.

Function The most prominent roles of mitochondria are to produce ATP (i.e., phosphorylation of ADP) through respiration, and to regulate cellular metabolism. The central set of reactions involved in ATP production are collectively known as the citric acid cycle, or the Krebs Cycle. However, the mitochondrion has many other functions in addition to the production of ATP.

Energy conversion

A dominant role for the mitochondria is the production of ATP, as reflected by the large number of proteins in the inner membrane for this task. This is done by oxidizing the major products of glucose, pyruvate, and NADH, which are produced in the cytosol. This process of cellular respiration, also known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is limited, the glycolytic products will be metabolized by anaerobic respiration, a process that is independent of the mitochondria. The production of ATP from glucose has an approximately 13-fold higher yield during aerobic respiration compared to anaerobic respiration. Recently it has been shown that plant mitochondria can produce a limited amount of ATP without oxygen by using the alternate substrate nitrite.

Pyruvate and the citric acid cycle

Each pyruvate molecule produced by glycolysis is actively transported across the inner mitochondrial membrane, and into the matrix where it is oxidized and combined with coenzyme A to form CO2, acetyl-CoA, and NADH.

The acetyl-CoA is the primary substrate to enter the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or Krebs cycle. The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process, produces reduced cofactors (three molecules of NADH and one molecule of FADH2) that are a source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted to an ATP).

NADH and FADH2: the electron transport chain

The electron transport chain in the mitochondrion is the site of oxidative phosphorylation in eukaryotes. The NADH and succinate generated in the citric acid cycle are oxidized, providing energy to power ATP synthase.

Photosynthetic electron transport chain of the thylakoid membrane

An electron transport chain (ETC) couples a reaction between an electron donor (such as NADH) and an electron acceptor (such as O2) to the transfer of H+ ions across a membrane, through a set of mediating biochemical reactions. These H+ ions are used to produce adenosine triphosphate (ATP), the main energy intermediate in living organisms, as they move back across the membrane. Electron transport chains are used for extracting energy from sunlight (photosynthesis) and from redox reactions such as the oxidation of sugars (respiration).

In chloroplasts, light drives the conversion of water to oxygen and NADP+ to NADPH and a transfer of H+ ions. NADPH is used as an electron donor for carbon fixation. In mitochondria, it is the conversion of oxygen to water, NADH to NAD+ and succinate to fumarate that drives the transfer of H+ ions. While some bacteria have electron transport chains similar to those in chloroplasts or mitochondria, other bacteria use different electron donors and acceptors. Both the respiratory and photosynthetic electron transport chains are major sites of premature electron leakage to oxygen, thus being major sites of superoxide production and drivers of oxidative stress.

Background The electron transport chain consists of a spatially separated series of redox reactions in which electrons are transferred from a donor molecule to an acceptor molecule. The underlying force driving these reactions is the Gibbs free energy of the reactants and products. The Gibbs free energy is the energy available ("free") to do work. Any reaction that decreases the overall Gibbs free energy of a system will proceed spontaneously. The transfer of electrons proceeds from an electron donor to an acceptor.

ATP synthase, an enzyme highly conserved among all domains of life, is powered by a transmembrane proton electrochemical gradient, which is the result of a series of redox reactions. The function of the electron transport chain is to produce this gradient.

The transmembrane electrochemical potential gradient may enable transport of molecules across membranes. It may also enable mechanical work, such as rotating bacterial flagella, or to produce ATP, which provides energy to power other cellular reactions. A small amount of ATP is available from substrate-level phosphorylation, for example, in glycolysis. In most organisms the majority of ATP is generated in electron transport chains, while only some obtain ATP by fermentation.

Electron transport chains in mitochondria Most eukaryotic cells contain mitochondria, which produce ATP from products of the Krebs cycle, fatty acid oxidation, and amino acid oxidation. At the mitochondrial inner membrane, electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. The electron chain comprises a series of electron acceptors, each more electronegative than the previous one. Oxygen is the most electronegative electron acceptor in the chain, hence its role as the terminal acceptor. This enzymatic series produces a proton gradient across the mitochondrial membrane, producing a thermodynamic state that has the potential to do work. A small percentage of electrons prematurely leak to oxygen, resulting in the formation of the toxic free-radical superoxide, a molecule thought to contribute to a number of diseases and aging.

Mitochondrial redox carriers

Stylized representation of the ETC. Energy obtained through the transfer of electrons (black arrows) down the ETC is used to pump protons (red arrows) from the mitochondrial matrix into the intermembrane space, creating an electrochemical proton gradient across the mitochondrial inner membrane (IMM) called ΔΨ. This electrochemical proton gradient allows ATP synthase (ATP-ase) to use the flow of H+

through the enzyme back into the matrix to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the Krebs cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes them to coenzyme Q (ubiquinone; labeled UQ), which also receives electrons from complex II (succinate dehydrogenase; labeled II). UQ passes electrons to complex III (cytochrome bc1 complex; labeled III), which passes them to cytochrome c (cyt c). Cyt c passes electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses the electrons and hydrogen ions to reduce molecular oxygen to water.

Four membrane-bound complexes have been identified in mitochondria. Each is an extremely complex transmembrane structure that is embedded in the inner membrane. Three of them are proton pumps. The structures are electrically connected by lipid-soluble electron carriers and water-soluble electron carriers. The overall electron transport chain

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2 ↑ Complex II

Complex I

In Complex I (NADH dehydrogenase, also called NADH:ubiquinone oxidoreductase; EC 1.6.5.3) two electrons are removed from NADH and transferred to a lipid-soluble carrier, ubiquinone (Q). The reduced product, ubiquinol (QH2) freely diffuses within the membrane, and Complex I translocates four protons (H+) across the membrane, thus producing a proton gradient. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of the main sites of production of harmful superoxide.

The pathway of electrons occurs as follows:

NADH is oxidized to NAD+, by reducing Flavin mononucleotide to FMNH2 in one two-electron step. FMNH2 is then oxidized in two one-electron steps, through a semiquinone intermediate. Each electron thus transfers from the FMNH2 to an Fe-S cluster, from the Fe-S cluster to ubiquinone (Q). Transfer of the first electron results in the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form, QH2. During this process, four protons are translocated from the mitochondrial matrix to the intermembrane space, creating a proton gradient that generates ATP through oxidative phosphorylation.

Complex II

In Complex II (succinate dehydrogenase; EC 1.3.5.1) additional electrons are delivered into the quinone pool (Q) originating from succinate and transferred (via FAD) to Q. Complex II consists of four protein subunits: SDHA, SDHB, SDHC, and SDHD. Other electron donors (e.g., fatty acids and glycerol 3-phosphate) also direct electrons into Q (via FAD).

Complex III

In Complex III (cytochrome bc1 complex; EC 1.10.2.2) two electrons are removed from QH2 at the QO site and sequentially transferred to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. The two other electrons sequentially pass across the protein to the Qi site where the quinone part of ubiquinone is reduced to quinol. A proton gradient is formed by two quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site. (in total six protons are translocated: two protons reduce quinone to quinol and four protons are released from two ubiquinol molecules). The bc1 complex does not 'pump' protons, but helps build the proton gradient by an asymmetric absorption/release of protons.

When electron transfer is reduced (by a high membrane potential or respiratory inhibitors such as antimycin A), Complex III may leak electrons to molecular oxygen, resulting in superoxide formation.

Complex IV

In Complex IV (cytochrome c oxidase; EC 1.9.3.1) four electrons are removed from four molecules of cytochrome c and transferred to molecular oxygen (O2), producing two molecules of water. At the same time, four protons are translocated across the membrane, contributing to the proton gradient. The activity of cytochrome c is inhibited by cyanide.

Coupling with oxidative phosphorylation

According to the chemiosmotic coupling hypothesis, proposed by Nobel Prize in Chemistry winner Peter D. Mitchell, the electron transport chain and oxidative phosphorylation are coupled by a proton gradient across the inner mitochondrial membrane. The efflux of protons from the mitochondrial matrix creates an electrochemical gradient (proton gradient). This gradient is used by the FOF1 ATP synthase complex to make ATP via oxidative phosphorylation. ATP synthase is sometimes described as Complex V of the electron transport chain. The FO component of ATP synthase acts as an ion channel that provides for a proton flux back into the mitochondrial matrix. This reflux releases free energy produced during the generation of the oxidized forms of the electron carriers (NAD+ and Q). The free energy is used to drive ATP synthesis, catalyzed by the F1 component of the complex. Coupling with oxidative phosphorylation is a key step for ATP production. However, in specific cases, uncoupling the two processes may be biologically useful. The uncoupling protein, thermogenin—present in the inner mitochondrial membrane of brown adipose tissue—provides for an alternative flow of protons back to the inner mitochondrial matrix. This alternative flow results in thermogenesis rather than ATP production and generates heat. Synthetic uncouplers (e.g., 2,4-dinitrophenol) also exist, and, at high doses, are lethal.

Summary

In the mitochondrial electron transport chain electrons move from an electron donor (NADH or QH2) to a terminal electron acceptor (O2) via a series of redox reactions. These reactions are coupled to the creation of a proton gradient across the mitochondrial inner membrane. There are three proton pumps: I, III, and IV. The resulting transmembrane proton gradient is used to make ATP via ATP synthase.

The reactions catalyzed by Complex I and Complex III work roughly at equilibrium. This means that these reactions are readily reversible, by increasing the concentration of the products relative to the concentration of the reactants (for example, by increasing the proton gradient). ATP synthase is also readily reversible. Thus ATP can be used to build a proton gradient, which in turn can be used to make NADH. This process of reverse electron transport is important in many prokaryotic electron transport chains.

Electron transport chains in bacteria In eukaryotes, NADH is the most important electron donor. The associated electron transport chain is

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2 where Complexes I, III and IV are proton pumps, while Q and cytochrome c are mobile electron carriers. The electron acceptor is molecular oxygen.

In prokaryotes (bacteria and archaea) the situation is more complicated, because there are several different electron donors and several different electron acceptors. The generalized electron transport chain in bacteria is:

Donor Donor Donor ↓ ↓ ↓ dehydrogenase → quinone → bc1 → cytochrome ↓ ↓ oxidase(reductase) oxidase(reductase) ↓ ↓ Acceptor Acceptor

Note that electrons can enter the chain at three levels: at the level of a dehydrogenase, at the level of the quinone pool, or at the level of a mobile cytochrome electron carrier. These levels correspond to successively more positive redox potentials, or to successively decreased potential differences relative to the terminal electron acceptor. In other words, they correspond to successively smaller Gibbs free energy changes for the overall redox reaction Donor → Acceptor.

Individual bacteria use multiple electron transport chains, often simultaneously. Bacteria can use a number of different electron donors, a number of different dehydrogenases, a number of different oxidases and reductases, and a number of different electron

acceptors. For example, E. coli (when growing aerobically using glucose as an energy source) uses two different NADH dehydrogenases and two different quinol oxidases, for a total of four different electron transport chains operating simultaneously.

A common feature of all electron transport chains is the presence of a proton pump to create a transmembrane proton gradient. Bacterial electron transport chains may contain as many as three proton pumps, like mitochondria, or they may contain only one or two. They always contain at least one proton pump.

Electron donors

In the present day biosphere, the most common electron donors are organic molecules. Organisms that use organic molecules as an energy source are called organotrophs. Organotrophs (animals, fungi, protists) and phototrophs (plants and algae) constitute the vast majority of all familiar life forms.

Some prokaryotes can use inorganic matter as an energy source. Such organisms are called lithotrophs ("rock-eaters"). Inorganic electron donors include hydrogen, carbon monoxide, ammonia, nitrite, sulfur, sulfide, and ferrous iron. Lithotrophs have been found growing in rock formations thousands of meters below the surface of Earth. Because of their volume of distribution, lithotrophs may actually outnumber organotrophs and phototrophs in our biosphere.

The use of inorganic electron donors as an energy source is of particular interest in the study of evolution. This type of metabolism must logically have preceded the use of organic molecules as an energy source.

Dehydrogenases

Bacteria can use a number of different electron donors. When organic matter is the energy source, the donor may be NADH or succinate, in which case electrons enter the electron transport chain via NADH dehydrogenase (similar to Complex I in mitochondria) or succinate dehydrogenase (similar to Complex II). Other dehydrogenases may be used to process different energy sources: formate dehydrogenase, lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, H2 dehydrogenase (hydrogenase), etc. Some dehydrogenases are also proton pumps; others simply funnel electrons into the quinone pool.

Most of dehydrogenases are synthesized only when needed. Depending on the environment in which they find themselves, bacteria select different enzymes from their DNA library and synthesize only those that are needed for growth.Enzymes that are synthesized only when needed are said to be 'inducible'.

Quinone carriers

Quinones are mobile, lipid-soluble carriers that shuttle electrons (and protons) between large, relatively immobile macromolecular complexes embedded in the membrane. Bacteria use ubiquinone (the same quinone that mitochondria use) and related quinones such as menaquinone.

Proton pumps

A proton pump is any process that creates a proton gradient across a membrane. Protons can be physically moved across a membrane; this is seen in mitochondrial Complexes I and IV. The same effect can be produced by moving electrons in the opposite direction. The result is the disappearance of a proton from the cytoplasm and the appearance of a proton in the periplasm. Mitochondrial Complex III uses this second type of proton pump, which is mediated by a quinone (the Q cycle).

Some dehydrogenases are proton pumps; others are not. Most oxidases and reductases are proton pumps, but some are not. Cytochrome bc1 is a proton pump found in many, but not all, bacteria (it is not found in E. coli). As the name implies, bacterial bc1 is similar to mitochondrial bc1 (Complex III).

Proton pumps are the heart of the electron transport process. They produce the transmembrane electrochemical gradient that supplies energy to the cell.

Cytochrome electron carriers

Cytochromes are pigments that contain iron. They are found in two very different environments.

Some cytochromes are water-soluble carriers that shuttle electrons to and from large, immobile macromolecular structures imbedded in the membrane. The mobile cytochrome electron carrier in mitochondria is cytochrome c. Bacteria use a number of different mobile cytochrome electron carriers.

Other cytochromes are found within macromolecules such as Complex III and Complex IV. They also function as electron carriers, but in a very different, intramolecular, solid-state environment.

Electrons may enter an electron transport chain at the level of a mobile cytochrome or quinone carrier. For example, electrons from inorganic electron donors (nitrite, ferrous iron, etc.) enter the electron transport chain at the cytochrome level. When electrons enter at a redox level greater than NADH, the electron transport chain must operate in reverse to produce this necessary, higher-energy molecule.

Terminal oxidases and reductases

When bacteria grow in aerobic environments, the terminal electron acceptor (O2) is reduced to water by an enzyme called an oxidase. When bacteria grow in anaerobic environments, the terminal electron acceptor is reduced by an enzyme called a reductase.

In mitochondria the terminal membrane complex (Complex IV) is cytochrome oxidase. Aerobic bacteria use a number of different terminal oxidases. For example, E. coli does not have a cytochrome oxidase or a bc1 complex. Under aerobic conditions, it uses two different terminal quinol oxidases (both proton pumps) to reduce oxygen to water.

Anaerobic bacteria, which do not use oxygen as a terminal electron acceptor, have terminal reductases individualized to their terminal acceptor. For example, E. coli can use fumarate reductase, nitrate reductase, nitrite reductase, DMSO reductase, or trimethylamine-N-oxide reductase, depending on the availability of these acceptors in the environment.

Most terminal oxidases and reductases are inducible. They are synthesized by the organism as needed, in response to specific environmental conditions.

Electron acceptors

Just as there are a number of different electron donors (organic matter in organotrophs, inorganic matter in lithotrophs), there are a number of different electron acceptors, both organic and inorganic. If oxygen is available, it is invariably used as the terminal electron acceptor, because it generates the greatest Gibbs free energy change and produces the most energy.

In anaerobic environments, different electron acceptors are used, including nitrate, nitrite, ferric iron, sulfate, carbon dioxide, and small organic molecules such as fumarate.

Since electron transport chains are redox processes, they can be described as the sum of two redox pairs. For example, the mitochondrial electron transport chain can be described as the sum of the NAD+/NADH redox pair and the O2/H2O redox pair. NADH is the electron donor and O2 is the electron acceptor.

Not every donor-acceptor combination is thermodynamically possible. The redox potential of the acceptor must be more positive than the redox potential of the donor. Furthermore, actual environmental conditions may be far different from standard conditions (1 molar concentrations, 1 atm partial pressures, pH = 7), which apply to standard redox potentials. For example, hydrogen-evolving bacteria grow at an ambient partial pressure of hydrogen gas of 10-4 atm. The associated redox reaction, which is thermodynamically favorable in nature, is thermodynamically impossible under “standard” conditions.

Summary

Bacterial electron transport pathways are, in general, inducible. Depending on their environment, bacteria can synthesize different transmembrane complexes and produce different electron transport chains in their cell membranes. Bacteria select their electron transport chains from a DNA library containing multiple possible dehydrogenases, terminal oxidases and terminal reductases. The situation is often summarized by saying that electron transport chains in bacteria are branched, modular, and inducible.

Photosynthetic electron transport chains In oxidative phosphorylation, electrons are transferred from a high-energy electron donor (e.g., NADH) to an electron acceptor (e.g., O2) through an electron transport chain. In photophosphorylation, the energy of sunlight is used to create a high-energy electron donor and an electron acceptor. Electrons are then transferred from the donor to the acceptor through another electron transport chain.

Photosynthetic electron transport chains have many similarities to the oxidative chains discussed above. They use mobile, lipid-soluble carriers (quinones) and mobile, water-soluble carriers (cytochromes, etc.). They also contain a proton pump. It is remarkable that the proton pump in all photosynthetic chains resembles mitochondrial Complex III.

Summary Electron transport chains are redox reactions that transfer electrons from an electron donor to an electron acceptor. The transfer of electrons is coupled to the translocation of protons across a membrane, producing a proton gradient. The proton gradient is used to produce useful work.

The coupling of thermodynamically favorable to thermodynamically unfavorable biochemical reactions by biological macromolecules is an example of an emergent property – a property that could not have been predicted, even given full knowledge of the primitive geochemical systems from which these macromolecules evolved. It is an open question whether such emergent properties evolve only by chance, or whether they necessarily evolve in any large biogeochemical system, given the underlying laws of physics.

Heat production

Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. The process is mediated by a proton channel called thermogenin, or UCP1. Thermogenin is a 33kDa protein first discovered in 1973. Thermogenin is

primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and decreases with age.

Storage of calcium ions

Mitochondria (M) within a chondrocyte stained for calcium as shown by electron microscopy.

The concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release makes them very good "cytosolic buffers" for calcium. The endoplasmic reticulum (ER) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by a calcium uniporter on the inner mitochondrial membrane. It is primarily driven by the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur via a sodium-calcium exchange protein or via "calcium-induced-calcium-release" pathways. This can initiate calcium spikes or calcium waves with large changes in the membrane potential. These can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells.

Additional functions

Mitochondria play a central role in many other metabolic tasks, such as:

• Regulation of the membrane potential

• Apoptosis-programmed cell death • Calcium signaling (including calcium-evoked apoptosis) • Cellular proliferation regulation • Regulation of cellular metabolism • Certain heme synthesis reactions • Steroid synthesis.

Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases.

Origin

Electron micrograph of a mitochondrion showing its mitochondrial matrix and membranes

The endosymbiotic theory concerns the origins of mitochondria, plastids (e.g. chloroplasts), and possibly other organelles of eukaryotic cells. According to this theory, certain organelles originated as free-living bacteria that were taken inside another cell as endosymbionts. Mitochondria developed from proteobacteria (in particular, Rickettsiales or close relatives) and chloroplasts from cyanobacteria.

History The endosymbiotic theory was first articulated by the Russian botanist Konstantin Mereschkowski in 1905. Mereschkowski was familiar with work by botanist Andreas Schimper, who had observed in 1883 that the division of chloroplasts in green plants closely resembled that of free-living cyanobacteria, and who had himself tentatively proposed (in a footnote) that green plants had arisen from a symbiotic union of two organisms. Ivan Wallin extended the idea of an endosymbiotic origin to mitochondria in the 1920s. These theories were initially dismissed or ignored. More detailed electron microscopic comparisons between cyanobacteria and chloroplasts (for example studies by Hans Ris), combined with the discovery that plastids and mitochondria contain their

own DNA (which by that stage was recognized to be the hereditary material of organisms) led to a resurrection of the idea in the 1960s.

The endosymbiotic theory was advanced and substantiated with microbiological evidence by Lynn Margulis in a 1967 paper, The Origin of Mitosing Eukaryotic Cells. In her 1981 work Symbiosis in Cell Evolution she argued that eukaryotic cells originated as communities of interacting entities, including endosymbiotic spirochaetes that developed into eukaryotic flagella and cilia. This last idea has not received much acceptance, because flagella lack DNA and do not show ultrastructural similarities to bacteria or archaea. According to Margulis and Dorion Sagan, "Life did not take over the globe by combat, but by networking" (i.e., by cooperation). The possibility that peroxisomes may have an endosymbiotic origin has also been considered, although they lack DNA. Christian de Duve proposed that they may have been the first endosymbionts, allowing cells to withstand growing amounts of free molecular oxygen in the Earth's atmosphere. However, it now appears that they may be formed de novo, contradicting the idea that they have a symbiotic origin.

It is believed that over millenia these endosymbionts transferred some of their own DNA to the host cell's nucleus during the evolutionary transition from a symbiotic community to an instituted eukaryotic cell (called "serial endosymbiosis"). This hypothesis is thought to be possible because it is known today from scientific observation that transfer of DNA occurs between bacteria species, even if they are not closely related. Bacteria can take up DNA from their surroundings and have a limited ability to incorporate it into their own genome.

Evidence Evidence that mitochondria and plastids arose from bacteria is as follows:

• New mitochondria and plastids are formed only through a process similar to binary fission. In some algae, such as Euglena, the plastids can be destroyed by certain chemicals or prolonged absence of light without otherwise affecting the cell. In such a case, the plastids will not regenerate.

• They are surrounded by two or more membranes, and the innermost of these shows differences in composition from the other membranes of the cell. The composition is like that of a bacterial cell membrane.

• Both mitochondria and plastids contain DNA that is different from that of the cell nucleus and that is similar to that of bacteria (in being circular in shape and in its size).

• DNA sequence analysis and phylogenetic estimates suggest that nuclear DNA contains genes that probably came from plastids.

• These organelles' ribosomes are like those found in bacteria (70s). • Proteins of organelle origin, like those of bacteria, use N-formylmethionine as the

initiating amino acid. • Much of the internal structure and biochemistry of plastids, for instance the

presence of thylakoids and particular chlorophylls, is very similar to that of

cyanobacteria. Phylogenetic estimates constructed with bacteria, plastids, and eukaryotic genomes also suggest that plastids are most closely related to cyanobacteria.

• Mitochondria have several enzymes and transport systems similar to those of bacteria.

• Some proteins encoded in the nucleus are transported to the organelle, and both mitochondria and plastids have small genomes compared to bacteria. This is consistent with an increased dependence on the eukaryotic host after forming an endosymbiosis. Most genes on the organellar genomes have been lost or moved to the nucleus. Most genes needed for mitochondrial and plastid function are located in the nucleus. Many originate from the bacterial endosymbiont.

• Plastids are present in very different groups of protists, some of which are closely related to forms lacking plastids. This suggests that if chloroplasts originated de novo, they did so multiple times, in which case their close similarity to each other is difficult to explain.

• Many of these protists contain "primary" plastids that have not yet been acquired from other plastid-containing eukaryotes.

• Among eukaryotes that acquired their plastids directly from bacteria (known as Primoplantae), the glaucophyte algae have chloroplasts that strongly resemble cyanobacteria. In particular, they have a peptidoglycan cell wall between the two membranes.

• Mitochondria and plastids are similar in size to bacteria.

Secondary endosymbiosis Primary endosymbiosis involves the engulfment of a bacterium by another free living organism. Secondary endosymbiosis occurs when the product of primary endosymbiosis is itself engulfed and retained by another free living eukaryote. Secondary endosymbiosis has occurred several times and has given rise to extremely diverse groups of algae and other eukaryotes. Some organisms can take opportunistic advantage of a similar process, where they engulf an alga and use the products of its photosynthesis, but once the prey item dies (or is lost) the host returns to a free living state. Obligate secondary endosymbionts become dependent on their organelles and are unable to survive in their absence. RedToL, the Red Algal Tree of Life Initiative funded by the National Science Foundation highlights the role red algae or Rhodophyta played in the evolution of our planet through secondary endosymbiosis.

One possible secondary endosymbiosis in process has been observed by Okamoto & Inouye (2005). The heterotrophic protist Hatena behaves like a predator until it ingests a green alga, which loses its flagella and cytoskeleton, while Hatena, now a host, switches to photosynthetic nutrition, gains the ability to move towards light and loses its feeding apparatus.

The process of secondary endosymbiosis left its evolutionary signature within the unique topography of plastid membranes. Secondary plastids are surrounded by three (in euglenophytes and some dinoflagellates) or four membranes (in haptophytes,

heterokonts, cryptophytes, and chlorarachniophytes). The two additional membranes are thought to correspond to the plasma membrane of the engulfed alga and the phagosomal membrane of the host cell. The endosymbiotic acquisition of a eukaryote cell is represented in the cryptophytes; where the remnant nucleus of the red algal symbiont (the nucleomorph) is present between the two inner and two outer plastid membranes.

Despite the diversity of organisms containing plastids, the morphology, biochemistry, genomic organisation, and molecular phylogeny of plastid RNAs and proteins suggest a single origin of all extant plastids – although this theory is still debated.

Problems • Neither mitochondria nor plastids can survive in oxygen or outside the cell,

having lost many essential genes required for survival. The standard counterargument points to the large timespan that the mitochondria/plastids have co-existed with their hosts. In this view, genes and systems that were no longer necessary were simply deleted, or in many cases, transferred into the host genome instead. (In fact these transfers constitute an important way for the host cell to regulate plastid or mitochondrial activity.)

• The transfer of genes from mitochondria and plastids to the “host genome” or cell nucleus raises a further problem: why were all genes not transferred? In other words, why do any genes at all remain in mitochondria and plastids? This problem is addressed by the CoRR Hypothesis, which proposes that genes and respiratory chain proteins are Co-located for Redox Regulation.

• A large cell, especially one equipped for phagocytosis, has vast energetic requirements, which cannot be achieved without the internalisation of energy production (due to the decrease in the surface area to volume ratio as size increases). This implies that, for the cell to gain mitochondria, it could not have been a eukaryote, and must have been a bacterium. This in turn implies that the emergence of the eukaryotes and the formation of mitochondria were achieved simultaneously. This may be explained by possibly a very close symbiotic relationship between two types of bacteria which eventually led to gene exchange and engulfing of the mitochondria precursors through partial fusion or engulfing by the host bacteria.

• Genetic analysis of small eukaryotes that lack mitochondria shows that they all still retain genes for mitochondrial proteins. This implies that all these eukaryotes once had mitochondria. This objection can be answered if, as suggested above, the origin of the eukaryotes coincided with the formation of mitochondria.

These last two problems are accounted for in the Hydrogen hypothesis.

Genome

Mitochondrial DNA

Electron microscopy reveals mitochondrial DNA in discrete foci. Bars: 200 nm. (A) Cytoplasmic section after immunogold labelling with anti-DNA; gold particles marking mtDNA are found near the mitochondrial membrane. (B) Whole mount view of cytoplasm after extraction with CSK buffer and immunogold labelling with anti-DNA; mtDNA (marked by gold particles) resists extraction. From Iborra et al., 2004.

Mitochondrial DNA (mtDNA) is the DNA located in organelles called mitochondria, structures within eukaryotic cells that convert the chemical energy from food into a form that cells can use, ATP. Most other DNA present in eukaryotic organisms is found in the cell nucleus.

Replication mtDNA is replicated by the DNA polymerase gamma complex which is composed of a 140 kDa catalytic DNA polymerase encoded by the POLG gene and a 55 kDa accessory subunit encoded by the POLG2 gene. During embryogenesis, replication of mtDNA is strictly down-regulated from the fertilized oocyte through the preimplantation embryo. At

the blastocyst stage, the onset of mtDNA replication is specific to the cells of the trophectoderm. In contrast, the cells of the inner cell mass restrict mtDNA replication until they receive the signals to differentiate to specific cell types.

Origin Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today's eukaryotic cells. This theory is called the endosymbiotic theory. Each mitochondrion is estimated to contain 2-10 mtDNA copies. In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution.

Mitochondrial inheritance In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include simple dilution (an egg contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000), degradation of sperm mtDNA in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism, this single parent (uniparental) pattern of mtDNA inheritance is found in most animals, most plants and in fungi as well.

Female inheritance

In sexual reproduction, mitochondria are normally inherited exclusively from the mother. The mitochondria in mammalian sperm are usually destroyed by the egg cell after fertilization. Also, most mitochondria are present at the base of the sperm's tail, which is used for propelling the sperm cells. Sometimes the tail is lost during fertilization. In 1999 it was reported that paternal sperm mitochondria (containing mtDNA) are marked with ubiquitin to select them for later destruction inside the embryo. Some in vitro fertilization techniques, particularly injecting a sperm into an oocyte, may interfere with this.

The fact that mitochondrial DNA is maternally inherited enables researchers to trace maternal lineage far back in time. (Y chromosomal DNA, paternally inherited, is used in an analogous way to trace the agnate lineage.) This is accomplished in humans by sequencing one or more of the hypervariable control regions (HVR1 or HVR2) of the mitochondrial DNA, as with a genealogical DNA test. HVR1 consists of about 440 base pairs. These 440 base pairs are then compared to the control regions of other individuals (either specific people or subjects in a database) to determine maternal lineage. Most often, the comparison is made to the revised Cambridge Reference Sequence. Vilà et al. have published studies tracing the matrilineal descent of domestic dogs to wolves. The concept of the Mitochondrial Eve is based on the same type of analysis, attempting to discover the origin of humanity by tracking the lineage back in time.

Because mtDNA is not highly conserved and has a rapid mutation rate, it is useful for studying the evolutionary relationships - phylogeny - of organisms. Biologists can determine and then compare mtDNA sequences among different species and use the comparisons to build an evolutionary tree for the species examined.

Because mtDNA is transmitted from mother to child (both male and female), it can be a useful tool in genealogical research into a person's maternal line.

Male inheritance

It has been reported that mitochondria can occasionally be inherited from the father in some species such as mussels. Paternally inherited mitochondria have additionally been reported in some insects such as fruit flies, honeybees, and periodical cicadas.

Evidence supports rare instances of male mitochondrial inheritance in some mammals as well. Specifically, documented occurrences exist for mice, where the male-inherited mitochondria was subsequently rejected. It has also been found in sheep, and in cloned cattle. It has been found in a single case in a human male and was linked to infertility.

While many of these cases involve cloned embryos or subsequent rejection of the paternal mitochondria, others document in vivo inheritance and persistence under lab conditions.

Structure In humans (and probably in metazoans in general), 100-10,000 separate copies of mtDNA are usually present per cell (egg and sperm cells are exceptions). In mammals, each double-stranded circular mtDNA molecule consists of 15,000-17,000 base pairs. The two strands of mtDNA are differentiated by their nucleotide content with the guanine rich strand referred to as the heavy strand, and the cytosine rich strand referred to as the light strand. The heavy strand encodes 28 genes, and the light strand encodes 9 genes for a total of 37 genes. Of the 37 genes, 13 are for proteins (polypeptides), 22 are for transfer RNA (tRNA) and two are for the small and large subunits of ribosomal RNA (rRNA). This pattern is also seen among most metazoans, although in some cases one or more of the 37 genes is absent and the mtDNA size range is greater. Even greater variation in mtDNA gene content and size exists among fungi and plants, although there appears to be a core subset of genes that are present in all eukaryotes (except for the few that have no mitochondria at all). Some plant species have enormous mtDNAs (as many as 2,500,000 base pairs per mtDNA molecule) but, surprisingly, even those huge mtDNAs contain the same number and kinds of genes as related plants with much smaller mtDNAs.

Genes

Transport chain

Many of the genes encode the transport chain:

Category Genes NADH dehydrogenase (complex I)

MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6

Coenzyme Q - cytochrome c reductase/Cytochrome b (complex III)

MT-CYB

cytochrome c oxidase (complex IV) MT-CO1, MT-CO2, MT-CO3

ATP synthase MT-ATP6, MT-ATP8

rRNA

Mitochondrial rRNA is encoded by MT-RNR1 (12S) and MT-RNR2 (16S).

tRNA

The following genes encode tRNA:

Amino Acid 3-Letter 1-Letter MT DNA Alanine Ala A MT-TA Arginine Arg R MT-TR

Asparagine Asn N MT-TN Aspartic acid Asp D MT-TD

Cysteine Cys C MT-TC Glutamic acid Glu E MT-TE

Glutamine Gln Q MT-TQ Glycine Gly G MT-TG Histidine His H MT-TH Isoleucine Ile I MT-TI Leucine Leu L MT-TL1, MT-TL2 Lysine Lys K MT-TK

Methionine Met M MT-TM Phenylalanine Phe F MT-TF

Proline Pro P MT-TP Serine Ser S MT-TS1, MT-TS2

Threonine Thr T MT-TT Tryptophan Trp W MT-TW

Tyrosine Tyr Y MT-TY Valine Val V MT-TV

Mutations

The involvement of mitochondrial DNA in several human diseases

Susceptibility

mtDNA is particularly susceptible to reactive oxygen species generated by the respiratory chain due to its close proximity. Though mtDNA is packaged by proteins and harbors significant DNA repair capacity, these protective functions are less robust than those operating on nuclear DNA and therefore thought to contribute to enhanced susceptibility of mtDNA to oxidative damage.

Genetic illness

Mutations of mitochondrial DNA can lead to a number of illnesses including exercise intolerance and Kearns-Sayre syndrome (KSS), which causes a person to lose full function of heart, eye, and muscle movements. Some evidence suggests that they might be major contributors to the aging process and age-associated pathologies.

Use in identification In humans, mitochondrial DNA spans 16,569 DNA building blocks (base pairs), representing a fraction of the total DNA in cells. Unlike nuclear DNA, which is inherited from both parents and in which genes are rearranged in the process of recombination, there is usually no change in mtDNA from parent to offspring. Although mtDNA also recombines, it does so with copies of itself within the same mitochondrion. Because of this and because the mutation rate of animal mtDNA is higher than that of nuclear DNA, mtDNA is a powerful tool for tracking ancestry through females (matrilineage) and has been used in this role to track the ancestry of many species back hundreds of generations.

Human mtDNA can also be used to help identify individuals. Forensic laboratories occasionally use mtDNA comparison to identify human remains, and especially to identify older unidentified skeletal remains. Although unlike nuclear DNA, mtDNA is not specific to one individual, it can be used in combination with other evidence (anthropological evidence, circumstantial evidence, and the like) to establish identification. mtDNA is also used to exclude possible matches between missing persons and unidentified remains. Many researchers believe that mtDNA is better suited to identification of older skeletal remains than nuclear DNA because the greater number of copies of mtDNA per cell increases the chance of obtaining a useful sample, and because a match with a living relative is possible even if numerous maternal generations separate the two. American outlaw Jesse James's remains were identified using a comparison between mtDNA extracted from his remains and the mtDNA of the son of the female-line great-granddaughter of his sister. Similarly, the remains of Alexandra Feodorovna (Alix of Hesse), last Empress of Russia, and her children were identified by comparison of their mitochondrial DNA with that of Prince Philip, Duke of Edinburgh, whose maternal grandmother was Alexandra’s sister Victoria of Hesse. Similarly to identify Emperor Nicholas II remains his mitochondrial DNA was compared with that of James Carnegie, 3rd Duke of Fife, whose maternal great-grandmother Alexandra of Denmark (Queen Alexandra) was sister of Nicholas II mother Dagmar of Denmark (Empress Maria Feodorovna).

The low effective population size and rapid mutation rate (in animals) makes mtDNA useful for assessing genetic relationships of individuals or groups within a species and also for identifying and quantifying the phylogeny among different species, provided they are not too distantly related. To do this, biologists determine and then compare the mtDNA sequences from different individuals or species. Data from the comparisons is used to construct a network of relationships among the sequences, which provides an estimate of the relationships among the individuals or species from which the mtDNAs

were taken. This approach has limits that are imposed by the rate of mtDNA sequence change. In animals, the high mutation rate makes mtDNA most useful for comparisons of individuals within species and for comparisons of species that are closely or moderately-closely related, among which the number of sequence differences can be easily counted. As the species become more distantly related, the number of sequence differences becomes very large; changes begin to accumulate on changes until an accurate count becomes impossible.

History Mitochondrial DNA was discovered in the 1960s by Margit M. K. Nass and Sylvan Nass by electron microscopy as DNase-sensitive thread inside mitochondria, and by Ellen Haslbrunner, Hans Tuppy and Gottfried Schatz by biochemical assays on highly purified mitochondrial fractions.

The human mitochondrial genome is a circular DNA molecule of about 16 kilobases. It encodes 37 genes: 13 for subunits of respiratory complexes I, III, IV and V, 22 for mitochondrial tRNA (for the 20 standard amino acids, plus an extra gene for leucine and serine), and 2 for rRNA. One mitochondrion can contain two to ten copies of its DNA.

As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats. Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved and polyadenylated to yield mature mRNAs. Not all proteins necessary for mitochondrial function are encoded by the mitochondrial genome; most are coded by genes in the cell nucleus and the corresponding proteins are imported into the mitochondrion. The exact number of genes encoded by the nucleus and the mitochondrial genome differs between species. In general, mitochondrial genomes are circular, although exceptions have been reported. In general, mitochondrial DNA lacks introns, as is the case in the human mitochondrial genome; however, introns have been observed in some eukaryotic mitochondrial DNA, such as that of yeast and protists, including Dictyostelium discoideum.

In animals the mitochondrial genome is typically a single circular chromosome that is approximately 16-kb long and has 37 genes. The genes while highly conserved may vary in location. Curiously this pattern is not found in the human body louse (Pediculus humanus). Instead this mitochondrial genome is arranged in 18 minicircular chromosomes each of which is 3–4 kb long and has one to three genes. This pattern is also found in other sucking lice but not in chewing lice. Recombination has been shown to occur between the minichromosomes. The reason for this difference is not known.

While slight variations on the standard code had been predicted earlier, none was discovered until 1979, when researchers studying human mitochondrial genes determined that they used an alternative code. Many slight variants have been discovered since, including various alternative mitochondrial codes. Further, the AUA, AUC, and AUU codons are all allowable start codons.

Exceptions to the universal genetic code (UGC) in mitochondria

Organism Codon Standard Novel

Mammalian AGA, AGG Arginine Stop codon AUA Isoleucine Methionine UGA Stop codon Tryptophan

Invertebrates AGA, AGG Arginine Serine AUA Isoleucine Methionine UGA Stop codon Tryptophan

Yeast AUA Isoleucine Methionine UGA Stop codon Tryptophan CUA Leucine Threonine

Some of these differences should be regarded as pseudo-changes in the genetic code due to the phenomenon of RNA editing, which is common in mitochondria. In higher plants, it was thought that CGG encoded for tryptophan and not arginine; however, the codon in the processed RNA was discovered to be the UGG codon, consistent with the universal genetic code for tryptophan. Of note, the arthropod mitochondrial genetic code has undergone parallel evolution within a phylum, with some organisms uniquely translating AGG to lysine.

Mitochondrial genomes have far fewer genes than the bacteria from which they are thought to be descended. Although some have been lost altogether, many have been transferred to the nucleus, such as the respiratory complex II protein subunits. This is thought to be relatively common over evolutionary time. A few organisms, such as the Cryptosporidium, actually have mitochondria that lack any DNA, presumably because all their genes have been lost or transferred. In Cryptosporidium, the mitochondria have an altered ATP generation system that renders the parasite resistant to many classical mitochondrial inhibitors such as cyanide, azide, and atovaquone.

Replication and inheritance Mitochondria divide by binary fission similar to bacterial cell division; unlike bacteria, however, mitochondria can also fuse with other mitochondria. The regulation of this division differs between eukaryotes. In many single-celled eukaryotes, their growth and division is linked to the cell cycle. For example, a single mitochondrion may divide synchronously with the nucleus. This division and segregation process must be tightly controlled so that each daughter cell receives at least one mitochondrion. In other eukaryotes (in mammals for example), mitochondria may replicate their DNA and divide mainly in response to the energy needs of the cell, rather than in phase with the cell cycle. When the energy needs of a cell are high, mitochondria grow and divide. When the energy use is low, mitochondria are destroyed or become inactive. In such examples, and

in contrast to the situation in many single celled eukaryotes, mitochondria are apparently randomly distributed to the daughter cells during the division of the cytoplasm.

An individual's mitochondrial genes are not inherited by the same mechanism as nuclear genes. At fertilization of an egg cell by a sperm, the egg nucleus and sperm nucleus each contribute equally to the genetic makeup of the zygote nucleus. In contrast, the mitochondria, and therefore the mitochondrial DNA, usually comes from the egg only. The sperm's mitochondria enter the egg but do not contribute genetic information to the embryo. Instead, paternal mitochondria are marked with ubiquitin to select them for later destruction inside the embryo. The egg cell contains relatively few mitochondria, but it is these mitochondria that survive and divide to populate the cells of the adult organism. Mitochondria are, therefore, in most cases inherited down the female line, known as maternal inheritance. This mode is seen in most organisms including all animals. However, mitochondria in some species can sometimes be inherited paternally. This is the norm among certain coniferous plants, although not in pine trees and yew trees. It has been suggested that it occurs at a very low level in humans.

Uniparental inheritance leads to little opportunity for genetic recombination between different lineages of mitochondria, although a single mitochondrion can contain 2–10 copies of its DNA. For this reason, mitochondrial DNA usually is thought to reproduce by binary fission. What recombination does take place maintains genetic integrity rather than maintaining diversity. However, there are studies showing evidence of recombination in mitochondrial DNA. It is clear that the enzymes necessary for recombination are present in mammalian cells. Further, evidence suggests that animal mitochondria can undergo recombination. The data are a bit more controversial in humans, although indirect evidence of recombination exists. If recombination does not occur, the whole mitochondrial DNA sequence represents a single haplotype, which makes it useful for studying the evolutionary history of populations.

Population genetic studies The near-absence of genetic recombination in mitochondrial DNA makes it a useful source of information for scientists involved in population genetics and evolutionary biology. Because all the mitochondrial DNA is inherited as a single unit, or haplotype, the relationships between mitochondrial DNA from different individuals can be represented as a gene tree. Patterns in these gene trees can be used to infer the evolutionary history of populations. The classic example of this is in human evolutionary genetics, where the molecular clock can be used to provide a recent date for mitochondrial Eve. This is often interpreted as strong support for a recent modern human expansion out of Africa. Another human example is the sequencing of mitochondrial DNA from Neanderthal bones. The relatively large evolutionary distance between the mitochondrial DNA sequences of Neanderthals and living humans has been interpreted as evidence for lack of interbreeding between Neanderthals and anatomically-modern humans.

However, mitochondrial DNA reflects the history of only females in a population and so may not represent the history of the population as a whole. This can be partially overcome by the use of paternal genetic sequences, such as the non-recombining region of the Y-chromosome. In a broader sense, only studies that also include nuclear DNA can provide a comprehensive evolutionary history of a population.

Dysfunction and disease

Mitochondrial diseases

With their central place in cell metabolism, damage — and subsequent dysfunction — in mitochondria is an important factor in a wide range of human diseases. Mitochondrial disorders often present as neurological disorders, but can manifest as myopathy, diabetes, multiple endocrinopathy, or a variety of other systemic manifestations. Diseases caused by mutation in the mtDNA include Kearns-Sayre syndrome, MELAS syndrome and Leber's hereditary optic neuropathy. In the vast majority of cases, these diseases are transmitted by a female to her children, as the zygote derives its mitochondria and hence its mtDNA from the ovum. Diseases such as Kearns-Sayre syndrome, Pearson's syndrome, and progressive external ophthalmoplegia are thought to be due to large-scale mtDNA rearrangements, whereas other diseases such as MELAS syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF), and others are due to point mutations in mtDNA.

In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial proteins. This is the case in Friedreich's ataxia, hereditary spastic paraplegia, and Wilson's disease. These diseases are inherited in a dominance relationship, as applies to most other genetic diseases. A variety of disorders can be caused by nuclear mutations of oxidative phosphorylation enzymes, such as coenzyme Q10 deficiency and Barth syndrome. Environmental influences may interact with hereditary predispositions and cause mitochondrial disease. For example, there may be a link between pesticide exposure and the later onset of Parkinson's disease.

Other pathologies with etiology involving mitochondrial dysfunction include schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus. A common thread thought to link these seemingly-unrelated conditions is cellular damage causing oxidative stress. How exactly mitochondrial dysfunction fits into the etiology of these pathologies is yet to be elucidated.

Possible relationships to aging

Given the role of mitochondria as the cell's powerhouse, there may be some leakage of the high-energy electrons in the respiratory chain to form reactive oxygen species. This can result in significant oxidative stress in the mitochondria with high mutation rates of mitochondrial DNA. A vicious cycle is thought to occur, as oxidative stress leads to mitochondrial DNA mutations, which can lead to enzymatic abnormalities and further

oxidative stress. A number of changes occur to mitochondria during the aging process. Tissues from elderly patients show a decrease in enzymatic activity of the proteins of the respiratory chain. Large deletions in the mitochondrial genome can lead to high levels of oxidative stress and neuronal death in Parkinson's disease. Hypothesized links between aging and oxidative stress are not new and were proposed over 50 years ago; however, there is much debate over whether mitochondrial changes are causes of aging or merely characteristics of aging. One notable study in mice demonstrated shortened lifespan but no increase in reactive oxygen species despite increasing mitochondrial DNA mutations, suggesting that mitochondrial DNA mutations can cause lifespan shortening by other mechanisms. As a result, the exact relationships between mitochondria, oxidative stress, and aging have not yet been settled.

Chapter- 4

Microbial Metabolism

Microbial metabolism is the means by which a microbe obtains the energy and nutrients (e.g. carbon) it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe’s ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.

Types of microbial metabolism

Flow chart to determine the metabolic characteristics of microorganisms

All microbial metabolisms can be arranged according to three principles:

1. How the organism obtains carbon for synthesising cell mass:

• autotrophic – carbon is obtained from carbon dioxide (CO2) • heterotrophic – carbon is obtained from organic compounds • mixotrophic – carbon is obtained from both organic compounds and by fixing

carbon dioxide

2. How the organism obtains reducing equivalents used either in energy conservation or in biosynthetic reactions:

• lithotrophic – reducing equivalents are obtained from inorganic compounds • organotrophic – reducing equivalents are obtained from organic compounds

3. How the organism obtains energy for living and growing:

• chemotrophic – energy is obtained from external chemical compounds • phototrophic – energy is obtained from light

In practice, these terms are almost freely combined. Typical examples are as follows:

• chemolithoautotrophs obtain energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide. Examples: Nitrifying bacteria, Sulfur-oxidizing bacteria, Iron-oxidizing bacteria, Knallgas-bacteria

• photolithoautotrophs obtain energy from light and carbon from the fixation of carbon dioxide, using reducing equivalents from inorganic compounds. Examples: Cyanobacteria (water (H2O) as reducing equivalent donor), Chlorobiaceae, Chromaticaceae (hydrogen sulfide (H2S) as reducing equivalent donor), Chloroflexus (hydrogen (H2) as reducing equivalent donor)

• chemolithoheterotrophs obtain energy from the oxidation of inorganic compounds, but cannot fix carbon dioxide (CO2). Examples: some Thiobacilus, some Beggiatoa, some Nitrobacter spp., Wolinella (with H2 as reducing equivalent donor), some Knallgas-bacteria, some sulfate-reducing bacteria

• chemoorganoheterotrophs obtain energy, carbon, and reducing equivalents for biosynthetic reactions from organic compounds. Examples: most bacteria, e. g. Escherichia coli, Bacillus spp., Actinobacteria

• photoorganoheterotrophs obtain energy from light, carbon and reducing equivalents for biosynthetic reactions from organic compounds. Some species are strictly heterotrophic, many others can also fix carbon dioxide and are mixotrophic. Examples: Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodomicrobium, Rhodocyclus, Heliobacterium, Chloroflexus (alternatively to photolithoautotrophy with hydrogen)

Heterotrophic microbial metabolism Most microbes are heterotrophic (more precisely chemoorganoheterotrophic), using organic compounds as both carbon and energy sources. Heterotrophic microbes live off

of nutrients that they scavenge from living hosts (as commensals or parasites) or find in dead organic matter of all kind (saprophages). Microbial metabolism is the main contribution for the bodily decay of all organisms after death. Many eukaryotic microorganisms are heterotrophic by predation or parasitism, properties also found in some bacteria such as Bdellovibrio (an intracellular parasite of other bacteria, causing death of its victims) and Myxobacteria such as Myxococcus (predators of other bacteria which are killed and lysed by cooperating swarms of many single cells of Myxobacteria). Most pathogenic bacteria can be viewed as heterotrophic parasites of humans or the other eukaryotic species they affect. Heterotrophic microbes are extremely abundant in nature and are responsible for the breakdown of large organic polymers such as cellulose, chitin or lignin which are generally indigestible to larger animals. Generally, the breakdown of large polymers to carbon dioxide (mineralization) requires several different organisms, with one breaking down the polymer into its constituent monomers, one able to use the monomers and excreting simpler waste compounds as by-products, and one able to use the excreted wastes. There are many variations on this theme, as different organisms are able to degrade different polymers and secrete different waste products. Some organisms are even able to degrade more recalcitrant compounds such as petroleum compounds or pesticides, making them useful in bioremediation.

Biochemically, prokaryotic heterotrophic metabolism is much more versatile than that of eukaryotic organisms, although many prokaryotes share the most basic metabolic models with eukaryotes, e. g. using glycolysis (also called EMP pathway) for sugar metabolism and the citric acid cycle to degrade acetate, producing energy in the form of ATP and reducing power in the form of NADH or quinols. These basic pathways are well conserved because they are also involved in biosynthesis of many conserved building blocks needed for cell growth (sometimes in reverse direction). However, many bacteria and archaea utilize alternative metabolic pathways other than glycolysis and the citric acid cycle. A well-studied example is sugar metabolism via the keto-deoxy-phosphogluconate pathway (also called ED pathway) in Pseudomonas. Moreover, there is a third alternative sugar-catabolic pathway used by some bacteria, the pentose phosphate pathway. The metabolic diversity and ability of prokaryotes to use a large variety of organic compounds arises from the much deeper evolutionary history and diversity of prokaryotes, as compared to eukaryotes. It is also noteworthy that the mitochondrion, the small membrane-bound intracellular organelle that is the site of eukaryotic energy metabolism, arose from the endosymbiosis of a bacterium related to obligate intracellular Rickettsia, and also to plant-associated Rhizobium or Agrobacterium. Therefore it is not surprising that all mitrochondriate eukaryotes share metabolic properties with these Proteobacteria. Most microbes respire (use an electron transport chain), although oxygen is not the only terminal electron acceptor that may be used. As discussed below, the use of terminal electron acceptors other than oxygen has important biogeochemical consequences.

Fermentation Fermentation is a specific type of heterotrophic metabolism that uses organic carbon instead of oxygen as a terminal electron acceptor. This means that these organisms do not

use an electron transport chain to oxidize NADH to NAD+ and therefore must have an alternative method of using this reducing power and maintaining a supply of NAD+ for the proper functioning of normal metabolic pathways (e.g. glycolysis). As oxygen is not required, fermentative organisms are anaerobic. Many organisms can use fermentation under anaerobic conditions and aerobic respiration when oxygen is present. These organisms are facultative anaerobes. To avoid the overproduction of NADH, obligately fermentative organisms usually do not have a complete citric acid cycle. Instead of using an ATPase as in respiration, ATP in fermentative organisms is produced by substrate-level phosphorylation where a phosphate group is transferred from a high-energy organic compound to ADP to form ATP. As a result of the need to produce high energy phosphate-containing organic compounds (generally in the form of CoA-esters) fermentative organisms use NADH and other cofactors to produce many different reduced metabolic by-products, often including hydrogen gas (H2). These reduced organic compounds are generally small organic acids and alcohols derived from pyruvate, the end product of glycolysis. Examples include ethanol, acetate, lactate, and butyrate. Fermentative organisms are very important industrially and are used to make many different types of food products. The different metabolic end products produced by each specific bacterial species are responsible for the different tastes and properties of each food.

Not all fermentative organisms use substrate-level phosphorylation. Instead, some organisms are able to couple the oxidation of low-energy organic compounds directly to the formation of a proton (or sodium) motive force and therefore ATP synthesis. Examples of these unusual forms of fermentation include succinate fermentation by Propionigenium modestum and oxalate fermentation by Oxalobacter formigenes. These reactions are extremely low-energy yielding. Humans and other higher animals also use fermentation to produce lactate from excess NADH, although this is not the major form of metabolism as it is in fermentative microorganisms.

Special metabolic properties

Methylotrophy

Methylotrophy refers to the ability of an organism to use C1-compounds as energy sources. These compounds include methanol, methyl amines, formaldehyde, and formate. Several other less common substrates may also be used for metabolism, all of which lack carbon-carbon bonds. Examples of methylotrophs include the bacteria Methylomonas and Methylobacter. Methanotrophs are a specific type of methylotroph that are also able to use methane (CH4) as a carbon source by oxidizing it sequentially to methanol (CH3OH), formaldehyde (CH2O), formate (HCOO-), and carbon dioxide CO2 initially using the enzyme methane monooxygenase. As oxygen is required for this process, all (conventional) methanotrophs are obligate aerobes. Reducing power in the form of quinones and NADH is produced during these oxidations to produce a proton motive force and therefore ATP generation. Methylotrophs and methanotrophs are not considered as autotrophic, because they are able to incorporate some of the oxidized methane (or other metabolites) into cellular carbon before it is completely oxidized to

CO2 (at the level of formaldehyde), using either the serine pathway (Methylosinus, Methylocystis) or the ribulose monophosphate pathway (Methylococcus), depending on the species of methylotroph.

In addition to aerobic methylotrophy, methane can also be oxidized anaerobically. This occurs by a consortium of sulfate-reducing bacteria and relatives of methanogenic Archaea working syntrophically (see below). Little is currently known about the biochemistry and ecology of this process.

Methanogenesis is the biological production of methane. It is carried out by methanogens, strictly anaerobic Archaea such as Methanococcus, Methanocaldococcus, Methanobacterium, Methanothermus, Methanosarcina, Methanosaeta and Methanopyrus. The biochemistry of methanogenesis is unique in nature in its use of a number of unusual cofactors to sequentially reduce methanogenic substrates to methane, such as coenzyme M and methanofuran.. These cofactors are responsible (among other things) for the establishment of a proton gradient across the outer membrane thereby driving ATP synthesis. Several types of methanogenesis occur, differing in the starting compounds oxidized. Some methanogens reduce carbon dioxide (CO2) to methane (CH4) using electrons (most often) from hydrogen gas (H2) chemolithoautotrophically. These methanogens can often be found in environments containing fermentative organisms. The tight association of methanogens and fermentative bacteria can be considered to be syntrophic (see below) because the methanogens, which rely on the fermentors for hydrogen, relieve feedback inhibition of the fermentors by the build-up of excess hydrogen that would otherwise inhibit their growth. This type of syntrophic relationship is specifically known as interspecies hydrogen transfer. A second group of methanogens use methanol (CH3OH) as a substrate for methanogenesis. These are chemoorganotrophic, but still autotrophic in using CO2 as only carbon source. The biochemistry of this process is quite different from that of the carbon dioxide-reducing methanogens. Lastly, a third group of methanogens produce both methane and carbon dioxide from acetate (CH3COO-) with the acetate being split between the two carbons. These acetate-cleaving organisms are the only chemoorganoheterotrophic methanogens. All autotrophic methanogens use a variation of the acetyl-CoA pathway to fix CO2 and obtain cellular carbon.

Syntrophy

Syntrophy, in the context of microbial metabolism, refers to the pairing of multiple species to achieve a chemical reaction that, on its own, would be energetically unfavorable. The best studied example of this process is the oxidation of fermentative end products (such as acetate, ethanol and butyrate) by organisms such as Syntrophomonas. Alone, the oxidation of butyrate to acetate and hydrogen gas is energetically unfavorable. However, when a hydrogenotrophic (hydrogen-using) methanogen is present the use of the hydrogen gas will significantly lower the concentration of hydrogen (down to 10-5 atm) and thereby shift the equilibrium of the butyrate oxidation reaction under standard conditions (ΔGº’) to non-standard conditions (ΔG’). Because the concentration of one product is lowered, the reaction is "pulled" towards the products and shifted towards net

energetically favorable conditions (for butyrate oxidation: ΔGº’= +48.2 kJ/mol, but ΔG' = -8.9 kJ/mol at 10-5 atm hydrogen and even lower if also the initially produced acetate is further metabolized by methanogens). Conversely, the available free energy from methanogenesis is lowered from ΔGº’= -131 kJ/mol under standard conditions to ΔG' = -17 kJ/mol at 10-5 atm hydrogen. This is an example of intraspecies hydrogen transfer. In this way, low energy-yielding carbon sources can be used by a consortium of organisms to achieve further degradation and eventual mineralization of these compounds. These reactions help prevent the excess sequestration of carbon over geologic time scales, releasing it back to the biosphere in usable forms such as methane and CO2.

Anaerobic respiration While aerobic organisms during respiration use oxygen as a terminal electron acceptor, anaerobic organisms use other electron acceptors. These inorganic compounds have a lower reduction potential than oxygen, meaning that respiration is less efficient in these organisms and leads to slower growth rates than aerobes. Many facultative anaerobes can use either oxygen or alternative terminal electron acceptors for respiration depending on the environmental conditions.

Most respiring anaerobes are heterotrophs, although some do live autotrophically. All of the processes described below are dissimilative, meaning that they are used during energy production and not to provide nutrients for the cell (assimilative). Assimilative pathways for many forms of anaerobic respiration are also known.

Denitrification - nitrate as electron acceptor

Denitrification is the utilization of nitrate (NO3-) as a terminal electron acceptor. It is a

widespread process that is used by many members of the Proteobacteria. Many facultative anaerobes use denitrification because nitrate, like oxygen, has a high reduction potential. Many denitrifying bacteria can also use ferric iron (Fe3+) and some organic electron acceptors. Denitrification involves the stepwise reduction of nitrate to nitrite (NO2

-), nitric oxide (NO), nitrous oxide (N2O), and dinitrogen (N2) by the enzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively. Protons are transported across the membrane by the initial NADH reductase, quinones, and nitrous oxide reductase to produce the electrochemical gradient critical for respiration. Some organisms (e.g. E. coli) only produce nitrate reductase and therefore can accomplish only the first reduction leading to the accumulation of nitrite. Others (e.g. Paracoccus denitrificans or Pseudomonas stutzeri) reduce nitrate completely. Complete denitrification is an environmentally significant process because some intermediates of denitrification (nitric oxide and nitrous oxide) are important greenhouse gases that react with sunlight and ozone to produce nitric acid, a component of acid rain. Denitrification is also important in biological wastewater treatment where it is used to reduce the amount of nitrogen released into the environment thereby reducing eutrophication.

Sulfate reduction - sulfate as electron acceptor

Sulfate reduction is a relatively energetically poor process used by many Gram negative bacteria found within the δ-Proteobacteria, Gram-positive organisms relating to Desulfotomaculum or the archaeon Archaeoglobus. Hydrogen sulfide (H2S) is produced as a metabolic end product. For sulfate reduction electron donors and energy are needed.

Electron donors

Many sulfate reducers are heterotrophic, using carbon compounds such as lactate and pyruvate (among many others) as electron donors, while others are autotrophic, using hydrogen gas (H2) as an electron donor. Some unusual autotrophic sulfate-reducing bacteria (e.g. Desulfotignum phosphitoxidans) can use phosphite (HPO3

-) as an electron donor whereas others (e.g. Desulfovibrio sulfodismutans, Desulfocapsa thiozymogenes, Desulfocapsa sulfoexigens) are capable of sulfur disproportionation (splitting one compound into two different compounds, in this case an electron donor and an electron acceptor) using elemental sulfur (S0), sulfite (SO2−3), and thiosulfate (S2O3

2-) to produce both hydrogen sulfide (H2S) and sulfate (SO2−4).

Energy for reduction

All sulfate-reducing organisms are strict anaerobes. Because sulfate is energetically stable, before it can be metabolized it must first be activated by adenylation to form APS (adenosine 5’-phosphosulfate) thereby consuming ATP. The APS is then reduced by the enzyme APS reductase to form sulfite (SO2− 3) and AMP. In organisms that use carbon compounds as electron donors, the ATP consumed is accounted for by fermentation of the carbon substrate. The hydrogen produced during fermentation is actually what drives respiration during sulfate reduction.

Acetogenesis - carbon dioxide as electron acceptor

Acetogenesis is a type of microbial metabolism that uses hydrogen (H2) as an electron donor and carbon dioxide (CO2) as an electron acceptor to produce acetate, the same electron donors and acceptors used in methanogenesis (see above). Bacteria that can autotrophically synthesize acetate are called homoacetogens. Carbon dioxide reduction in all homoacetogens occurs by the acetyl-CoA pathway. This pathway is also used for carbon fixation by autotrophic sulfate-reducing bacteria and hydrogenotrophic methanogens. Often homoacetogens can also be fermentative, using the hydrogen and carbon dioxide produced as a result of fermentation to produce acetate, which is secreted as an end product.

Other inorganic electron acceptors

Ferric iron (Fe3+) is a widespread anaerobic terminal electron acceptor both for autotrophic and heterotrophic organisms. Electron flow in these organisms is similar to those in electron transport, ending in oxygen or nitrate, except that in ferric iron-reducing

organisms the final enzyme in this system is a ferric iron reductase. Model organisms include Shewanella putrefaciens and Geobacter metallireducens. Since some ferric iron-reducing bacteria (e.g. G. metallireducens) can use toxic hydrocarbons such as toluene as a carbon source, there is significant interest in using these organisms as bioremediation agents in ferric iron-rich contaminated aquifers.

Although ferric iron is the most prevalent inorganic electron acceptor, a number of organisms (including the iron-reducing bacteria mentioned above) can use other inorganic ions in anaerobic respiration. While these processes may often be less significant ecologically, they are of considerable interest for bioremediation, especially when heavy metals or radionuclides are used as electron acceptors. Examples include:

• Manganic ion (Mn4+) reduction to manganous ion (Mn2+) • Selenate (SeO4

2-) reduction to selenite (SeO32-) and selenite reduction to inorganic

selenium (Se0) • Arsenate (AsO4

3-) reduction to arsenite (AsO33-)

• Uranyl ion ion (UO22+) reduction to uranium dioxide (UO2)

Organic terminal electron acceptors

A number of organisms, instead of using inorganic compounds as terminal electron acceptors, are able to use organic compounds to accept electrons from respiration. Examples include:

• Fumarate reduction to succinate • Trimethylamine N-oxide (TMAO) reduction to trimethylamine (TMA) • Dimethyl sulfoxide (DMSO) reduction to Dimethyl sulfide (DMS) • Reductive dechlorination

TMAO is a chemical commonly produced by fish, and when reduced to TMA produces a strong odor. DMSO is a common marine and freshwater chemical which is also odiferous when reduced to DMS. Reductive dechlorination is the process by which chlorinated organic compounds are reduced to form their non-chlorinated endproducts. As chlorinated organic compounds are often important (and difficult to degrade) environmental polutants, reductive dechlorination is an important process in bioremediation.

Chemolithotrophy Chemolithotrophy is a type of metabolism where energy is obtained from the oxidation of inorganic compounds. Most chemolithotrophic organisms are also autotrophic. There are two major objectives to chemolithotrophy: the generation of energy (ATP) and the generation of reducing power (NADH).

Hydrogen oxidation

Many organisms are capable of using hydrogen (H2) as a source of energy. While several mechanisms of anaerobic hydrogen oxidation have been mentioned previously (e.g. sulfate reducing- and acetogenic bacteria), hydrogen can also be used as an energy source aerobically. In these organisms, hydrogen is oxidized by a membrane-bound hydrogenase causing proton pumping via electron transfer to various quinones and cytochromes. In many organisms, a second cytoplasmic hydrogenase is used to generate reducing power in the form of NADH, which is subsequently used to fix carbon dioxide via the Calvin cycle. Hydrogen-oxidizing organisms, such as Cupriavidus necator (formerly Ralstonia eutropha), often inhabit oxic-anoxic interfaces in nature to take advantage of the hydrogen produced by anaerobic fermentative organisms while still maintaining a supply of oxygen.

Sulfur oxidation

Sulfur oxidation involves the oxidation of reduced sulfur compounds (such as sulfide (H2S), inorganic sulfur (S0), and thiosulfate (S2O3

2-) to form sulfuric acid (H2SO4). A classic example of a sulfur-oxidizing bacterium is Beggiatoa, a microbe originally described by Sergei Winogradsky, one of the founders of environmental microbiology. Another example is Paracoccus. Generally, the oxidation of sulfide occurs in stages, with inorganic sulfur being stored either inside or outside of the cell until needed. This two step process occurs because energetically sulfide is a better electron donor than inorganic sulfur or thiosulfate, allowing for a greater number of protons to be translocated across the membrane. Sulfur-oxidizing organisms generate reducing power for carbon dioxide fixation via the Calvin cycle using reverse electron flow, an energy-requiring process that pushes the electrons against their thermodynamic gradient to produce NADH. Biochemically, reduced sulfur compounds are converted to sulfite (SO2− 3) and subsequently converted to sulfate (SO2− 4) by the enzyme sulfite oxidase. Some organisms, however, accomplish the same oxidation using a reversal of the APS reductase system used by sulfate-reducing bacteria (see above). In all cases the energy liberated is transferred to the electron transport chain for ATP and NADH production. In addition to aerobic sulfur oxidation, some organisms (e.g. Thiobacillus denitrificans) use nitrate (NO3

2-) as a terminal electron acceptor and therefore grow anaerobically.

Ferrous iron (Fe2+) oxidation

Ferrous iron is a soluble form of iron that is stable at extremely low pHs or under anaerobic conditions. Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe3+) form and is hydrolyzed abiotically to insoluble ferric hydroxide (Fe(OH)3). There are three distinct types of ferrous iron-oxidizing microbes. The first are acidophiles, such as the bacteria Acidithiobacillus ferooxidans and Leptospirrillum ferrooxidans, as well as the archaeon Ferroplasma. These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage. The second type of microbes oxidize ferrous iron at cirum-neutral pH. These

micro-organisms (for example Gallionella ferruginea or Leptothrix ochracea) live at the oxic-anoxic interfaces and are microaerophiles. The third type of iron-oxidizing microbes are anaerobic photosynthetic bacteria such as Rhodopsuedomonas, which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation. Biochemically, aerobic iron oxidation is a very energetically poor process which therefore requires large amounts of iron to be oxidized by the enzyme rusticyanin to facilitate the formation of proton motive force. Like sulfur oxidation, reverse electron flow must be used to form the NADH used for carbon dioxide fixation via the Calvin cycle.

Nitrification

Nitrification is the process by which ammonia (NH3) is converted to nitrate (NO3-).

Nitrification is actually the net result of two distinct processes: oxidation of ammonia to nitrite (NO2

-) by nitrosifying bacteria (e.g. Nitrosomonas) and oxidation of nitrite to nitrate by the nitrite-oxidizing bacteria (e.g. Nitrobacter). Both of these processes are extremely energetically poor leading to very slow growth rates for both types of organisms. Biochemically, ammonia oxidation occurs by the stepwise oxidation of ammonia to hydroxylamine (NH2OH) by the enzyme ammonia monooxygenase in the cytoplasm, followed by the oxidation of hydroxylamine to nitrite by the enzyme hydroxylamine oxidoreductase in the periplasm.

Electron and proton cycling are very complex but as a net result only one proton is translocated across the membrane per molecule of ammonia oxidized. Nitrite reduction is much simpler, with nitrite being oxidized by the enzyme nitrite oxidoreductase coupled to proton translocation by a very short electron transport chain, again leading to very low growth rates for these organisms. Oxygen is required in both ammonia and nitrite oxidation, meaning that both nitrosifying and nitrite-oxidizing bacteria are aerobes. As in sulfur and iron oxidation, NADH for carbon dioxide fixation using the Calvin cycle is generated by reverse electron flow, thereby placing a further metabolic burden on an already energy-poor process.

Anammox

Anammox stands for anaerobic ammonia oxidation and the organisms responsible were relatively recently discovered, in the late 1990s. This form of metabolism occurs in members of the Planctomycetes (e.g. Candidatus Brocadia anammoxidans) and involves the coupling of ammonia oxidation to nitrite reduction. As oxygen is not required for this process these organisms are strict anaerobes. Amazingly, hydrazine (N2H4 - rocket fuel) is produced as an intermediate during anammox metabolism. To deal with the high toxicity of hydrazine, anammox bacteria contain a hydrazine-containing intracellular organelle called the anammoxasome, surrounded by highly compact (and unusual) ladderane lipid membrane. These lipids are unique in nature, as is the use of hydrazine as a metabolic intermediate. Anammox organisms are autotrophs although the mechanism for carbon dioxide fixation is unclear. Because of this property, these organisms could be used in industry to remove nitrogen in wastewater treatment processes. Anammox has

also been shown have widespread occurrence in anaerobic aquatic systems and has been speculated to account for approximately 50% of nitrogen gas production in the ocean.

Phototrophy Many microbes (phototrophs) are capable of using light as a source of energy to produce ATP and organic compounds such as carbohydrates, lipids, and proteins. Of these, algae are particularly significant because they are oxygenic, using water as an electron donor for electron transfer during photosynthesis. Phototrophic bacteria are found in the phyla Cyanobacteria, Chlorobi, Proteobacteria, Chloroflexi, and Firmicutes. Along with plants these microbes are responsible for all biological generation of oxygen gas on Earth. Because chloroplasts were derived from a lineage of the Cyanobacteria, the general principles of metabolism in these endosymbionts can also be applied to chloroplasts. In addition to oxygenic photosynthesis, many bacteria can also photosynthesize anaerobically, typically using sulfide (H2S) as an electron donor to produce sulfate. Inorganic sulfur (S0), thiosulfate (S2O3

2-) and ferrous iron (Fe2+) can also be used by some organisms. Phylogenetically, all oxygenic photosynthetic bacteria are Cyanobacteria, while anoxygenic photosynthetic bacteria belong to the purple bacteria (Proteobacteria), Green sulfur bacteria (e.g. Chlorobium), Green non-sulfur bacteria (e.g. Chloroflexus), or the heliobacteria (Low %G+C Gram positives). In addition to these organisms, some microbes (e.g. the Archaeon Halobacterium or the bacterium Roseobacter, among others) can utilize light to produce energy using the enzyme bacteriorhodopsin, a light-driven proton pump. However, there are no known Archaea that carry out photosynthesis.

As befits the large diversity of photosynthetic bacteria, there are many different mechanisms by which light is converted into energy for metabolism. All photosynthetic organisms locate their photosynthetic reaction centers within a membrane, which may be invaginations of the cytoplasmic membrane (Proteobacteria), thylakoid membranes (Cyanobacteria), specialized antenna structures called chlorosomes (Green sulfur and non-sulfur bacteria), or the cytoplasmic membrane itself (heliobacteria). Different photosynthetic bacteria also contain different photosynthetic pigments, such as chlorophylls and carotenoids, allowing them to take advantage of different portions of the electromagnetic spectrum and thereby inhabit different niches. Some groups of organisms contain more specialized light-harvesting structures (e.g. phycobilisomes in Cyanobacteria and chlorosomes in Green sulfur and non-sulfur bacteria), allowing for increased efficiency in light utilization.

Biochemically, anoxygenic photosynthesis is very different from oxygenic photosynthesis. Cyanobacteria (and by extension, chloroplasts) use the Z scheme of electron flow in which electrons eventually are used to form NADH. Two different reaction centers (photosystems) are used and proton motive force is generated both by using cyclic electron flow and the quinone pool. In anoxygenic photosynthetic bacteria, electron flow is cyclic, with all electrons used in photosynthesis eventually being transferred back to the single reaction center. A proton motive force is generated using only the quinone pool. In heliobacteria, Green sulfur, and Green non-sulfur bacteria,

NADH is formed using the protein ferredoxin, an energetically favorable reaction. In purple bacteria, NADH is formed by reverse electron flow due to the lower chemical potential of this reaction center. In all cases, however, a proton motive force is generated and used to drive ATP production via an ATPase.

Most photosynthetic microbes are autotrophic, fixing carbon dioxide via the Calvin cycle. Some photosynthetic bacteria (e.g. Chloroflexus) are photoheterotrophs, meaning that they use organic carbon compounds as a carbon source for growth. Some photosynthetic organisms also fix nitrogen.

Nitrogen fixation Nitrogen is an element required for growth by all biological systems. While extremely common (80% by volume) in the atmosphere, dinitrogen gas (N2) is generally biologically inaccessible due to its high activation energy. Throughout all of nature, only specialized bacteria and Archaea are capable of nitrogen fixation, converting dinitrogen gas into ammonia (NH3), which is easily assimilated by all organisms. These prokaryotes, therefore, are very important ecologically and are often essential for the survival of entire ecosystems. This is especially true in the ocean, where nitrogen-fixing cyanobacteria are often the only sources of fixed nitrogen, and in soils, where specialized symbioses exist between legumes and their nitrogen-fixing partners to provide the nitrogen needed by these plants for growth.

Nitrogen fixation can be found distributed throughout nearly all bacterial lineages and physiological classes but is not a universal property. Because the enzyme nitrogenase, responsible for nitrogen fixation, is very sensitive to oxygen which will inhibit it irreversibly, all nitrogen-fixing organisms must possess some mechanism to keep the concentration of oxygen low. Examples include:

• heterocyst formation (cyanobacteria e.g. Anabaena) where one cell does not photosynthesize but instead fixes nitrogen for its neighbors which in turn provide it with energy

• root nodule symbioses (e.g. Rhizobium) with plants that supply oxygen to the bacteria bound to molecules of leghaemoglobin

• anaerobic lifestyle (e.g. Clostridium pasteurianum) • very fast metabolism (e.g. Azotobacter vinelandii)

The production and activity of nitrogenases is very highly regulated, both because nitrogen fixation is an extremely energetically expensive process (16-24 ATP are used per N2 fixed) and due to the extreme sensitivity of the nitrogenase to oxygen.

Chapter- 5

Nitrogen Cycle

Schematic representation of the flow of nitrogen through the environment. The importance of bacteria in the cycle is immediately recognized as being a key element in the cycle, providing different forms of nitrogen compounds assimilable by higher organisms.

The nitrogen cycle is the process by which nitrogen is converted between its various chemical forms. This transformation can be carried out via both biological and non-biological processes. Important processes in the nitrogen cycle include fixation, mineralization, nitrification, and denitrification. The majority of Earth's atmosphere (approximately 78%) is nitrogen, making it the largest pool of nitrogen. However,

atmospheric nitrogen is unavailable for biological use, leading to a scarcity of usable nitrogen in many types of ecosystems. The nitrogen cycle is of particular interest to ecologists because nitrogen availability can affect the rate of key ecosystem processes, including primary production and decomposition. Human activities such as fossil fuel combustion, use of artificial nitrogen fertilizers, and release of nitrogen in wastewater have dramatically altered the global nitrogen cycle.

Ecological function Nitrogen is essential for many processes; it is crucial for any life on Earth. It is in all amino acids, is incorporated into proteins, and is present in the bases that make up nucleic acids, such as DNA and RNA. In plants, much of the nitrogen is used in chlorophyll molecules, which are essential for photosynthesis and further growth. Although earth’s atmosphere is an abundant source of nitrogen, most is relatively unusable by plants. Chemical processing, or natural fixation (through processes such as bacterial conversion), are necessary to convert gaseous nitrogen into forms usable by living organisms. This makes nitrogen a crucial part of food production. The abundance or scarcity of this "fixed" form of nitrogen, (also known as reactive nitrogen), dictates how much food can be grown on a piece of land.

The processes of the nitrogen cycle Nitrogen is present in the environment in a wide variety of chemical forms including organic nitrogen, ammonium (NH4

+), nitrate (NO3-), and nitrogen gas (N2). The processes

of the nitrogen cycle transform nitrogen from one chemical form to another. Many of the processes are carried out by microbes either to produce energy or to accumulate nitrogen in the form needed for growth. The diagram above shows how these processes fit together to form the nitrogen cycle.

Nitrogen fixation

Nitrogen fixation is the natural process, either biological or abiotic, by which nitrogen (N2) in the atmosphere is converted into ammonia (NH3). This process is essential for life because fixed nitrogen is required to biosynthesize the basic building blocks of life, e.g., nucleotides for DNA and RNA and amino acids for proteins. Nitrogen fixation also refers to other abiological conversions of nitrogen, such as its conversion to nitrogen dioxide.

Nitrogen fixation is utilized by numerous prokaryotes, including bacteria, actinobacteria, and certain types of anaerobic bacteria. Microorganisms that fix nitrogen are called diazotrophs. Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Nitrogen fixation also occurs as a result of non-biological processes. These include lightning, industrially through the Haber-Bosch Process, and combustion. Biological nitrogen fixation was discovered by the Dutch microbiologist Martinus Beijerinck.

Biological nitrogen fixation

Schematic representation of the nitrogen cycle. Abiotic nitrogen fixation has been omitted.

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. The formula for BNF is:

N2 + 8 H+ + 6 e− → 2 NH3 + H2

The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one molecule of H2. In free-living diazotrophs, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway.

Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. (In fact, many bacteria cease production of the enzyme in the presence of oxygen). Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as Leghemoglobin.

Plants that contribute to nitrogen fixation include the legume family – Fabaceae – with taxa such as clover, soybeans, alfalfa, lupines, peanuts, and rooibos. They contain symbiotic bacteria called Rhizobia within nodules in their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. When the plant dies, the fixed nitrogen is released, making it available to other plants and this helps

to fertilize the soil The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many traditional and organic farming practices, fields are rotated through various types of crops, which usually includes one consisting mainly or entirely of clover or buckwheat (family Polygonaceae), which were often referred to as "green manure."

Non-leguminous nitrogen-fixing plants

A sectioned alder tree root nodule

A whole alder tree root nodule.

Although by far the majority of nitrogen-fixing plants are in the legume family Fabaceae, there are a few non-leguminous plants, such as why does it say legumes and not vegitales alder, that can also fix nitrogen. These plants, referred to as "actinorhizal plants", consist of 24 genera of woody shrubs or trees distributed among in 8 plant families. The ability to fix nitrogen is not universally present in these families. For instance, of 122 genera in the Rosaceae, only 4 genera are capable of fixing nitrogen. All these families belong to the orders Cucurbitales, Fagales, and Rosales, which together with the Fabales form a clade of eurosids. In this clade, Fabales were the first lineage to branch off; thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant; however, it may be that the basic genetic and physiological requirements were present in an incipient state in the last common ancestors of all these plants, but only evolved to full function in some of them:

Family: Genera

Betulaceae: Alnus (alders)

Cannabaceae:

Coriariaceae: Coriaria

Datiscaceae: Datisca

Myricaceae:

Comptonia Morella arborea Myrica

Rhamnaceae:

Ceanothus Colletia Discaria Kentrothamnus

Rosaceae:

Cercocarpus (mountain mahoganies) Chamaebati

Trema

Casuarinaceae:

Allocasuarina Casuarina Ceuthostoma Gymnostoma

Elaeagnaceae:

Elaeagnus (silverberries) Hippophae (sea-buckthorns) Shepherdia (buffaloberries)

Retanilla Talguenea Trevoa

a (mountain miseries) Dryas Purshia/Cowania (bitterbrushes/cliffroses)

There are also several nitrogen-fixing symbiotic associations that involve cyanobacteria (such as Nostoc):

• Some lichens such as Lobaria and Peltigera • Mosquito fern (Azolla species) • Cycads • Gunnera

Microorganisms that fix nitrogen (Diazotrophs) • Cyanobacteria • Azotobacteraceae • Rhizobia • Frankia

Nitrogen fixation by cyanobacteria

Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria are able to utilize a variety of inorganic and organic sources of combined nitrogen, like nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacterial strains are also capable of diazotrophic growth. Genome sequencing has provided a large amount of information on the genetic basis of nitrogen metabolism and its control in different cyanobacteria. Comparative genomics, together with functional studies, has led to a significant advance in this field over the past years. 2-Oxoglutarate has turned out to be the central signalling molecule reflecting the carbon/nitrogen balance of cyanobacteria. Central players of nitrogen control are the global transcriptional factor NtcA, which controls the expression of many genes involved in nitrogen metabolism, as well as the PII signalling protein, which fine-tunes cellular activities in response to changing C/N conditions. These two proteins are sensors of the cellular 2-oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to nitrogen starvation involves heterogeneous responses in different strains. Nitrogen fixation by cyanobacteria in coral reefs can fix twice the amount of nitrogen than on land–around 1.8 kg of nitrogen is fixed per hectare per day.

Chemical nitrogen fixation Nitrogen can also be artificially fixed for use in fertilizers, explosives, or in other products. The most common method is the Haber process. Artificial fertilizer production is now the largest source of human-produced fixed nitrogen in the Earth's ecosystem.

The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400 °C), routine conditions for industrial catalysis. This highly efficient process uses natural gas as a hydrogen source and air as a nitrogen source.

Research on catalytic nitrogen-fixation

Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing the energy required for this conversion. However, such research has thus far failed to even approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen under ambient conditions. For example, lithium metal converts to lithium nitride under an atmosphere of nitrogen. Treatment of the resulting nitride gives ammonia.

The first dinitrogen complex was reported in 1965 based on ammonia coordinated to ruthenium ([Ru(NH3)5(N2)]2+), research in chemical fixation focused on transition metal complexes. Since then, a large number of transition metal compounds that contain dinitrogen as a ligand have been discovered. The dinitrogen ligand can either be bound to a single metal or bridge two (or more) metals. The coordination chemistry of dinitrogen is complex and currently under intense investigation. This research may lead to new ways of using dinitrogen in synthesis and on an industrial scale.

The first example of homolytic cleavage of dinitrogen under mild conditions was published in 1995. Two equivalents of a molybdenum complex reacted with one equivalent of dinitrogen, creating a triple bonded MoN complex. Since then, this triple bounded complex has been used to make nitriles. The first catalytic system converting nitrogen to ammonia at room temperature and pressure was discovered in 2003 and is based on another molybdenum compound, a proton source, and a strong reducing agent. However, this catalytic reduction fixates only a few nitrogen molecules.

Conversion of N2

The conversion of nitrogen (N2) from the atmosphere into a form readily available to plants and hence to animals and humans is an important step in the nitrogen cycle, which distributes the supply of this essential nutrient. There are four ways to convert N2 (atmospheric nitrogen gas) into more chemically reactive forms:

1. Biological fixation: some symbiotic bacteria (most often associated with leguminous plants) and some free-living bacteria are able to fix nitrogen as organic nitrogen. An example of mutualistic nitrogen fixing bacteria are the Rhizobium bacteria, which live in legume root nodules. These species are diazotrophs. An example of the free-living bacteria is Azotobacter.

2. Industrial N-fixation: Under great pressure, at a temperature of 600 C, and with the use of an iron catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH3). In the Haber-Bosch process, N2 is converted together with hydrogen gas (H2) into ammonia (NH3), which is used to make fertilizer and explosives.

3. Combustion of fossil fuels: automobile engines and thermal power plants, which release various nitrogen oxides (NOx).

4. Other processes: In addition, the formation of NO from N2 and O2 due to photons and especially lightning, can fix nitrogen.

Assimilation

Some plants get nitrogen from the soil, and by absorption of their roots in the form of either nitrate ions or ammonium ions. All nitrogen obtained by animals can be traced back to the eating of plants at some stage of the food chain.

Plants can absorb nitrate or ammonium ions from the soil via their root hairs. If nitrate is absorbed, it is first reduced to nitrite ions and then ammonium ions for incorporation into amino acids, intense nucleic acids, and chlorophyll. In plants that have a mutualistic relationship with rhizobia, some nitrogen is assimilated in the form of ammonium ions directly from the nodules. Animals, fungi, and other heterotrophic organisms absorb nitrogen as amino acids, nucleotides and other small organic molecules.

Ammonification

When a plant dies, an animal dies, or an animal expels waste, the initial form of nitrogen is organic. Bacteria, or in some cases, fungi, convert the organic nitrogen within the remains back into ammonium (NH4

+), a process called ammonification or mineralization. Enzymes Involved:

• GS: Gln Synthetase (Cytosolic & PLastid) • GOGAT: Glu 2-oxoglutarate aminotransferase (Ferredoxin & NADH dependent) • GDH: Glu Dehydrogenase:

o Minor Role in ammonium assimilation. o Important in amino acid catabolism.

Nitrification

The conversion of ammonium to nitrate is performed primarily by soil-living bacteria and other nitrifying bacteria. The primary stage of nitrification, the oxidation of ammonium (NH4

+) is performed by bacteria such as the Nitrosomonas species, which converts ammonia to nitrites (NO2

-). Other bacterial species, such as the Nitrobacter, are responsible for the oxidation of the nitrites into nitrates (NO3

-)..It is important for the nitrites to be converted to nitrates because accumulated nitrites are toxic to plant life.

Due to their very high solubility, nitrates can enter groundwater. Elevated nitrate in groundwater is a concern for drinking water use because nitrate can interfere with blood-oxygen levels in infants and cause methemoglobinemia or blue-baby syndrome. Where groundwater recharges stream flow, nitrate-enriched groundwater can contribute to eutrophication, a process leading to high algal, especially blue-green algal populations and the death of aquatic life due to excessive demand for oxygen. While not directly toxic to fish life like ammonia, nitrate can have indirect effects on fish if it contributes to this eutrophication. Nitrogen has contributed to severe eutrophication problems in some water bodies. As of 2006, the application of nitrogen fertilizer is being increasingly controlled in Britain and the United States. This is occurring along the same lines as control of

phosphorus fertilizer, restriction of which is normally considered essential to the recovery of eutrophied waterbodies.

Denitrification

Denitrification is a microbially facilitated process of nitrate reduction that may ultimately produce molecular nitrogen (N2) through a series of intermediate gaseous nitrogen oxide products. This respiratory process reduces oxidized forms of nitrogen in response to the oxidation of an electron donor such as organic matter. The preferred nitrogen electron acceptors in order of most to least thermodynamically favorable include nitrate (NO3

−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O). In terms of the

general nitrogen cycle, denitrification completes the cycle by returning N2 to the atmosphere. The process is performed primarily by heterotrophic bacteria (such as Paracoccus denitrificans and various pseudomonads), although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans). Denitrifiers are represented in all main phylogenetic groups. Generally several species of bacteria are involved in the complete reduction of nitrate to molecular nitrogen, and more than one enzymatic pathway have been identified in the reduction process.

Direct reduction from nitrate to ammonium, a process known as dissimilatory nitrate reduction to ammonium or DNRA, is also possible for organisms that have the nrf-gene. This is less common than denitrification in most ecosystems as a means of nitrate reduction. Other genes known in microorganisms which denitrify include nir (nitrate reductase) and nos (nitrous oxide reductase) among others; organisms identified as having these genes include Alcaligenes faecalis, Alcaligenes xylosoxidans, many in the Pseudomonas genus, Bradyrhizobium japonicum, and Blastobacter denitrificans.

Nutrient limitation

All organisms require certain nutrients in their surroundings (available to them) for survival. Depending upon the ecosystem, nitrogen is most likely the limiting nutrient, although phosphorus is the other primary limiting nutrient and these two elements interact chemically. Some organisms appear to be able to denitrify and remove phosphorus. The triple bond of N2 makes this a very stable compound; most organisms (i.e. plants) depend upon others to break this down to make it available for biochemical reactions. Symbiotic relationships between Rhizobium species and legumes are well-documented.

Conditions required

Denitrification takes place under special conditions in both terrestrial and marine ecosystems. In general, it occurs where oxygen, a more energetically favourable electron acceptor, is depleted, and bacteria respire nitrate as a substitute terminal electron acceptor. Due to the high concentration of oxygen in our atmosphere, denitrification only takes place in environments where oxygen consumption exceeds the rate of oxygen

supply, such as in some soils and groundwater, wetlands, poorly ventilated corners of the ocean, and in seafloor sediments.

Denitrification generally proceeds through some combination of the following intermediate forms:

NO3− → NO2

− → NO + N2O → N2 (g)

The complete denitrification process can be expressed as a redox reaction:

2 NO3− + 10 e− + 12 H+ → N2 + 6 H2O

This reaction shows a fractionation in isotope composition. Lighter isotopes of nitrogen are preferred in the reaction, leaving the heavier nitrogen isotopes in the residual matter. The process can cause delta-values of up to −40, where delta is a representation of the difference in isotopic composition. This can be used to identify denitrification processes in nature.

Deliberate use of process

Denitrification is commonly used to remove nitrogen from sewage and municipal wastewater. It is also an instrumental process in wetlands and riparian zones for the removal of excess nitrate from groundwater resulting from excessive agricultural or residential fertilizer usage.

Reduction under anoxic conditions can also occur through process called anaerobic ammonia oxidation (anammox):

NH4+ + NO2

− → N2 + 2 H2O

In some wastewater treatment plants, small amounts of methanol, ethanol, acetate or proprietary products like MicroCg or MicroCglycerin are added to the wastewater to provide a carbon source for the denitrification bacteria. Denitrification processes are also used in the treatment of industrial wastes.

Influence on global climate change

Increasing carbon dioxide levels within the atmosphere will influence global nutrient cycling, yet it is difficult to predict what those interactions might be. Chemical interactions between soils and the atmosphere will be influenced by changes in atmospheric composition. There are indications that increased fertilization of soils with nitrogen causes a decrease in carbon sequestration.

Jake Beaulieu, a postdoctoral researcher the Environmental Protection Agency in Cincinnati, Ohio and Jennifer Tank, Galla Professor of Biological Sciences at the University of Notre Dame, are lead authors of new paper demonstrating that streams and

rivers receiving nitrogen inputs from urban and agricultural land uses are a significant source of nitrous oxide to the atmosphere.

Anaerobic ammonium oxidation

In this biological process, nitrite and ammonium are converted directly into dinitrogen gas. This process makes up a major proportion of dinitrogen conversion in the oceans.

Human influences on the nitrogen cycle As a result of extensive cultivation of legumes (particularly soy, alfalfa, and clover), growing use of the Haber-Bosch process in the creation of chemical fertilizers, and pollution emitted by vehicles and industrial plants, human beings have more than doubled the annual transfer of nitrogen into biologically available forms. In addition, humans have significantly contributed to the transfer of nitrogen trace gases from Earth to the atmosphere, and from the land to aquatic systems. Human alterations to the global nitrogen cycle are most intense in developed countries and in Asia, where vehicle emissions and industrial agriculture are highest.

N2O (nitrous oxide) has risen in the atmosphere as a result of agricultural fertilization, biomass burning, cattle and feedlots, and other industrial sources. N2O has deleterious effects in the stratosphere, where it breaks down and acts as a catalyst in the destruction of atmospheric ozone. N2O in the atmosphere is a greenhouse gas, currently the third largest contributor to global warming, after carbon dioxide and methane. While not as abundant in the atmosphere as carbon dioxide, for an equivalent mass, nitrous oxide is nearly 300 times more potent in its ability to warm the planet.

Ammonia (NH3) in the atmosphere has tripled as the result of human activities. It is a reactant in the atmosphere, where it acts as an aerosol, decreasing air quality and clinging on to water droplets, eventually resulting in acid rain. Fossil fuel combustion has contributed to a 6 or 7 fold increase in NOx flux to the atmosphere. NO2 actively alters atmospheric chemistry, and is a precursor of tropospheric (lower atmosphere) ozone production, which contributes to smog, acid rain, damages plants and increases nitrogen inputs to ecosystems. Ecosystem processes can increase with nitrogen fertilization, but anthropogenic input can also result in nitrogen saturation, which weakens productivity and can kill plants. Decreases in biodiversity can also result if higher nitrogen availability increases nitrogen-demanding grasses, causing a degradation of nitrogen-poor, species diverse heathlands.

Wastewater treatment

Onsite sewage facilities such as septic tanks and holding tanks release large amounts of nitrogen into the environment by discharging through a drainfield into the ground. Microbial activity consumes the nitrogen and other contaminants in the wastewater.

However, in certain areas, the soil is unsuitable to handle some or all of the wastewater, and, as a result, the wastewater with the contaminants enters the aquifers. These contaminants accumulate and eventually end up in drinking water. One of the contaminants concerned about the most is nitrogen in the form of nitrates. A nitrate concentration of 10 ppm (parts per million) or 10 milligrams per liter is the current EPA limit for drinking water and typical household wastewater can produce a range of 20–85 ppm.

The health risk associated with drinking water (with >10 ppm nitrate) is the development of methemoglobinemia and has been found to cause blue baby syndrome. Several states have now started programs to introduce advanced wastewater treatment systems to the typical onsite sewage facilities. The result of these systems is an overall reduction of nitrogen, as well as other contaminants in the wastewater.

Environmental impacts

Additional risks posed by increased availability of inorganic nitrogen in aquatic ecosystems include water acidification; eutrophication of fresh and saltwater systems; and toxicity issues for animals, including humans. Eutrophication often leads to lower dissolved oxygen levels in the water column, including hypoxic and anoxic condtions, which can cause cause death of aquatic fauna. Relatively sessile benthos, or bottom-dwelling creatures, are particularly vulnerable because of their lack of mobility, though large fish kills are not uncommon. Oceanic dead zones near the mouth of the Mississippi in the Gulf of Mexico are a well known example of algal bloom-induced hypoxia.

Ammonia (NH3) is highly toxic to fish and the water discharge level of ammonia from wastewater treatment facilities must often be closely monitored. To prevent fish deaths, nitrification prior to discharge is often desirable. Land application can be an attractive alternative to the mechanical aeration needed for nitrification.

Chapter- 6

Phototroph and Photophosphorylation

Phototroph

Terrestrial and aquatic phototrophs: Plants grow on a fallen log floating in algae rich water.

Phototrophs (Gk: φωτο = light, τροϕή = nourishment) are the organisms (usually plants) that carry out photosynthesis to acquire energy. They use the energy from sunlight to convert carbon dioxide and water into organic materials to be utilized in cellular functions such as biosynthesis and respiration.

Most phototrophs are autotrophs, also known as photoautotrophs, and can fix carbon. They can be contrasted with chemoautotrophs that obtain their energy by the oxidation of electron donors in their environments. Photoheterotrophs produce ATP through photophosphorylation but use organic compounds to build structures. Some phototrophs are organotrophs, also known as photo-organotrophs.

In an ecological context, phototrophs provide nutrition for all other forms of life (besides other autotrophs such as chemotrophs). In terrestrial environments plants are the predominant variety, while aquatic environments include a range of phototrophic organisms such as algae (e.g. kelp), other protists (such as euglena), phytoplankton and bacteria (such as cyanobacteria).

One product of this process is starch, which is a storage or reserve form of carbon, which can be used when light conditions are too poor to satisfy the immediate needs of the organism. Photosynthetic bacteria have a substance called bacteriochlorophyll, live in lakes and pools, and use the hydrogen from hydrogen sulfide instead of from water, for the chemical process. (The bacteriochlorophyll pigment absorbs light in the extreme UV and infra-red parts of the spectrum which is outside the range used by normal chlorophyll). Cyanobacteria live in fresh water, seas, soil and lichen, and use a plant-like photosynthesis.

A photolithotrophic autotroph is an autotrophic organism that uses light energy, and an inorganic electron donor (e.g., H2O, H2, H2S), and CO2 as its carbon source. Examples include plants.

The depth to which sunlight or artificial light can penetrate into water, so that photosynthesis may occur, is known as the photic zone.

Flowchart

Flowchart to determine if a species is autotroph, heterotroph, or a subtype

• Autotroph o Chemoautotroph o Photoautotroph

• Heterotroph o Chemoheterotroph o Photoheterotroph

Photophosphorylation

Photophosphorylation through light-dependent reactions of photosynthesis at the thylakoid membrane

The production of ATP using the energy of sunlight is called photophosphorylation. Only two sources of energy are available to living organisms: sunlight and oxidation-reduction (redox) reactions. All organisms produce ATP, which is the universal energy currency of life.

In photophosphorylation, light energy is used to create a high-energy electron donor and a lower-energy electron acceptor. Electrons then move spontaneously from donor to acceptor through an electron transport chain.

Background ATP is made by an enzyme called ATP synthase. Both the structure of this enzyme and its underlying gene are remarkably similar in all known forms of life.

ATP synthase is powered by a transmembrane electrochemical potential gradient, usually in the form of a proton gradient. The function of the electron transport chain is to produce this gradient. In all living organisms, a series of redox reactions is used to produce a transmembrane electrochemical potential gradient, or a so-called proton motive force (pmf).

Redox reactions are chemical reactions in which electrons are transferred from a donor molecule to an acceptor molecule. The underlying force driving these reactions is the

Gibbs free energy of the reactants and products. The Gibbs free energy is the energy available (“free”) to do work. Any reaction that decreases the overall Gibbs free energy of a system will proceed spontaneously (given that the system is isobaric and also adiabatic)

The transfer of electrons from a high-energy molecule (the donor) to a lower-energy molecule (the acceptor) can be spatially separated into a series of intermediate redox reactions. This is an electron transport chain.

The fact that a reaction is thermodynamically possible does not mean that it will actually occur. A mixture of hydrogen gas and oxygen gas does not spontaneously ignite. It is necessary either to supply an activation energy or to lower the intrinsic activation energy of the system, in order to make most biochemical reactions proceed at a useful rate. Living systems use complex macromolecular structures to lower the activation energies of biochemical reactions.

It is possible to couple a thermodynamically favorable reaction (a transition from a high-energy state to a lower-energy state) to a thermodynamically unfavorable reaction (such as a separation of charges, or the creation of an osmotic gradient), in such a way that the overall free energy of the system decreases (making it thermodynamically possible), while useful work is done at the same time. Biological macromolecules that catalyze a thermodynamically favorable reaction if and only if a thermodynamically unfavorable reaction occurs simultaneously underlie all known forms of life.

Electron transport chains (most known as ETC) produce energy in the form of a transmembrane electrochemical potential gradient. This energy is used to do useful work. The gradient can be used to transport molecules across membranes. It can be used to do mechanical work, such as rotating bacterial flagella. It can be used to produce ATP and NADPH, high-energy molecules that are necessary for growth.

Cyclic photophosphorylation In cyclic electron flow, the electron begins in a pigment complex called photosystem I, passes from the primary acceptor to plastoquinone, then to cytochrome b6f (a similar complex to that found in mitochondria), and then to plastocyanin before returning to chlorophyll. This transport chain produces a proton-motive force, pumping H+ ions across the membrane; this produces a concentration gradient that can be used to power ATP synthase during chemiosmosis. This pathway is known as cyclic photophosphorylation, and it produces neither O2 nor NADPH. Unlike non-cyclic photophosphorylation, NADP+ does not accept the electrons, but they are sent back to photosystem I. NADPH is NOT produced in cyclic photophosphorylation. In bacterial photosynthesis, a single photosystem is used, and therefore is involved in cyclic photophosphorylation. It is favoured in anaerobic conditions and conditions of high irradiance and CO2 compensation point.

Noncyclic photophosphorylation The other pathway, noncyclic photophosphorylation, is a two-stage process involving two different chlorophyll photosystems. Being a light reaction, Noncyclic photophosphorylation occurs on thylakoid membranes inside chloroplasts. First, a water molecule is broken down into 2H+ + 1/2 O2 + 2e- by a process called photolysis (or light-splitting). The two electrons from the water molecule are kept in photosystem II, while the 2H+ and 1/2O2 are left out for further use. Then a photon is absorbed by chlorophyll pigments on surrounding the reaction core center of the photosystem. The light excites the electrons of each pigment, causing a chain reaction that eventually transfers energy to the core of photosystem II, exciting the two electrons that are transferred to the primary electron acceptor, pheophytin. The deficit of electrons is replenished by taking electrons from another molecule of water. The electrons transfer from pheophytin to plastoquinone, then to plastocyanin, providing the energy for hydrogen ions (H+) to be pumped into the thylakoid space. This creates a gradient, making H+ ions flow back into the stroma of the chloroplast, providing the energy for the regeneration of ATP.

The photosystem II complex replaced its lost electrons from an external source; however, the two other electrons are not returned to photosystem II as they would in the analogous cyclic pathway. Instead, the still-excited electrons are transferred to a photosystem I complex, which boosts their energy level to a higher level using a second solar photon. The highly excited electrons are transferred to the acceptor molecule, but this time are passed on to an enzyme called Ferredoxin- NADP reductase|NADP+ reductase, for short FNR, which uses them to catalyse the reaction (as shown):

NADP+ + 2H+ + 2e- → NADPH + H+

This consumes the H+ ions produced by the splitting of water, leading to a net production of 1/2O2, ATP, and NADPH+H+ with the consumption of solar photons and water.

The concentration of NADPH in the chloroplast may help regulate which pathway electrons take through the light reactions. When the chloroplast runs low on ATP for the Calvin cycle, NADPH will accumulate and the plant may shift from noncyclic to cyclic electron flow.

Chapter- 7

Chloroplast

The simplified internal structure of a chloroplast

Chloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.

The word chloroplast (χλωροπλάσ69ς) is derived from the Greek words chloros (χλωρός), which means green, and plastis (πλάστης), which means "the one who forms". Chloroplasts are members of a class of organelles known as plastids.

Evolutionary origin

Chloroplasts visible in the cells of Plagiomnium affine — Many-fruited Thyme-moss

A model chloroplast

Chloroplasts are one of the many different types of organelles in the plant cell. In general, they are considered to have originated from cyanobacteria through endosymbiosis. This was first suggested by Mereschkowsky in 1905 after an observation by Schimper in 1883 that chloroplasts closely resemble cyanobacteria. All chloroplasts are thought to derive directly or indirectly from a single endosymbiotic event (in the Archaeplastida), except for Paulinella chromatophora, which has recently acquired a photosynthetic cyanobacterial endosymbiont which is not closely related to chloroplasts of other eukaryotes. In that they derive from an endosymbiotic event, chloroplasts are similar to mitochondria, but chloroplasts are found only in plants and protista. The chloroplast is surrounded by a double-layered composite membrane with an intermembrane space; further, it has reticulations, or many infoldings, filling the inner spaces. The chloroplast has its own DNA, which codes for redox proteins involved in electron transport in photosynthesis; this is termed the plastome.

In green plants, chloroplasts are surrounded by two lipid-bilayer membranes. They are believed to correspond to the outer and inner membranes of the ancestral cyanobacterium. Chloroplasts have their own genome, which is considerably reduced compared to that of free-living cyanobacteria, but the parts that are still present show clear similarities with the cyanobacterial genome. Plastids may contain 60-100 genes whereas cyanobacteria often contain more than 1500 genes. Many of the missing genes are encoded in the nuclear genome of the host. The transfer of nuclear information has been estimated in tobacco plants at one gene for every 16000 pollen grains.

In some algae (such as the heterokonts and other protists such as Euglenozoa and Cercozoa), chloroplasts seem to have evolved through a secondary event of endosymbiosis, in which a eukaryotic cell engulfed a second eukaryotic cell containing chloroplasts, forming chloroplasts with three or four membrane layers. In some cases, such secondary endosymbionts may have themselves been engulfed by still other eukaryotes, thus forming tertiary endosymbionts. In the alga Chlorella, there is only one chloroplast, which is bell-shaped.

In some groups of mixotrophic protists such as the dinoflagellates, chloroplasts are separated from a captured alga or diatom and used temporarily. These klepto chloroplasts may only have a lifetime of a few days and are then replaced.

Structure Chloroplasts are observable as flat discs usually 2 to 10 micrometers in diameter and 1 micrometer thick. In land plants, they are, in general, 5 μm in diameter and 2.3 μm thick. The chloroplast is contained by an envelope that consists of an inner and an outer phospholipid membrane. Between these two layers is the intermembrane space. A typical parenchyma cell contains about 10 to 100 chloroplasts.

Chloroplast ultrastructure: 1. outer membrane 2. intermembrane space 3. inner membrane (1+2+3: envelope) 4. stroma (aqueous fluid) 5. thylakoid lumen (inside of thylakoid) 6. thylakoid membrane 7. granum (stack of thylakoids) 8. thylakoid (lamella)

9. starch 10. ribosome 11. plastidial DNA 12. plastoglobule (drop of lipids)

The material within the chloroplast is called the stroma, corresponding to the cytosol of the original bacterium, and contains one or more molecules of small circular DNA. It also contains ribosomes; however most of its proteins are encoded by genes contained in the host cell nucleus, with the protein products transported to the chloroplast.

TEM image of a chloroplast

Within the stroma are stacks of thylakoids, the sub-organelles, which are the site of photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum). A thylakoid has a flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes place on the thylakoid membrane; as in mitochondrial oxidative phosphorylation, it involves the coupling of cross-membrane fluxes with biosynthesis via the dissipation of a proton electrochemical gradient.

In the electron microscope, thylakoid membranes appear as alternating light-and-dark bands, each 0.01 μm thick. Embedded in the thylakoid membrane are antenna complexes, each of which consists of the light-absorbing pigments, including chlorophyll and

carotenoids, as well as proteins that bind the pigments. This complex both increases the surface area for light capture, and allows capture of photons with a wider range of wavelengths. The energy of the incident photons is absorbed by the pigments and funneled to the reaction centre of this complex through resonance energy transfer. Two chlorophyll molecules are then ionised, producing an excited electron, which then passes onto the photochemical reaction centre.

Recent studies have shown that chloroplasts can be interconnected by tubular bridges called stromules, formed as extensions of their outer membranes. Chloroplasts appear to be able to exchange proteins via stromules, and thus function as a network.

Transplastomic plants Recently, chloroplasts have caught attention by developers of genetically modified plants. In most flowering plants, chloroplasts are not inherited from the male parent, although in plants such as pines, chloroplasts are inherited from males. Where chloroplasts are inherited only from the female, transgenes in these plastids cannot be disseminated by pollen. This makes plastid transformation a valuable tool for the creation and cultivation of genetically modified plants that are biologically contained, thus posing significantly lower environmental risks. This biological containment strategy is therefore suitable for establishing the coexistence of conventional and organic agriculture. While the reliability of this mechanism has not yet been studied for all relevant crop species, recent results in tobacco plants are promising, showing a failed containment rate of transplastomic plants at 3 in 1,000,000.

advanced concepts of energy transformation in metabolism biochemistry

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