Microbial physiology Unit 2Bacterial Respiration

• When pyruvate is oxidized to CO2, a far higheryield of ATP is possible.

• Oxidation using O2 as the terminal electronacceptor is called aerobic respiration;oxidation using other acceptors under anoxicconditions is called anaerobic respiration.

Oxidation-Reduction Reactions

• Oxidation is the removal of electrons (e-) from an atom or molecule, areaction that often produces energy.

• An example of an oxidation in which molecule A loses an electron tomolecule B. Molecule A has undergone oxidation (meaning that it has lostone or more electrons), whereas molecule B has undergone reduction(meaning that it has gained one or more electrons).

• Oxidation and reduction reactions are always coupled; in other words,each time one substance is oxidized, another is simultaneously reduced.The pairing of these reactions is called oxidation-reduction or a redoxreaction.

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The Generation of ATP

• Much of the energy released during oxidation-reduction reactions is trapped within the cell by the formation of ATP. Specifically, a phosphate group, is added to ADP with the input of energy to form ATP:

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• The symbol ~ designates a "high-energy" bond-that is, one that can readily be broken to releaseusable energy.

• The high-energy bond that attaches the third P ina sense contains the energy stored in thisreaction.

• When this P is removed, usable energy isreleased. The addition of P to a chemicalcompound is called phosphorylation.

• Organisms use three mechanisms ofphosphorylation to generate ATP from ADP.

Substrate-level Phosphorylation

• In substrate-level phosphorylation. ATP isusually generated when a high-energy P isdirectly transferred from a phosphorylatedcompound (a substrate) to ADP.

• Generally, the P has acquired its energy duringan earlier reaction in which the substrate itselfwas oxidized.

• The following example shows only the carbonskeleton and the P of a typical substrate:

Oxidative Phosphorylation

• In oxidative phosphorylation, electrons are transferred fromorganic compounds to one group of electron carriers (usually toNAD+ and FAD). Then, the electrons are passed through a seriesof different electron carriers to molecules of oxygen (02) or otheroxidized inorganic and organic molecules.

• This process occurs in the plasma membrane of prokaryotes andin the inner mitochondrial membrane of eukaryotes.

• The sequence of electron carriers used in oxidativephosphorylation is called an electron transport chain (system).

• The transfer of electrons from one electron carrier to the nextreleases energy, some of which is used to generate ATP fromADP through a process called chemiosmosis.

Cellular Respiration

• After glucose has been broken down to pyruvicacid, the pyruvic acid can be channeled into thenext step Cellular respiration or simplyrespiration;

• Defined as an ATP-generating process in whichmolecules are oxidized and the final electronacceptor is (almost always) an inorganicmolecule.

• An essential feature of respiration is theoperation of an electron transport chain.

Aerobic Respiration

• An electron transport chain (system) consists ofa sequence of carrier molecules that are capableof oxidation and reduction.

• As electrons are passed through the chain, thereoccurs a step vise release of energy, which is usedto drive the chemiosmotic generation of ATP.

• The final oxidation is irreversible.

• In prokaryotic cells, the electron transport chainis contained in the plasma membrane.

Carrier molecules in ETS

• There are three classes of carrier molecules in electron transportchains.

• The first are Flavoproteins.• These proteins contain flavin, a coenzyme derived from riboflavin

(vitamin B2) and are capable of performing alternating oxidationsand reductions. One important flavin coenzyme is flavinmononucleotide (FMN).

• The second class of carrier molecules are cytochromes, proteinswith an iron-containing group (heme) capable of existing alternatelyas a reduced form (Fe+2) and an oxidized form (Fe+3).

• The cytochromes involved in electron transport chains includecytochrome b (cyt b), cytochrome c1 (cyt c1), cytochrome c (cyt c) ,cytochrome a (cyt a), and cytochrome a (cyt a3).

• The third class is known as ubiquinones, or coenzyme Q.symbolized Q; these are small nonprotein carriers.

An electron transport chain

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Steps involved

• The first step in the mitochondrial electron transport chain involvesthe transfer of high-energy electrons from NADH to FMN, the firstcarrier in the chain.

• This transfer actually involves the passage of a hydrogen atom with two electrons to FMN, which then picks up an additional H+ from the surrounding aqueous medium.

• As a result of the first transfer, NADH is oxidized to NAD+, and FMNis reduced to FMNH2.

• In the second step in the electron transport chain, FMNH2 passes2H+ to the other side of the mitochondrial membrane and passestwo electrons to Q.

• As a result, FMNH2 is oxidized to FMN. Q also picks up an additional2H+ from the surrounding aqueous medium and releases it on theother side of the membrane.

• The next part of the electron transport chain involvesthe cytochromes.

• Electrons are passed successively from Q tocytochrome b (cyt b), cytochrome c1 (cyt c1),cytochrome c (cyt c) , cytochrome a (cyt a), andcytochrome a (cyt a3).

• Each cytochrome in the chain is reduced as it picks upelectrons and is oxidized as it gives up electrons.

• The last cytochrome, cyt a3, passes its electrons tomolecular oxygen (02), which becomes negativelycharged and then picks up protons from thesurrounding medium to form H20.

• An important feature of the electron transport chain is thepresence of some carriers, such as FMN and Q, that acceptand release protons as well as electrons, and other carriers,such as cytochromes, that transfer electrons only.

• Electron flow down the chain is accompanied at severalpoints by the active transport (pumping) of protons fromthe matrix side of the inner mitochondrial membrane tothe opposite side of the membrane.

• The result is a buildup of protons on one side of themembrane. Just as water behind a dam stores energy thatcan be used to generate electricity, this build up of protonsprovides energy for the generation of ATP by thechemiosmotic mechanism.

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The Chemiosmotic Mechanism of ATP Generation

• The mechanism of ATP synthesis using the electrontransport chain is called chemiosmosis.

• Substances diffuse passively across membranes from areasof high concentration to areas of low concentration; thisdiffusion yields energy.

• And the movement of substances against such aconcentration gradient requires energy and that, in such anactive transport of molecules or ions across biologicalmembranes, the required energy is usually provided by ATP.

• In chemiosmosis, the energy released when a substancemoves along a gradient is used to synthesize ATP. The"substance" in this case refers to protons.

The steps of chemiosmosis

1. As energetic electrons from NADH pass down the electron transportchain, some of the carriers in the chain pump- actively transport-protons across the membrane. Such carrier molecules are called protonpumps.

2. The phospholipid membrane is normally impermeable to protons, so thisone-directional pumping establishes a proton gradient (a difference inthe concentrations of protons on the two sides of the membrane). Inaddition to a concentration gradient, there is an electrical chargegradient. The excess H+ on one side of the membrane makes that sidepositively charged compared with the other side. The resultingelectrochemical gradient has potential energy, called the proton motiveforce.

3. The protons on the side of the membrane with the higher protonconcentration can diffuse across the membrane only through specialprotein channels that contain an enzyme called ATP synthase. When thisflow occurs, energy is released and is used by the enzyme to synthesizeATP from ADP and Pi.

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A Summary of Aerobic Respiration

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A summary of aerobic respirationin prokaryotes. Glucose is brokendown completely to carbon dioxideand water, and ATP is generated.This process has three major phases:glycolysis, the Krebs cycle, and theelectron transport chain. Thepreparatory step is betweenglycolysis and the Krebs cycle. Thekey event in aerobic respiration isthat electrons are picked up fromintermediates of glycolysis and theKrebs cycle by NAD+ or FAD andare carried by NADH or FADH2 to theelectron transport chain. NADH isalso produced during the conversionof pyruvic acid to acetyl CoA Most ofthe ATP generated by aerobicrespiration is made by thechemiosmotic mechanism during theelectron transport chain phase: thisis called oxidative phosphorylation.

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inhibition of electron transport chain

Electron transport inhibitors:

• Rotenone

• Antimycin

• Cyanide

• Malonate

• Carbon monoxide (CO)

• Oligomycin (inhibitor of oxidative phosphorylation)

CompoundsUse

Effect on oxidative phosphorylation

CyanideCarbon monoxideAzideHydrogen sulfide

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.

CCCP2,4-Dinitrophenol

Poisons 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 the ubiquinone-binding site.

Malonate and oxaloacetate Poisons Competitive inhibitors of succinatedehydrogenase

Antimycin A Piscicide Binds to the Qi site of cytochrome c reductase, thereby inhibiting the oxidation of ubiquinol.

heterotrophic and chemolithotrophic bacteria

• Organisms able to use inorganic chemicals aselectron donors are called chemolithotrophs.

• Examples of relevant inorganic electron donorsinclude H2S, hydrogen gas (H2), Fe2+, and NH3.

• Chemolithotrophic metabolism is typicallyaerobic and begins with the oxidation of theinorganic electron donor.

• Electrons from the inorganic donor enter anelectron transport chain and a proton motiveforce is formed inexactly the same way as forchemoorganotrophs.

• However, one important distinction between chemolithotrophs andchemoorganotrophs, besides their electron donors, is their sourceof carbon for biosynthesis.

• Chemoorganotrophs use organic compounds (glucose, acetate, andthe like) as carbon sources. By contrast, chemolithotrophs usecarbon dioxide (CO2) as a carbon source and are thereforeautotrophs.

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Sources of Energy for Microorganisms

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The Three Stages of Catabolism. Ageneral diagram of aerobiccatabolism in achemoorganoheterotroph showingthe three stages in this processand the central position of thetricarboxylicacid cycle. Although there aremany different proteins,polysaccharides, and lipids, theyare degraded through theactivity of a few commonmetabolic pathways. The dashedlines show the flow of electrons,carried by NADHand FADH2, to the electrontransport chain.

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Anaerobic Respiration

• Electrons derived from sugars and other organicmolecules are usually donated either to endogenousorganic electron acceptors or to molecular O2 by wayof an electron transport chain.

• However, many bacteria have electron transport chainsthat can operate with exogenous electron acceptorsother than O2. This energy-yielding process is calledanaerobic respiration.

• The major electron acceptors are nitrate, sulfate, andCO2, but metals and a few organic molecules can alsobe reduced.

• Some bacteria can use nitrate as the electronacceptor at the end of their electron transportchain and still produce ATP.

• Often this process is called dissimilatorynitrate reduction.

• Nitrate may be reduced to nitrite by nitratereductase, which replaces cytochromeoxidase.

• However, reduction of nitrate to nitrite is not a particularlyeffective way of making ATP, because a large amount ofnitrate is required for growth.

• The nitrite formed is also quite toxic. Therefore nitrate often isfurther reduced all the way to nitrogen gas, a process knownas denitrification. Each nitrate will then accept five electrons,and the product will be nontoxic.

• There is considerable evidence that denitrification is amultistep process with four enzymes participating: nitratereductase, nitrite reductase, nitric oxide reductase, andnitrous oxide reductase.

• Interestingly, one of the intermediates is nitric oxide (NO). In mammals thismolecule acts as a neurotransmitter, helps regulate blood pressure, and is used bymacrophages to destroy bacteria and tumor cells.

• Two types of bacterial nitrite reductases catalyze the formation of NO in bacteria.One contains cytochromes c and d1 (e.g., Paracoccus and Pseudomonasaeruginosa), and the other is a copper protein (e.g., Alcaligenes).

• Nitrite reductase seems to be periplasmic in gram-negative bacteria.

• Nitric oxide reductase catalyzes the formation of nitrous oxide from NO and is amembrane-bound cytochrome bc complex.

• A well studied example of denitrification is gram-negative soil bacteriumParacoccus denitrificans, which reduces nitrate to N2 anaerobically.

• Its chain contains membrane-bound nitrate reductase and nitric oxide reductase,whereas nitrite reductase and nitrous oxide reductase are periplasmic.

• The four enzymes use electrons from coenzyme Q and c-type cytochromes toreduce nitrate and generate PMF.

• Denitrification is carried out by some members ofthe genera Pseudomonas, Paracoccus, andBacillus. They use this route as an alternative tonormal aerobic respiration and may beconsidered facultative anaerobes.

• If O2 is present, these bacteria use aerobicrespiration (the synthesis of nitrate reductase isrepressed by O2).

• Denitrification in anaerobic soil results in the lossof soil nitrogen and adversely affects soil fertility.

• Two other major groups of bacteria employinganaerobic respiration are obligate anaerobes.

• Those using CO2 or carbonate as a terminalelectron acceptor are called methanogensbecause they reduce CO2 to methane.

• Sulfate also can act as the final acceptor inbacteria such as Desulfovibrio. It is reduced tosulfide (S2- or H2S), and eight electrons areaccepted.

• Anaerobic respiration is not as efficient in ATPsynthesis as aerobic respiration—that is, not asmuch ATP is produced by oxidativephosphorylation with nitrate, sulfate, or CO2 asthe terminal acceptors.

• Reduction in ATP yield arises from the fact thatthese alternate electron acceptors have lesspositive reduction potentials than O2.

• The reduction potential difference between adonor like NADH and nitrate is smaller than thedifference between NADH and O2.

• Because energy yield is directly related to themagnitude of the reduction potential difference,less energy is available to make ATP in anaerobicrespiration.

• Nevertheless, anaerobic respiration is usefulbecause it is more efficient than fermentationand allows ATP synthesis by electron transportand oxidative phosphorylation in the absence ofO2.

• Anaerobic respiration is very prevalent in oxygen-depleted soils and sediments.

• Often one will see a succession of microorganisms in anenvironment when several electron acceptors are present.

• For example, if O2, nitrate, manganese ion, ferric ion,sulfate, and CO2 are available in a particular environment, apredictable sequence of oxidant use takes place when anoxidizable substrate is available to the microbial population.

• Oxygen is employed as an electron acceptor first because itinhibits nitrate use by microorganisms capable ofrespiration with either O2 or nitrate.

• While O2 is available, sulfate reducers and methanogensare inhibited because these groups are obligate anaerobes.

• Once the O2 and nitrate are exhausted, competition for useof other oxidants begins.

• Manganese and iron will be used first, followed bycompetition between sulfate reducers and methanogens.

• This competition is influenced by the greater energy yieldobtained with sulfate as an electron acceptor. The sulfatereducer Desulfovibrio grows rapidly and uses the availablehydrogen at a faster rate than Methanobacterium.

• When the sulfate is exhausted, Desulfovibrio no longeroxidizes hydrogen, and the hydrogen concentration rises.

• The methanogens finally dominate the habitat and reduceCO2 to methane.

References

• Reading

• Brock biology ofmicroorgamism (13th

edition) by Madigan,Martinko, Stahl, Clark

• Microbiology (10th

edition) by Tortora,Funke and Case

• Microbiology (5th

edition) by Prescott

• Images

• 1-10: Microbiology (10th

edition) by Tortora, Funke and Case