ELECTRON TRANSPORT CHAIN Energy-rich molecules, such as glucose, are

metabolized by a series of oxidation reactions

ultimately yielding CO2 and water (Figure 6.6). The

metabolic intermediates of these reactions donate

electrons to specific coenzymes—nicotinamide

adenine dinucleotide (NAD+) and flavin adenine

dinucleotide (FAD)—to form the energy-rich

reduced coenzymes, NADH and FADH2. These

reduced coenzymes can, in turn, each donate a pair

of electrons to a specialized set of electron carriers,

collectively called the electron transport chain.

• As electrons are passed down the electron

transport chain, they lose much of their free

energy. Part of this energy can be captured

and stored by the production of ATP from ADP

and inorganic phosphate (Pi). This process is

called oxidative phosphorylation

A. Mitochondrion

The electron transport chain is present in the

inner mitochondrial membrane and is the final

common pathway by which electrons derived

from different fuels of the body flow to oxygen.

Electron transport and ATP synthesis by

oxidative phosphorylation proceed

continuously in all tissues that contain

mitochondria.

1. Structure of the mitochondrion: The

components of the electron transport chain are

located in the inner membrane. Although the

outer membrane contains special pores, making

it freely permeable to most ions and small

molecules, the inner mitochondrial membrane

is a specialized structure that is impermeable to

most small ions, including H+, Na+, and K+,

small molecules such as ATP, ADP, pyruvate,

and other metabolites important to

mitochondrial function (Figure 6.7).

Specialized carriers or transport systems are

required to move ions or molecules across this

membrane. The inner mitochondrial

membrane is unusually rich in protein, half of

which is directly involved in electron transport

and oxidative phosphorylation. The inner

mitochondrial membrane is highly convoluted.

The convolutions, called cristae, serve to

greatly increase the surface area of the

membrane.

• Aerobic organisms are able to capture a far

greater proportion of the available free energy of

respiratory substrates than anaerobic organisms.

Most of this takes place inside mitochondria,

which have been termed the "powerhouses" of

the cell. Respiration is coupled to the generation

of the high-energy intermediate, ATP, by

oxidative phosphorylation.

2. ATP synthase complexes:

These complexes of proteins are referred

to as inner membrane particles and are

attached to the inner surface of the inner

mitochondrial membrane. They appear as

spheres that protrude into the

mitochondrial matrix.

3. Matrix of the mitochondrion: This gel-like solution

in the interior of mitochondria is fifty percent

protein. These molecules include the enzymes

responsible for the oxidation of pyruvate, amino

acids, fatty acids (by β-oxidation), and those of the

tricarboxylic acid (TCA) cycle. The synthesis of urea

and heme occur partially in the matrix of

mitochondria.

In addition, the matrix contains NAD+

and FAD (the oxidized forms of the two

coenzymes that are required as hydrogen

acceptors)and ADP and Pi, which are

used to produce ATP. [Note: The matrix

also contains mitochondrial RNA and

DNA (mtRNA and mtDNA) and

mitochondrial ribosomes.]

B. Organization of the chain

The inner mitochondrial membrane can be

disrupted into five separate enzyme complexes,

called complexes I,II,III, IV, and V.

Complexes I to IV each contain part of the

electron transport chain (Figure 6.8), whereas

complex V catalyzes ATP synthesis .

Each complex accepts or donates electrons to

relatively mobile electron carriers, such as

coenzyme Q and cytochrome c

Each carrier in the electron transport chain

can receive electrons from an electron donor,

and can subsequently donate electrons to the

next carrier in the chain. The electrons

ultimately combine with oxygen and protons to

form water. This requirement for oxygen makes

the electron transport process the respiratory

chain, which accounts for the greatest portion

of the body's use of oxygen.

C. Reactions of the electron transport

chain

With the exception of coenzyme Q, all

members of this chain are proteins. These may

function as enzymes as is the case with the

dehydrogenases, they may contain iron as part

of an iron-sulfur center, they may be

coordinated with a porphyrin ring as in the

cytochromes, or they may contain copper, as

does the cytochrome a + a3 complex.

1. Formation of NADH:

NAD+ is reduced to NADH by

dehydrogenases that remove two hydrogen

atoms from their substrate. (For examples of

these reactions, see the discussion of the

dehydrogenases found in the TCA cycle. Both

electrons but only one proton (that is a hydride

ion, :H ) are transferred to the NAD+, forming

NADH plus a free proton, H+.

2. NADH dehydrogenase:

The free proton plus the hydride ion carried by

NADH are next transferred to NADH

dehydrogenase, an enzyme complex (complex

I)embedded in the inner mitochondrial membrane.

This complex has a tightly bound molecule of flavin

mononucleotide (FMN, a coenzyme structurally

related to FAD, that accepts the two hydrogen

atoms 2_e~ + 2H+) becoming FMNH2.

• NADH dehydrogenase also contains several

iron atoms paired with sulfur atoms to make

iron-sulfur centers (Figure 6.9). These are

necessary for the transfer of the hydrogen

atoms to the next member of the chain,

ubiquinone (known as coenzyme Q).

3. Coenzyme Q: Coenzyme Q is a quinone

derivative with a long isoprenoid tail. It

is also called ubiquinone because it is

ubiquitous in biologic systems. Coenzyme Q

can accept hydrogen atoms both from FMNH2,

produced by NADH dehydrogenase, and from

FADH2 (Complex II), which is produced by

succinate dehydrogenase and

acyl CoA dehydrogenase .

4. Cytochromes: The remaining members of the electron transport chain are cytochromes. Each contains a heme group made of a porphyrin ring containing an atom of iron (see p. 277). Unlike the heme groups of hemoglobin, the cytochrome iron atom is reversibly converted from its ferric (Fe+ 3) to its ferrous( Fe+2) form as a normal part of its function as a reversible carrier of electrons. Electrons are passed along the chain from coenzyme Q to cytochromes b and c (Complex III) and a +a3 (Complex IV, see Figure 6.8)

5. Cytochrome a +a3 This cytochrome

complexes the only electron

carrier in which the heme iron has a free

ligand that can react directly with molecular

oxygen. At this site, the transported electrons,

molecular oxygen, and free protons are

brought together to produce water (see Figure

6.8). Cytochrome a + a3(also called

cytochrome oxidase) contains bound copper

atoms that are required for this complex

reaction to occur.

• Q Accepts Electrons Via Complex I and

Complex II.

• The Q Cycle Couples Electron Transfer to

Proton Transport in Complex III.

• Molecular Oxygen Is Reduced to Water Via

Complex IV.

6. Site-specific inhibitors: Site-specific inhibitors of electron transport have been identified and are illustrated in Figure (6.10)

These compounds prevent the passage of electrons by binding to a component of the chain, blocking the oxidation/reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Because electron transport and oxidative phosphorylation are tightly coupled, site-specific inhibition of the electron transport chain also inhibits ATP synthesis.]

Sequence of redox system in

respiratory chain

• The arrangement of component enzyme and

coenzyme in the respiratory chain depend on

the redox potential, the most negative

potential is that of NAD so it ś the first member

of the respiratory chain , which can receive H

from substrate and become reduced.

C. Release of free energy during electron

transport

Free energy is released as electrons are

transferred along the electron transport chain

from an electron donor (reducing agent or

reductant) to an electron acceptor (oxidizing

agent or oxidant). The (electrons can be

transferred in different forms, for example, as

hydride ions (:H-) to NAD+, as hydrogen

atoms (-H) to FMN, coenzyme Q, and FAD, or

as electrons –e-) to cytochromes.

Site of ATP ProductionEach pair of electrons when it enter the chain

from the beginning till reach with oxygen then

one high energy product (ATP) is produced in 3

sites between NADH and FP, CoQ or ubiquinone

and cytochrome b , cytochrome a a3 &O2 .

Therefore 3molecule of ATP are produced from

the cycle when started from the beginning (NAD)

to the end. While when the electrons enter the

respiratory chain from CoQ then 2ATP are

formed as it miss or by pass the first sites of

synthesis of ATP.

site1 site2

ADP+Pi ATP ADP+Pi ATP

NAD FP CoQ Cyto b

site3

ATP ADP+Pi

O2 Cyto aa3 Cyto c Cyto c1

OXIDATIVE PHOSPHORYLATION

• The transfer of electrons down the electron

transport chain is energetically favored

because NADH is a strong electron donor and

molecular oxygen is an avid electron acceptor.

Mechanism of oxidative

phosphorylation

A. Chemiosmotic hypothesis

• The chemiosmotic hypothesis (also known as

the Mitchell hypothesis) explains how the free

energy generated by the transport of electrons

by the electron transport chain is used to

produce ATP from ADP +Pi.

1. Proton pump:

Electron transport is coupled to the

phosphorylation of ADP by the transport of

protons (H+) across the inner mitochondrial

membrane from the matrix to the

intermembrane space. This process creates

across the inner mitochondrial membrane an

electrical gradient (with more positive charges

on the outside of the membrane than on the

inside) and a pH gradient (the outside of the

membrane is at a lower pH than the inside;

Figure 6.13).

• The energy generated by this proton

gradient is sufficient to drive ATP

synthesis. Thus, the proton gradient

serves as the common intermediate that

couples oxidation to phosphorylation.

2. ATP synthase:

The enzyme complex ATP synthase (complex V,

see Figure (6.13 ) synthesizes ATP, using the

energy of the proton gradient generated by the

electron transport chain. [Note: It is also

called ATPase, because the isolated enzyme

also catalyzes the hydrolysis of ATP to ADP and

inorganic phosphate.]

• The chemiosmotic hypothesis proposes that

after protons have been transferred to the

cytosolic side of the inner mitochondrial

membrane, they reenter the mitochondrial

matrix by passing through a channel in the

ATP synthase complex, resulting in the

synthesis of ATP from ADP + Pi .

A.Oligomycin: This drug binds to the stalk of

ATP synthase, closing the H+ channel, and

preventing reentry of protons into the

mitochondrial matrix. Electron transport stops

because of the difficulty of pumping any more

protons against the steep gradients. Electron

transport and phosphorylation are, therefore,

again shown to be tightly coupled processes—

inhibition of phosphorylation inhibits

oxidation.

b. Uncoupling proteins (UCP):

UCPs occur in the inner mitochondrial membrane of

mammals, including humans. These proteins

create a "proton leak," that is, they allow protons

to reenter the mitochondrial matrix without energy

being captured as ATP (Figure 6.14). [Note:

Energy is released in the form of heat.]UCP1, also

called thermogenin, is responsible for the

activation of fatty acid oxidation and heat

production in the brown adipocytes of mammals.

Brown fat, unlike the more abundant white fat,

wastes a most ninety percent of its respiratory

energy for thermogensis in response to cold, at

birth, and during arousal in hibernating

animals. However humans have little brown fat

(except in the newborn), and UCP1 does not

appear to play a major role in energy balance.

Other uncoupling proteins (UCP2, UCP3)

have been found in humans, but their

significance remains controversial.

c. Synthetic uncouplers: Electron transport and

phosphorylation can be uncoupled by compounds

that increase the permeability of the inner

mitochondrial membrane to protons. The classic

example is a 2,4-dinitrophenol, lipophilic proton

carrier that readily diffuses through the

mitochondial membrane. This uncoupler causes

electron transport to proceed at a rapid rate without

establishing a proton gradient, much as do the

UCPs (see Figure 6.14). The energy produced by the

transport of electrons is released as heat rather than

being used to synthesize ATP.

P/O Ratio

The P/O ratio is the ratio of the number of moles

of ATP generated to the number of atoms of

oxygen reduced in the electron transport

chain. It is used to express the efficiency of

oxidative phosphorylation of the system.

There are 3 sites of generation of ATP in the

electron transport chain. Thus P/O ratio is 3 if

reducing equivalents enter electron transport

chain through NAD. On the other hand for the

FAD requiring reactions P/O ratio is 2.

Transport of reducing equivalents

The inner mitochondrial membrane lacks an

NADH transport protein, and NADH produced

in the cytosol cannot directly penetrate into

mitochondria. However, two electrons of

NADH (also called reducing equivalents) are

transported from the cytosol into the

mitochondria using shuttle mechanisms.

❖glycerophosphate shuttle

Through the glycerophosphate shuttle electrons are

transported to the mitochondrial electron

transport chain via FAD.

In the first step, 3-phosphoglycerol dehydrogenase

with the help of dihydroxyacetone phosphate,

catalyzes the oxidation of cytosolic NADH to

NAD+ which re-enters glycolysis.

• In the next step, electrons from 3-phosphoglycerol (glycerophosphate ) are transferred to a flavoprotein . This oxidation of 3-phosphoglycerol results in the reduction of FAD to FADH2. Since flavoprotein dehydrogenase is situated on the outer surface of the inner mitochondrial membrane, it supplies electrons directly to the electron transport chain and results in the reoxidation of FADH2 to FAD within the mitochondria. The overall process thus results in the transport of the cytosolicreducing equivalents directly into the mitochondrial electron transport chain (Figure 13.12).

❖Malate- Aspartate ShuttleIn Malate Aspartate shuttle, cytosolic

oxaloacetate is converted either to aspartatethrough the action of aspartateaminotransferase or to malate by malatedehydrogenase.

With the use of reducing equivalents (from the cytosolic NADH), oxaloacetate is reduced (by malate dehydrogenase) to malate and gets transported into the mitochondria. After its reoxidation in the matrix by the same enzyme, malate releases its reducing equivalents and reoxidized to oxaloacetate.

• Oxaloacetae since is impermeable through the

mitochondrial membrane, it reacts with

glutamate. Glutamate oxaloacetate

transaminase transminates oxaloacetate to

aspartate, and glutamate to α-ketoglutarate.

Aspartate and α-ketoglutarate are transported

through the mitochondrial membrane to the

cytosol by the α-ketoglutarate transporter and

the Glutamate- Aspartate transporter,

respectively. In the cytosol, Aspartate is

reconverted to oxaloacetate.

• As both, aspartate as well as malate are

freely permeable through the inner

mitochondrial membrane, reducing

equivalents which are accepted by NAD+

and originated in the cytosol, are thus

transported to the mitochondria for their

entry into the electron transport

chain(Figure 13.13).