Electron Transport and Oxidative Phosphorylation

I. The Role of Electron Transport in Metabolism

1. The importance of mitochondrial structure in ATP production

■ The production of ATP by oxidative phosphorylation (endergonic process) is separate from electron transport to oxygen (exergonic process) the reactions are strongly linked to one another and are tightly coupled to the synthesis of ATP by phosphorylation of ADP.

■ The operation of the electron transport chain leads to pumping of protons (H+) across the inner mitochondrial membrane, creating a pH gradient (proton gradient).

■ This proton gradient represents stored potential energy and provides the basis of the coupling mechanism. Chemiosmotic coupling is the name given to this mechanism.

■ NADH and FADH2 (glycolysis and the citric

acid cycle) transfer electrons to O2 in the series of reactions known collectively as the electron transport chain. O2 (ultimate electron acceptor) is reduced

to H2O. 2.5 moles of ATP / 1 mole NADH;

1.5 moles of ATP / 1 mole FADH2. NADH and FADH2 pass electrons to

coenzyme Q, providing an alternative mode of entry into the electron transport chain.

Electrons are then passed from coenzyme Q to a series of proteins called cytochromes and, eventually, to O2.

II. Reduction Potentials in the Electron Transport Chain

■ Each carrier in the electron transport chain each can exist in an oxidized or a reduced form.

■ Electron carriers how would we know whether electrons would be more likely to be transferred from A to B?

■ Determined by a reduction potential for each of the carriers. ■ A molecule with a high reduction potential tends to be reduced if it is

paired with a molecule with a lower reduction potential.

1. Reduction potentials

III. Organization of Electron Transport Complexes

■ Respiratory complexes: multienzyme systems. ■ Each of the respiratory complexes can carry out the reactions of a portion

of the electron transport chain.

1. Electron transport chain reactions in the respiratory complexes

Complex I

■ NADH-CoQ oxidoreductase

(1) Catalyzes the first steps of electron transport from NADH to coenzyme Q (CoQ).

(2) An integral part of the inner mitochondrial membrane.

(3) Contain an Fe-S cluster and the flavoprotein that oxidizes NADH.

(4) Flavoprotein has a Flavin coenzyme (flavin mononucleotide, or FMN).

■ The reaction occurs in several steps, with successive oxidation and reduction of the flavoprotein and the iron-sulfur moiety. (1) Transfer of electrons from NADH to the flavin portion of the flavoprotein: (2) Reduced flavoprotein reoxidized;

Oxidized form of the Fe-S protein reduced. (3) Reduced Fe-S protein donates its electrons to coenzyme Q

(ubiquinone) reduced to CoQH2.

NADH + H+ + E—FMN NAD+ + E—FMNH2

E—FMNH2 + 2Fe—Soxidized E—FMN + 2Fe—Sreduced + 2H+ 2Fe—Sreduced + CoQ + 2H+ 2Fe—Soxidized + CoQH2 Overall reaction: NADH + H+ + CoQ NAD+ + CoQH2

(4) This reaction is one of the three responsible for the proton pumping: strongly exergonic (ΔG°′ = -81 kJ/mol). (5) Coenzyme Q (final electron receptor of complex I) mobile (free to move in the membrane) pass the electrons to the third complex for further transport to O2.

Complex II

■ Succinate-CoQ oxidoreductase

(1) Catalyzes the transfer of electrons to coenzyme Q. (2) Substrate: succinate (citric acid cycle) oxidized to fumarate.

Succinate + E—FAD Fumarate + E—FADH2 (3) The flavin group is reoxidized; Fe-S protein is reduced:

E—FADH2 + Fe—Soxidized E—FAD + Fe—Sreduced (4) This reduced Fe-S protein donates its electrons to oxidized CoQ CoQ is reduced.

Fe—Sreduced + CoQ + 2H+ Fe—Soxidized + CoQH2

Overall reaction: Succinate + CoQ Fumarate + CoQH2

(5) An integral part of the inner mitochondrial membrane. (6) Exergonic reaction (ΔG°′ = -13.5 kJ/mol) not enough energy to drive ATP production. no hydrogen ions are pumped out of the matrix during this step.

Complex Ill

■ CoQH2-cytochrome c oxidoreductase (also called cytochrome reductase)

(1) Catalyzes the oxidation of reduced coenzyme Q (CoQH2). (2) Electrons passed along to cytochrome c in a multistep process. (3) Overall reaction:

CoQH2 + 2 Cyt c[Fe(III)] CoQ + 2 Cyt c[Fe(II)] + 2H+

(4) Complex include cytochrome b (actually two b type cytochromes,

cytochrome bH and bL), cytochrome c1, and several Fe—S proteins.

(5) Cytochromes can carry electrons, but not hydrogens. (6) H+ leave the matrix: [CoQH2 CoQ] H+ pass out on the other side of

the membrane.

(7) An integral part of the inner mitochondrial membrane.

(8) Coenzyme Q is soluble in the lipid component of the mitochondrial membrane.

(9) Cytochrome c itself is not part of the complex but is loosely bound to the outer surface of the inner mitochondrial membrane, facing the intermembrane space.

(10) Two important electron carriers (CoQ, Cyt c) not part of the respiratory complexes but can move freely in the membrane.

■ The flow of electrons from reduced CoQ to the other components of the complex does not take a simple, direct path.

(1) A cyclic flow of electrons involves CoQ twice. (2) As a quinone, CoQ can exist in 3 forms semiquinone form: crucial importance.

(3) Crucial involvement of CoQ called Q cycle.

■ In part of the Q cycle (1) 1 electron is passed:

CoQH2 Fe—S Cyt c1 Cyt c; leaving coenzyme Q in the semiquinone form.

(2) A second molecule of coenzyme Q is involved. (3) Each of the 2 molecules of coenzyme Q

involved in the Q cycle loses 1 electron. (4) Net result same as if 1 molecule of CoQ had

lost 2 electrons. (5) 1 molecule of CoQH2 is regenerated, and 1 is

oxidized to CoQ. (6) Most important:

the Q cycle provides a mechanism for electrons to be transferred 1 at a time from coenzyme Q to cytochrome.

■ Proton pumping occurs as a result of the reactions of this complex. The Q cycle is implicated in the process. ΔG°′ = –34.2 kJ/mol: supplies enough energy to drive the production of ATP.

Complex IV

■ Cytochrome c oxidase

(1) Transfer of electrons from cytochrome c to O2. (2) The overall reaction is

2 Cyt c[Fe (II)] + 2H+ + 1/2 O2 2 Cyt c[Fe (III)] + H2O

(3) Proton pumping also takes place as a result of this reaction. (4) An integral part of the inner mitochondrial membrane. (5) Contains cytochromes a and a3, as well as 2 Cu2+ ions (intermediate

electron acceptors) that are involved in the electron transport process. Cyt c Cyt a Cu2+ Cyt a3 O2

(6) Cytochromes a and a3 taken together form the complex known as

cytochrome oxidase. (7) Reduced cytochrome oxidase oxidized by O2; O2 reduced to H2O.

The half reaction for the reduction of O2 (oxidizing agent) is 1/2 O2 + 2H+ + 2e- H2O

2. Summary of electron transport chain reaction energetics

3. The Redox of the heme in cytochromes ■ The cytochromes are macromolecules. ■ Found in all types of organisms ; In eukaryotes, located in inner

mitochondrial membrane, also occur in the endoplasmic reticulum. ■ All cytochromes contain the heme group. ■ The iron of the heme group does not bind to oxygen; involved in the

series of redox reactions. (1) differences in the side chains of the heme group of the cytochromes

involved in the various stages of electron transport. (2) variations in the polypeptide chain and in the way the polypeptide chain

is attached to the heme. (1)+(2) account for the differences in properties among the cytochromes

in the electron transport chain.

■ Nonheme iron proteins (1) Do not contain a heme group. (2) Many of the most important proteins in this

category contain sulfur. (3) The iron is usually bound to cysteine or to S2-.

IV. The Connection between Electron Transport and

Phosphorylation

■ The energy-releasing oxidation reactions proton pumping pH gradient

across the inner mitochondrial membrane.

■ A voltage difference across the membrane is generated by the concentration

differences of ions inside and out.

■ The energy of the electrochemical potential (voltage drop) across the

membrane is converted to the chemical energy of ATP by the coupling

process.

1. The coupling factor in oxidative phosphorylation

■ A coupling factor is needed to link oxidation and phosphorylation.

■ A complex protein oligomer, separate from the electron transport complexes,

serves this function.

■ ATP synthase

■ Compounds known as uncouplers inhibit the phosphorylation of ADP

without affecting electron transport.

(1) Uncoupler: 2,4-dinitrophenol, valinomycin, gramicidin A,

(2) When an uncoupler is present, O2 is still reduced to H2O, but ATP is not

produced.

V. The Mechanism of Coupling in Oxidative Phosphorylation

■ Several mechanisms have been proposed to account for the coupling of

electron transport and ATP production.

■ The point of departure: chemiosmotic coupling, which was later modified to

include a consideration of conformational coupling.

1. Chemiosmotic coupling mechanism

■ Based on the difference in proton concentration (proton gradient) between the intermembrane space and the matrix of an actively respiring mitochondrion.

■ Proton gradient various electron carriers proteins are not symmetrically oriented; nor do they react in the same way.

■ In the process of electron transport, the proteins of the respiratory complexes take up protons from the matrix

to transfer them in redox reactions these electron carriers subsequently release protons into the

intermembrane space when they are reoxidized, creating the proton gradient.

The proton gradient in turn can drive the production of ATP that occurs when the protons flow back into the matrix.

■ Experimental evidence

1. A system with definite inside and outside compartments (closed vesicles)

is essential for oxidative phosphorylation does not occur in soluble preparations or in membrane fragments without compartmentalization.

2. Submitochondrial preparations that contain closed vesicles carry out

oxidative phosphorylation the asymmetrical orientation of the respiratory complexes with respect to the membrane can be demonstrated.

3. A model system for oxidative phosphorylation:

reconstituted membrane vesicles, mitochondrial ATP synthase, and a proton pump (bacteriorhodopsin, a protein found in the membrane of halobacteria) the proton pumping takes place when the

protein is illuminated. 4. The existence of the pH gradient has been

demonstrated and confirmed experimentally. ■ Production of ATP depends on ion

channels: ATP synthase. 1. Protons flow back into the matrix

through ion channels (F0) in the ATP synthase.

2. Formation of ATP (F1 unit). 3. The unique feature of

chemiosmotic coupling is the direct linkage of the proton gradient to the phosphorylation reaction.

2. Conformational coupling mechanism

■ The proton gradient leads to conformational changes in the ATP synthase itself.

■ Recent evidence the proton gradient is involved in

the release of tightly bound ATP from the synthase as a result of the conformational change.

(1) 3 sites for substrate on the synthase and 3 possible

conformational states: open (O), with low affinity for substrate; loose-binding (L), not catalytically active; and tight-binding (T), catalytically active.

(2) At any given time, each site is in one of three different conformational states. These states interconvert as a result of the proton flux through the synthase.

(3) ATP already formed by the synthase is bound at a site in the T conformation, while ADP and Pi bind at a site in the L conformation.

(4) A proton flux converts the site in the T conformation

to the O conformation, releasing the ATP. (5) The site at which ADP and Pi are bound assumes

the T conformation, which can then give rise to ATP.

VI. Respiratory Inhibitors Can Be Used to Study Electron Transport

■ The compounds that come after the blockage point will lack electrons and will tend to be found in the oxidized form.

■ Respiratory inhibitors gather additional evidence to establish the order of components in the electron transport pathway.

Do respiratory inhibitors have a connection with respiratory complexes?

■ Specialized spectroscopic techniques the individual types of cytochromes

can be identified by the wavelength at which the peak appears, and the relative amounts can be determined from the intensities of the peaks.

VII. Shuttle Mechanisms

■ NADH (produced by glycolysis) in the cytosol cannot cross the inner mitochondrial membrane.

■ The electrons can be transferred to a carrier that can cross the membrane.

1. Glycerol-phosphate shuttle

(1) Insect flight muscle, mammalian muscle and brain. (1) Uses the presence on the outer face of the inner mitochondrial

membrane of an FAD-dependent enzyme that oxidizes glycerol phosphate. (2) Reduction of dihydroxyacetone phosphate glycerol phosphate;

NADH is oxidized to NAD+. (3) Oxidizing agent: FAD FADH2. (4) FADH2 passes electrons through the electron transport chain

1.5 moles of ATP for each mole of cytosolic NADH.

2. Malate-aspartate shuttle

(1) Mammalian kidney, liver, and heart. (2) Uses the fact that malate can cross the mitochondrial membrane, while

OAA cannot. (3) Noteworthy point: NADH in the cytosol NADH in the mitochondrion. (4) Cytosol: OAA malate (NADH NAD+) crosses the mitochondrial

membrane; mitochondrion: malate (NAD+ NADH) OAA aspartate

cross the mitochondrial membrane OAA in the cytosol. (5) NADH 2.5 moles of ATP are produced.

VIII. The ATP Yield from Complete Oxidation of Glucose