Electron Transport and Oxidative Phosphorylation

Chapter 20 Electron Transport and Oxidative Phosphorylation Biochemistry by Reginald Garrett and Charles Grisham

Garrett and Grisham, Biochemistry, Third Edition

Essential Question • How do cells oxidize NADH and [FADH2] and convert their reducing potential into the chemical energy of ATP?


Outline • Where in the Cell Are Electron Transport and Oxidative Phosphorylation Carried Out? • What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? • How Is the Electron-Transport Chain Organized? • What Are the Thermodynamic Implications of Chemiosmotic Coupling? • How Does a Proton Gradient Drive the Synthesis of ATP? • What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation? • How Are the Electrons of Cytosolic NADH Fed into Electron Transport? Garrett and Grisham, Biochemistry, Third Edition

20.1 - Where in the Cell Are Electron Transport and Oxidative Phosphorylation Carried Out? • Electron Transport: Electrons carried by reduced coenzymes are passed through a chain of proteins and coenzymes to drive the generation of a proton gradient across the inner mitochondrial membrane • Oxidative Phosphorylation: The proton gradient runs downhill to drive the synthesis of ATP • It all happens in or at the inner mitochondrial membrane Garrett and Grisham, Biochemistry, Third Edition

Figure 20.1 (a) A drawing of a mitochondrion with components labeled. (b) Tomography of a rat liver mitochondrion. The tubular structures in red, yellow, green, purple, and aqua represent individual cristae formed from the inner mitochondrial membrane. (b,Frey, T.G., and Mannella, C.A.,2000. The internal structure of mitochondria. Trends in Biochemical Sciences 25:319-324.)


20.2 – What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? High Eo' indicates a strong tendency to be reduced • Crucial equation: ∆Go' = -nF∆Eo' ∆Eo' = Eo'(acceptor) - Eo'(donor) • Electrons are donated by the half reaction with the more negative reduction potential and are accepted by the reaction with the more positive reduction potential: ∆Eo ' positive, ∆Go' negative • If a given reaction is written so the reverse is true, then the ∆Eo' will be a negative number and ∆Go' will be positive Garrett and Grisham, Biochemistry, Third Edition

Figure 20.2 Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the acetaldehyde/ethanol couple, (b) the fumarate/succinate couple, (c) the Fe3+/Fe2+ couple.


20.3 – How Is the Electron Transport Chain Organized? Figures 20.3 and 20.4 hold the secrets • Four protein complexes in the inner mitochondrial membrane • A lipid soluble coenzyme (UQ, CoQ) and a water soluble protein (cyt c) shuttle between protein complexes • Electrons generally fall in energy through the chain - from complexes I and II to complex IV Garrett and Grisham, Biochemistry, Third Edition

Figure 20.3 Eo΄ and E values for the components of the mitochondrial electrontransport chain. Values indicated are consensus values for animal mitochondria. Black bars represent Eo΄; red bars, E.


Figure 20.4 An overview of the complexes and pathways in the mitochondrial electron-transport chain. (Adapted from Nicholls,D.G., and Ferguson,S.J., 2002. Bioenergetics 3. London: Academic Press.)


Figure 20.5 (a) The three oxidation states of coenzyme Q. (b) A space-filling model of coenzyme Q.


Complex I Oxidizes NADH and Reduces Coenzyme Q NADH-CoQ Reductase • Electron transfer from NADH to CoQ • More than 30 protein subunits - mass of 850 kD • Path: NADH → FMN → Fe-S → UQ→ FeS → UQ

• Four H+ transported out per 2 eGarrett and Grisham, Biochemistry, Third Edition

Figure 20.6 Proposed structure and electron transport pathway for Complex I. Three protein complexes have been isolated, including the flavoprotein (FP), iron-sulfur protein (IP), and hydrophobic protein (HP). FP contains three peptides (of mass 51, 24, and 10 kD) and bound FMN and has 2 Fe-S centers (a 2Fe-2S center and a 4Fe-4S center). IP contains six peptides and at least 3 Fe-S centers. HP contains at least seven peptides and one Fe-S center.


Complex II Oxidizes Succinate and Reduces Coenzyme Q • • • • • •

Succinate-CoQ Reductase aka succinate dehydrogenase (from TCA cycle!) aka flavoprotein 2 (FP2) - FAD covalently bound four subunits, including 2 Fe-S proteins Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S Path: succinate → FADH2 → 2Fe2+ → UQH2 Net reaction: succinate + UQ → fumarate + UQH2 Garrett and Grisham, Biochemistry, Third Edition

Figure 20.7 The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).


Figure 20.8 A probable scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to coenzyme Q (UQ). Proton transport does not occur in this complex.


Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c • • • • • •

CoQ-Cytochrome c Reductase CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle The principal transmembrane protein in complex III is the b cytochrome - with hemes bL and bH Cytochromes, like Fe in Fe-S clusters, are oneelectron transfer agents Study Figure 20.12 - the Q cycle UQH2 is a lipid-soluble electron carrier cyt c is a water-soluble electron carrier Garrett and Grisham, Biochemistry, Third Edition

Figure 20.9 Typical visible absorption spectra of cytochromes. (a) Cytochrome c, reduced spectrum; (b) cytochrome c, oxidized spectrum; (c) the difference spectrum: (a) minus (b); (d) beef heart mitochondrial particles: room temperature difference (reduced minus oxidized) spectrum; (e) beef heart submitochondrial particles: same as (d) but at 77 K. α- and βbands are labeled, and in (d) and (e) the bands for cytochromes a, b and c are indicated.


Figure 20.10 The structures of iron protoporphyrin IX, heme c, and heme a.


Figure 20.11 The structure of UQ-cyt c reductase, also known as the cytochrome bc1 complex. The α-helices of cytochrome b (pale green) define the transmembrane domain of the protein. The bottom of the structure as shown extends approximately 75 Å into the mitochondrial matrix, and the top of the structure as shown extends about 38 Å into the intermembrane space. (Photograph kindly provided by Di Xia and Johann Deisenhofer [From Xia, D., Yu,C.-A., Kim, H., Xia, J.-Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J., 1997. The crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277:60-66.])


Figure 20.12 The Q cycle in mitochondria. (a) The electron transfer pathway following oxidation of the first UQH2 at the Qp site near the cytosolic face of the membrane. (b) The pathway following oxidation of a second UQH2.


Figure 20.13 The structure of mitochondrial cytochrome c. The heme is shown at the center of the structure, covalently linked to the protein via its two sulfur atoms (yellow). A third sulfur from a methionine residue coordinates the iron.


Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side • • • • •

Cytochrome c Oxidase Electrons from cyt c are used in a fourelectron reduction of O2 to produce 2H2O Oxygen is thus the terminal acceptor of electrons in the electron transport pathway - the end! Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites Structure is now known - mostly! Complex IV also transports H+ Garrett and Grisham, Biochemistry, Third Edition

Figure 20.15 Molecular graphic image of subunits I,II, and III of cytochrome c oxidase.


Figure 20.16 Molecular graphic image of cytochrome c oxidase. Seven of the 10 nuclear DNA-derived subunits (IV, VIa, VIc, VIIa, VIIb, VIIc, and VIII) possess transmembrane segments. Three (Va, Vb, and VIb) do not. Subunits IV and VIc are transmembrane and dumbbellshaped. Subunit Va is globular and bound to the matrix side of the complex, whereas VIb is a globular subunit on the cytosolic side of the membrane complex. Vb is globular and matrix-side associated as well, but it has an N-terminal extended domain. VIa has a transmembrane helix and a small globular domain. Subunit VIIa consists of a tilted transmembrane helix, with another short helical segment on the matrix side of the membrane. Subunits VIIa, VIIb, and VIII consist of transmembrane segments with short extended regions outside the membrane.


Figure 20.17 The electron transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centers to reduce O2 on the matrix side of the membrane.


Figure 20.18 (a) The CuA site of cytochrome oxidase. Copper ligands include two histidine imidazole groups and two cysteine side chains from the protein. (b) The coordination of histidine imidazole ligands to the iron atom in the heme a center of cytochrome oxidase.


Figure 20.19 The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the CuB and Fe of heme a3 metal sites.


Figure 20.20 A model for the mechanism of O2 reduction by cytochrome oxidase.

(Adapted from Nicholls, D. G., and Ferguson, S. J., 1992. Bioenergetics 2. London: Academic Press; and Babcock, G. T., and Wikström, M., 1992. Nature 356:301-309.)


20.4 – What Are the Thermodynamic Implications of Chemiosmotic Coupling? • • • •

This coupling was a mystery for many years Many biochemists squandered careers searching for the elusive "high energy intermediate" Peter Mitchell proposed a novel idea - a proton gradient across the inner membrane could be used to drive ATP synthesis Mitchell was ridiculed, but the chemiosmotic hypothesis eventually won him a Nobel prize Be able to calculate the ∆G for a proton gradient (Equation 20.24) Garrett and Grisham, Biochemistry, Third Edition

Figure 20.21 A model for the electron transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cytochrome c are mobile electron carriers and function by transferring electrons between the complexes. The proton transport driven by Complexes I, III, and IV is indicated.


20.5 – How Does a Proton Gradient Drive the Synthesis of ATP? Proton diffusion through the protein drives ATP synthesis! • Two parts: F1 and F0 (latter was originally "F0" for its inhibition by oligomycin) • See Figure 20.25 and Table 20.3 for details • Racker & Stoeckenius confirmed Mitchell’s hypothesis using vesicles containing the ATP synthase and bacteriorhodopsin • Paul Boyer’s binding change mechanism won a share of the 1997 Nobel in Chemistry Garrett and Grisham, Biochemistry, Third Edition

Figure 20.24 Molecular graphic images (a) side view and (b) top view of the F1ATP synthase showing the individual component peptides. The -subunit is the pink structure visible in the center of view (b).


Figure 20.25 A model of the F1 and F0 components of the ATP synthase, a rotating molecular motor. The and subunits constitute the stator of the motor, and the c, , and subunits form the rotor. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in and that synthesize ATP.


Figure 20.26 ATP production in the presence of a proton gradient and ATP/ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of 18O in phosphate as shown.


Figure 20.27 The binding change mechanism for ATP synthesis by ATP synthase. This model assumes that F1 has three interacting and conformationally distinct active sites. The open (O) conformation is inactive and has a low affinity for ligands; the L conformation (with “loose” affinity for ligands) is also inactive; the tight (T) conformation is active and has a high affinity for ligands. Synthesis of ATP is initiated (step 1) by binding of ADP and Pi to an L site. In the second step, an energy-driven conformational change converts the L site to a T conformation and also converts T to O and O to L. In the third step, ATP is synthesized at the T site and released from the O site. Two additional passes through this cycle produce two more ATPs and return the enzyme to its original state.


Figure 20.28 The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis.


Inhibitors of Oxidative Phosphorylation Reveal Insights About the Mechanism • Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish! • Cyanide, azide and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 • Oligomycin and DCCD are ATP synthase inhibitors


Figure 20.29 The structures of several inhibitors of electron transport and oxidative phosphorylation.


Figure 20.30 The sites of action of several inhibitors of electron transport and/or oxidative phosphorylation.


Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase Uncoupling e- transport and oxidative phosphorylation • Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient • Uncouplers are hydrophobic molecules with a dissociable proton • They shuttle back and forth across the membrane, carrying protons to dissipate the gradient Garrett and Grisham, Biochemistry, Third Edition

Figure 20.31 Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.


ATP-ADP Translocase Mediates the Movement of ATP and ADP Across the Mitochondrial Membrane

ATP must be transported out of the mitochondria • ATP out, ADP in - through a "translocase" • ATP movement out is favored because the cytosol is "+" relative to the "-" matrix • But ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in • So every ATP transported out costs one H+ • One ATP synthesis costs about 3 H+ • Thus, making and exporting 1 ATP = 4H+ Garrett and Grisham, Biochemistry, Third Edition

Figure 20.32 Outward transport of ATP (via the ATP/ADP translocase) is favored by the membrane electrochemical potential.


20.6 - What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation? • • • • •

i.e., How many ATP made per electron pair through the chain? e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen 4 H+ flow back into matrix per ATP to cytosol 10/4 = 2.5 for electrons entering as NADH For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen 6/4 = 1.5 for electrons entering as succinate Garrett and Grisham, Biochemistry, Third Edition

20.7 – How Are the Electrons of Cytosolic NADH Fed into Electron Transport? • • • •

Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane What to do?? "Shuttle systems" effect electron movement without actually carrying NADH Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane Garrett and Grisham, Biochemistry, Third Edition

Figure 20.33 The glycerophosphate shuttle (also known as the glycerol phosphate shuttle) couples the cytosolic oxidation of NADH with mitochondrial reduction of [FAD].


Figure 20.34 The malate (oxaloacetate)aspartate shuttle, which operates across the inner mitochondrial membrane.


The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used • • • •

See Table 20.4! 30 ATP per glucose if glycerol-3-P shuttle used 32 ATP per glucose if malate-Asp shuttle used In bacteria - no mitochondria - no extra H+ used to export ATP to cytosol, so: – 10/3 = ~3ATP/NADH – 6/3 = ~ 2ATP/FADH2 Garrett and Grisham, Biochemistry, Third Edition
