III. Metabolism Oxidative Phosphorylation

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1 Department of Chemistry and Biochemistry University of Lethbridge Biochemistry 3020 III. Metabolism Oxidative Phosphorylation Biochemical Anatomy of Mitochondria Transmembrane channels allow small molecules (< 5 kd) and ions to pass through the outer membrane. Convolutions of the inner membrane provides large surface area. depending on the tissue they are more or less profuse Specific transporter carry pyruvate, fatty acids and amino acids into the matrix for access to the citric acid cycle. 1

2 Universal Electron Acceptors Collect Electrons Electrons channeled into the respiratory chain are collected from dehydrogenases of the catabolic by universal electron acceptors. Electron Carriers Nicotinamide Adenine Dinucleotide Optical Test NAD + / NADP + can accept a hydride (2e - ) and a proton 2

3 Electron Carriers - Flavin Mono Nucleotide FMN (Flavin Mono Nucleotide) is a prosthetic group of some flavoproteins. It is similar in structure to FAD (Flavin Adenine Dinucleotide), but lacking the adenine nucleotide. When free in solution, FMN (like FAD) can accept 2 e H + to form FMNH 2. Electron Carriers - Flavin Mono Nucleotide FMN, when bound at the active site of some enzymes, can accept 1 e - to form the halfreduced semiquinone radical. The semiquinone can accept a 2nd e - to yield FMNH 2. Since it can accept/donate 1 or 2 e -, FMN has an important role mediating e - transfer between carriers that transfer 2e - (e.g., NADH) & those that can accept only 1e - (e.g., Fe +++ ). 3

4 Electron Carriers Ubiquinone Coenzyme Q (CoQ, Q, ubiquinone) is very hydrophobic. It dissolves in the hydrocarbon core of a membrane. The structure of CoQ includes a long isoprenoid tail, with multiple units having a carbon skeleton comparable to that of the compound isoprene. Most often n = 10. The isoprene tail of Q 10 is longer than the width of a lipid bilayer, but may be folded to yield a more compact shape. Electron Carriers Ubiquinone The quinone ring of coenzyme Q can be reduced to the quinol in a 2e - reaction: Q + 2 e + 2 H + QH 2. When bound to special sites in respiratory complexes, CoQ can accept 1 e to form a semiquinone radical (Q ). Thus CoQ, like FMN, can mediate between 1 e & 2 e donors/acceptors. Coenzyme Q functions as a mobile e - carrier within the mitochondrial inner membrane. 4

5 Electron Carriers - Cytochromes Heme is a prosthetic group of cytochromes. Heme contains an iron atom in a porphyrin ring system. The Fe is bonded to 4 N atoms of the porphyrin ring. Hemes in the 3 classes of cytochrome (a, b, c) differ slightly in substituents on the porphyrin ring system. A common feature is 2 propionate side-chains. Only heme c is covalently linked to the protein cytochrome c via thioether bonds to cysteine residues. Electron Carriers - Cytochromes Heme a is unique in having a long farnesyl side-chain that includes 3 isoprenoid units. 5

6 Electron Carriers - Cytochromes Cytochrome c The heme iron can undergo a 1 e - transition between ferric and ferrous states: Met80 Fe e - Fe ++ The porphyrin ring is planar. His18 The heme Fe is usually bonded to 2 axial ligands, above & below the heme plane (X,Y) in addition to 4 N of porphyrin. PDBid 5CYT Electron Carriers - Cytochromes Cytochrome c N X N Fe Met80 N Y N Axial ligands may be S or N atoms of amino acid sidechains. His18 Axial ligands in cyt c are Met S (yellow) and His N (blue). PDBid 5CYT A heme that binds O 2 may have an open (empty) axial ligand position. 6

7 Electron Carriers - Cytochromes Cytochromes are proteins with heme prosthetic groups. They absorb light at characteristic wavelengths. Absorbance changes upon oxidation/reduction of the heme iron provide a basis for monitoring the redox state of the heme. Some cytochromes are part of large integral membrane complexes, each consisting of several polypeptides and including multiple electron carriers. Cytochrome c is instead a small, water-soluble protein with a single heme group. Electron Carriers Iron-sulfur Centers Iron-sulfur centers (Fe-S) are prosthetic groups containing 1-4 iron atoms complexed to elemental & cysteine S atoms. Electron transfer proteins may contain multiple Fe-S centers. 4-Fe centers have a tetrahedral structure, with Fe & S atoms alternating as vertices of a cube. 7

8 Electron Carriers Iron-sulfur Centers Iron-sulfur centers transfer only one electron, even if they contain two or more iron atoms, because of the close proximity of the iron atoms. E.g., a 4-Fe center might cycle between redox states: Fe +++ 3, Fe++ 1 (oxidized) + 1 e- Fe +++ 2, Fe++ 2 (reduced) Iron-sulfur proteins where one Fe atom is coordinated by two His residues are named Rieske iron-sulfur proteins. Electron Carriers 8

9 Respiratory Chain Most constitutents of the respiratory chain are embedded in the inner mitochondrial membrane (or in the cytoplasmic membrane of aerobic bacteria). Composition of Respiratory Chain Complexes 9

10 Respiratory Chain Electron transfer from NADH to O 2 involves multi-subunit inner membrane complexes I, III & IV, plus CoQ & cyt c. Within each complex, electrons pass sequentially through a series of electron carriers. CoQ is located in the lipid core of the membrane. There are also binding sites for CoQ within protein complexes. Cytochrome c resides in the intermembrane space. It alternately binds to complex III or IV during e - transfer. Respiratory Chain The standard reduction potentials of constituent e - carriers are consistent with the e - transfers observed. 10

11 Effect of Inhibitors on Electron Transport Respiratory chain inhibitors include: Rotenone (a rat poison) & Amytal block complex I. Antimycin A blocks electron transfer in complex III. CN - & CO inhibit complex IV. Inhibition at any of these sites will block e - transfer from NADH to O 2. Experimental setup? Effect of Inhibitors on Electron Transport The oxygen electrode 11

12 Effect of Inhibitors on Electron Transport The Experiment: A buffered solution of mitochondria containing excess ADP and Pi is equilibrated in the reaction vessel of an oxygen electrode. Reagents are injected into the chamber and the O 2 consumption is monitored. Effect of Inhibitors on Electron Transport The Experiment: A buffered solution of mitochondria containing excess ADP and Pi is equilibrated in the reaction vessel of an oxygen electrode. Reagents are injected into the chamber and the O 2 consumption is monitored. Injection of Rotenone 12

13 Effect of Inhibitors on Electron Transport The Experiment: A buffered solution of mitochondria containing excess ADP and Pi is equilibrated in the reaction vessel of an oxygen electrode. Reagents are injected into the chamber and the O 2 consumption is monitored. Effect of Inhibitors on Electron Transport The Experiment: A buffered solution of mitochondria containing excess ADP and Pi is equilibrated in the reaction vessel of an oxygen electrode. Reagents are injected into the chamber and the O 2 consumption is monitored. 13

14 Complex I catalyzes oxidation of NADH, with reduction of coenzyme Q: Complex I Bovine complex I at 17 Å resolution. NADH + H + + Q NAD + + QH 2 And the transfer of 4 H + across the membrane: Grigorieff, N. (1998). J. Mol. Biol., 277, NADH + 5H + N + Q NAD+ + QH 2 + 4H + P Complex I is therefor aproton pump that uses the energy of electron transfer for the vectorial movement of protons across the membrane. Complex I is L-shaped and contains six iron sulfur centers and FMNcontaining protein. A high-resolution crystal structure is not yet available for this large complex that in mammals includes at least 46 proteins. Complex I The domain where NADH interacts protrudes into the mitochondrial matrix. Coenzyme Q binds within the membrane domain. Fe-S centers are in the NADH-binding domain & in a connecting domain closer to the membrane segment. The initial electron transfers are: NADH + H + + FMN NAD + + FMNH 2 FMNH 2 + (Fe-S) ox FMNH + (Fe-S) red + H + 14

15 Complex I After Fe-S is reoxidized by transfer of the electron to the next iron-sulfur center in the pathway: FMNH + (Fe-S) ox FMN + (Fe-S) red + H + Electrons pass through a series of iron-sulfur centers in complex I, eventually to coenzyme Q. Coenzyme Q accepts 2 e and picks up 2 H + to yield the fully reduced QH 2. Complex II Succinate Dehydrogenase of the Krebs Cycle is also called complex II or Succinate-CoQ Reductase. FAD is the initial electron receptor. FAD is reduced to FADH 2 during oxidation of succinate to fumarate. FADH 2 is then reoxidized by transfer of electrons through a series of three iron-sulfur centers to Coenzyme Q, yielding QH 2. 15

16 Complex II X-ray crystallographic analysis of E. coli complex II indicates a linear arrangement of electron carriers within complex II, consistent with the predicted sequence of electron transfers: FAD FeS 1 FeS 2 FeS 3 CoQ In this crystal structure oxaloacetate (OAA) is bound in place of succinate. PDBid 1NEK Path of Electrons to Ubiquinone Other substrates for mitochondrial dehydrogenases pass their e - into the respiratory chain at the level of ubiquinon, but not through complex II. Acetyl-CoA DH (β oxidation) transfers e - to electron transferring flavoprotein (ETF), from which they pass to Q via ETF:ubiquinone oxidoreductase. 16

17 β Oxidation Mitochondria contain four acyl-coa DH with different specificities: Glu376 short (C 4 to C 6 ) medium (C 6 to C 10 ) long (between medium & verylong) very long (C 12 to C 18 ) fatty acyl-coas PDBid 3MDE The FADH 2 is reoxidized by the mitochondrial electron transport chain. Complex III Complex III (cytochrome bc 1 complex) accepts electrons from coenzyme QH 2 that is generated by electron transfer in complexes I & II. It couples the transfer of electrons to cytochrome c with the vectorial transport of protons from the matrix to the inermembrane space. Cytochrome c 1, a prosthetic group within complex III, reduces cytochrome c, which is the electron donor to complex IV. 17

18 Complex III The Q cycle Complex III The Q cycle The Q cycle depends on: mobility of CoQ in the lipid bilayer existence of binding sites for CoQ within the complex that stabilize the semiquinone radical, Q. 18

19 Complex III The Q cycle It takes 2 cycles for CoQ bound at a site near the matrix to be reduced to QH 2, as 2e are transferred from the b hemes, and 2H + are extracted from the matrix compartment. In 2 cycles, 2 QH 2 enter the pathway & one is regenerated. Complex III Membrane Cytochrome c 1 Rieske protein Heme b L Heme b H The Rieske iron-sulfur center (Fe-S) has a flexible link to the rest of the complex. It changes position during e transfer. Fe-S extracts an e from CoQ, & then moves closer to heme c 1, to which it transfers the e. Complex III is an obligate homodimer. Fe-S in one half of the dimer interacts with bound CoQ & heme c 1 in the other half of the dimer. PDBid 1BE3 19

20 Complex IV Cytochrome oxidase (complex IV) carries out the irreversible reaction: O H e - 2 H 2 O The four electrons are transferred into the complex one at a time from cytochrome c. Large enzyme (13 SU; 204,000 D) Bacteria contain a form that is much simpler (3-4 SU). Comparison of the two forms suggests that three are critical to the function. Complex IV Mitochondrial SU II contains two Cu ions complexed with SH groups of two Cys residues. SU I contains two heme groups (a & a 3 ) Heme a 3 and Cu B form binuclear center accepts electrons from heme a and transfers them to O 2 The overall reaction: 4 cyt c (red) + 8 H + N + O 2 4 cyt c (ox) + 4H+ P + 2H 2 O 20

21 Metal Center Ligands in Complex IV PDB file 1OCC Heme axial ligands are His N atoms. Heme a is held in place between 2 transmembrane α-helices by its axial His ligands. Liganding of Heme a in Cytochrome Oxidase Metal Center Ligands in Complex IV Heme a 3, which sits adjacent to Cu B, has only one axial ligand. Cu ligands consist of His N, & in the case of Cu A also Cys S, Met S, & a Glu backbone O. Electrons transferred from cyt c enter complex IV through Cu A & heme a. heme a 3 binuclear center His ligands They then pass to the binuclear center where the chemical reaction takes place. O 2 binds at the open axial ligand position of heme a 3, adjacent to Cu B. PDB 1OCC Cu B The open axial ligand position of heme a 3 makes it susceptible to binding of CN, CO, or the radical signal molecule NO. All inhibit cytochrome oxidase activity. 21

22 Summary Electrons reach Q through Complexes I and II, QH 2 serves as mobile carrier of electrons end protons. QH 2 passes electrons to Complex III, which passes them to the mobile carrier cytochrome c. Complex IV transfers electrons from cytochrom c to O 2 Electron flow through Complexes I, III and IV is coupled to H + intermembranespace. flow into the Energy is Conserved in a Proton Gradient Transfer of two electrons from NADH through the respiratory chain: NADH + H + + 1/2O 2 NAD + + H 2 O E 0 = 1.14 V 22

23 Energy is Conserved in a Proton Gradient The standard free-energy change is: G 0 = - n F E 0 = -2(96.5 kj/v mol)(1.14v) = -220 kj/mol In the cell where the actual [NADH]/[NAD + ] ratio is kept above 1 the real free-energy change is substantially more negative. much of the energy is used to pump protons out of the matrix Energy is Conserved in a Proton Gradient For each pair of electrons transferred to O 2 protons are pumped, 4 H + by Complex I 4 H + by Complex III, and 2 H + by Complex IV Total 10 H + per e - pair formation of a proton gradient 23

24 Energy is Conserved in a Proton Gradient Energy stored in such a gradient can be termed proton-motive force. It has two components: (1) Chemical potential energy due to concentration difference (2) Electrical potential energy due to charge separation In Actively respiring mitochondria ψ = V ph = 0.75 =(5.70 kj/mol) ph + (96.5 kj/v mol) ψ Given that the free-energy change for pumping protons outward is about 20 kj/mol (H + ) 200 kj/mol for 10 H + The Exception Eastern skunk cabbage The mitochondria of plants, fungi, and unicellular eukaryotes have electrontransfer systems that are essentially the same as those in in animals. But they also contain alternative enzymes e - are directly transferred to O 2 no H + are translocated Energy is released as heat 24

25 The Chemiosmotic Model When electrons flow spontaneously down the electrochemical gradient, energy is made available to do work. ATP synthesis There is enough free energy stored in the proton gradient to drive the synthesis of ATP (50 kj/mol) But what is the chemical mechanism that couples the two processes? The Chemiosmotic Model The proton-motive force drives the synthesis of ATP as protons flow into the matrix through a proton pore associated with an ATP synthase. ADP + P i + n H + P ATP + H 2 O + n H+ N How would you try to test / measure this hypothesis? 25

26 Testing the Chemiosmotic Model The energy of substrate oxidation is used to generate a proton gradient, that drives the ATP synthesis inhibitors of the electron transport chain influence ATP synthesis Testing the Chemiosmotic Model But How do you explain this? 26

27 Testing the Chemiosmotic Model An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. Mechansim of ATP Synthesis F 1 F o ATP Synthase of mitochondria, chloroplasts, bacteria: When the electrochemical H + gradient is favorable, F 1 F o couples ATP synthesis to spontaneous H + flux toward the side of the membrane where F 1 protrudes (e.g., toward the mitochondrial matrix). Kinetic studies revealed: Enz-ATP (Enz-ADP+P i ) Keq = k -1 /k 1 = 24 s -1 /10s -1 =2.4 If there is no ph or ψ to drive the forward reaction, K favors the reverse, ATP hydrolysis (ATPase). 27

28 ATP Synthase Has Two Functional Domains By EM with negative staining, F 1 appears as "lollipops" on the inner mitochondrial membrane, facing the matrix. Urea wash ATP Synthase Has Two Functional Domains SMP Roles of major subunits were established in studies of submitochondrial particles (SMP). If mitochondria are treated with ultrasound, the inner membrane fragments and reseals as vesicles, with F 1 on the outside. Since F 1 of intact mitochondria faces the interior matrix space, these SMP are said to be inside out (inverted vesicles). 28

29 ATP Synthase Has Two Functional Domains Inverted membrane vesicles from the inner mito membrane still contain the intact respiratory chain. catalyze electron transfer SMP F 1, the catalytic subunit, if separated from SMP catalyzes ATP hydrolysis the spontaneous reaction in the absence of an energy input. If F 1 is removed from SMP electron transfer from NADH to O 2 continues but no H + gradient is produced. How can that be explained? The membrane still containing F o and becomes leaky to H +. Fo is proton pore. Adding back F 1 restores normal low permeability to H +. Inhibitors Inhibitors of F 1 F o, that block H + transport coupled to ATP synthesis or hydrolysis, include: oligomycin, an antibiotic DCCD (dicyclohexylcarbodiimide), a reagent that reacts with carboxyl groups in hydrophobic environments, forming a covalent adduct. Either oligomycin or DCCD blocks the H + leak in membranes depleted of F 1. Thus oligomycin and DCCD inhibit the ATP Synthase by interacting with F o. 29

30 The Structure of Mitochondrial F 1 The complete subunit composition of the ATP Synthase was first established in E. coli, which has an operon that encodes genes for all subunits. F 1 in E. coli consists of 5 polypeptides with stoichiometry α 3, β 3, γ, δ, e (named in order of decreasing mol. weights). α & β subunits (513 & 460 aa in E. coli) are homologous. There are three nucleotide-binding catalytic sites, located at αβ interfaces but predominantly involving residues of the β subunits. Each of the three α subunits contains a tightly bound ATP, but is inactive in catalysis. Adenine nucleotides bind to α & β subunits with Mg ++. The Structure of Mitochondrial F 0 F o is a complex of integral membrane proteins. The stoichiometry of subunits in E. coli F o is a, b 2, c 10. Mammalian F 1 F o is slightly more complex than the bacterial enzyme. Since names were originally assigned based only on apparent MW, some subunits were given different names in different organisms. Bovine δ subunit is homologous to E. coli ε subunit. Bovine "OSCP" is homologous to E. coli δ subunit. Bovine ε subunit is unique. 30

31 Mitochondrial ATP Synthase Complex Bovine mitochondrial F 1 Yeast mitochondrial F o PDBid 1BMF PDBid 1QO1 The Binding Change Mechanism The binding change mechanism of energy coupling was proposed by Paul Boyer. He shared the Nobel prize for this model that accounts for the existence of 3 catalytic sites in F 1. For simplicity, only the catalytic β subunits are shown. It is proposed that an irregularly shaped shaft linked to F o rotates relative to the ring of 3 β subunits. The rotation is driven by flow of H + through F o. 31

32 The Binding Change Mechanism The conformation of each b subunit changes sequentially as it interacts with the rotating shaft. Each β subunit is in a different stage of the catalytic cycle at any time. E.g., the upper subunit sequentially changes to: a loose conformation in which the active site can loosely bind ADP + P i a tight conformation in which substrates are tightly bound and ATP is formed an open conformation that favors ATP release. Supporting Evidence Bovine F 1 (DCCDtreated) 90 PDBid 1E79 Crystal structure of F 1 was solved by J. E. Walker, who shared the Nobel Prize. The γ subunit includes a bent helical loop that constitutes a "shaft" within the ring of a & b subunits. Shown is bovine F 1 treated with DCCD to yield crystals in which more of the stalk is ordered, allowing structure determination. Colors: α, β, γ, δ, ε. 32

33 Supporting Evidence Bovine F 1 (DCCDtreated) 90 PDBid 1E79 Note the wide base of the rotary shaft, including part of γ as well as δ and ε subunits. Recall that the bovine δ subunit, which is at the base of the shaft, is equivalent to ε of bacterial F1. Supporting Evidence 90 PDBid 1COW In crystals of F 1 not treated with DCCD, less of the shaft structure is solved, but ligand binding may be observed under more natural conditions. The 3 β subunits are found to differ in conformation & bound ligand. 33

34 Supporting Evidence Bound to one β subunit is a non-hydrolyzable ATP analog (assumed to be the tight conformation). Bound to another β subunit is ADP (loose). The third β subunit has an empty active site (open). This is consistent with the binding change model, which predicts that each β subunit, being differently affected by the irregularly shaped rotating shaft, will be in a different one of 3 stages of the catalytic cycle. ATP ATP ATP ADP ATP PDBid 1COW 34

35 Supporting Evidence - Rotation of the γ Shaft 2. Rotation of the γ shaft relative to the ring of α & β subunits was demonstrated by Noji, H. et al., Nature 386, (1997). β subunits of F 1 were tethered to a glass surface. A fluorescent-labeled actin filament was attached to the protruding end of the γ subunit. Video recordings showed the actin filament rotating like a propeller. The rotation was ATP-dependent. Supporting Evidence - Rotation of the γ Shaft The rotation is ATP-dependent. stepping 20 nm ATP 200 nm ATP 35

36 Supporting Evidence - Rotation of the γ Shaft The rotation also load dependent The larger the actin filament. the slower the rotation Rotation of the γ Shaft Studies using varied techniques have shown ATP-induced rotation to occur in discrete 120 steps, with intervening pauses. Some observations indicate that each 120 step consists of 90 & 30 substeps, with a brief intervening pause. How could that be explained? Proposals have been made correlating these substeps with particular stages of the reaction cycle, such as ATP binding and P i release. 36

37 Subunit Arrangement in the F 1 F O Complex Mitochondrial ATP Synthase E. coli ATP Synthase Each of the 2 F o b subunits is predicted to include 1 trans-membrane α-helix & a very polar, charged α-helical domain that extends out from the membrane. Coupling between ATP Synthesis and Proton Flow The a subunit of F o (271 amino acid residues in E. coli) is predicted from hydropathy plots, to include several transmembrane α-helices. It has been proposed that the a-subunit forms 2 half- channels or proton wires (each a series of protonatable groups or embedded waters), that allow passage of protons between the two membrane surfaces & the bilayer interior. 37

38 Proton Transfer The c subunit of F o has a structure with 2 transmembrane α-helices & a short connecting loop. The small c subunit (79 aa in E. coli) is also called proteolipid, because of its hydrophobicity. One α-helix includes an Asp or Glu residue whose carboxyl reacts with DCCD (Asp61 in E. coli). Mutation studies have shown that this DCCDreactive carboxyl, in the middle of the bilayer, is essential for H + transport through F o. PDB 1A91 Asp61 F o subunit c An essential arginine residue on one of the trans-membrane a-subunit α- helices has been identified as the group that accepts a proton from Asp61 and passes it to the exit channel. Proton Transfer As the ring of 10 c subunits rotates, the c- subunit carboxyls relay protons between the 2 a-subunit half-channels. This allows H + gradient-driven H + flux across the membrane to drive the rotation. 38

39 Proton-Motive Force - Part II The proton-motive force also drives transport processes. The inner mitochondrial membrane is generally impermeable for charged species But ADP and P i are needed in the matrix and ATP is used outside! The adenine nucleotide translocase. integral inner membrane complex binds ADP 3- in the intermembrane space and exchanges it simultaneously with ATP 4-. (antiporter) Why is it driven by the proton motive force? Antiporter moves 3 neg. charges in and 4 neg. charges out favoured by the electrochemical gradient. 39

40 Proton-Motive Force - Part II A second membran transport system essential to oxidative phosphorylation: phosphate translocase It facilitates the synport of H 2 PO 4- and H + into the matrix. By transporting one H + across the membrane some energy of the electron transfer is used. The Path of NADH Into the Mitochondrium NADH generated by dehydrogenases in the cytosol (Glycolysis) has to be transported into the mito matrix. The malate-aspartate shuttle Most active in liver, kidney & hart. 40

41 The Path of NADH Into the Mitochondrium Skeletal muscle and brain use the glycerol 3-phosphate shuttle. Mitochondria of plants have an externally oriented NADH dehydrogenase. transfers e - directly to ubiquinone. What is the problem / difference here? Inhibition of F 1 F o ATP Hydrolysis When a cell is deprived of oxygen the transfer of electrons to O 2 ceases. What could happen? e - dependen proton pumping ends the proton-motive force soon collapses ATP synthase could start to hydrolyze ATP. This is prevented by a small (84 aa) protein (IF 1 ) binds simultaneously to two ATP synthase molecules. 41

42 Inhibition of F 1 F o ATP Hydrolysis This is prevented by a small (84 aa) protein (IF 1 ) binds simultaneously to two ATP synthase molecules. IF 1 is only inhibitory in its dimeric form favoured at lower ph Why can that be used to regulate inhibition? Under oxygen starvation pyruvic and lactatic acid is formed lowers ph in the cytosol and the mito matrix 42

43 Some Questions Antimycin A blocks electron transfer between cytochromes b and c 1. If intact mitochondria were incubated with antimycin A, excess NADH, and an adequate supply of O 2, which of the following would be found in the oxidized state? A) Coenzyme Q B) Cytochrome a 3 C) Cytochrome b D) Cytochrome e E) Cytochrome f B Some Questions 2,4-Dinitrophenol and oligomycin inhibit mitochondrial oxidative phosphorylation. 2,4-Dinitrophenol is an uncoupling agent; oligomycin blocks the ATP synthesis reaction itself. Therefore, 2,4-dinitrophenol will: A) allow electron transfer in the presence of oligomycin. B) allow oxidative phosphorylation in the presence of oligomycin. C) block electron transfer in the presence of oligomycin. D) diminish O 2 consumption in the presence of oligomycin E) do none of the above. A 43

44 Some Questions Compound X is an inhibitor of mitochondrial ATP synthesis. It was observed that when compound X was added to cells, the NAD + /NADH ratio decreased. Would you expect X to be an uncoupling agent or an inhibitor of respiratory electron transfer? Explain in 30 words or less. It is an inhibitor of electron transfer; its addition lowers the NAD+/NADH ratio because NADH produced by oxidative reactions in mitochondria can no longer be reoxidized by electron flow to O 2. Reading Chapter 19 Photosynthesis 44