Name________KEY_______________________________________ SSN__________________________________________2395G Exam 311 Dec 2002100 points total 1. One-carbon transfers are mediated by the coenzyme tetrahydrofolate (H4folate) in mostorganisms. However, some Archaebacteria use the structurally related coenzyme,tetrahydromethanopterin (H4MPT) for the same transfers. An important reaction involving thesecofactors is the reduction of the bound one-carbon unit from the CH2 (methylene) oxidation state tothe CH3 (methyl) oxidation state, catalyzed by a specific reductase (see reactions A and B). In themammalian enzyme (reaction A), NADPH is the electron donor for the reduction; in theArchaebacteria enzyme (reaction B), a different cofactor, F420H2 serves as electron donor.A: CH2-H4folate + NADPH + H+ <==> CH3-H4folate + NADP+ ∆G°' = -39.8 kJ/molB: CH2-H4MPT + F420H2 <==> CH3-H4MPT + F420∆G°' = -5.5 kJ/mol E°' (V) NADP+ + 2H+ + 2 e- <==> NADPH + H+-0.320F420 + 2H+ + 2 e- <==> F420H2 -0.350F = 96494 J.volt-1.mol-1R = 8.314 J.K-1.mol-110 pts. a. Using the values given, calculate the standard redox potential (E°') for the CH2/CH3 couple foreach system (i.e. CH2-H4folate/CH3-H4folate vs. CH2-H4MPT/CH3-H4MPT).First calculate ∆E°' for each reaction: ∆G°' = -nF∆E°' ‡ ∆E°' = ∆G°'/-nFA: ∆E°' = -39.8 kJ/mol/(-2)(96.494 kJ/V/Mol) = 0.206 voltsB: ∆E°' = -5.5 kJ/mol/(-2)(96.494 kJ/V/Mol) = 0.028 voltsNow calculate E°' from ∆E°' = E°'acceptor - E°'donorA: CH2-H4folate = acceptor; NADPH = donor0.206 V = E°'acceptor - (-0.320 V)E°'acceptor = 0.206 - 0.320 = - 0.114 VB: CH2-H4MPT = acceptor; F420H2 = donor0.028 V = E°'acceptor - (-0.350 V)E°'acceptor = 0.028 - 0.350 = - 0.322 VName________KEY_______________________________________ SSN__________________________________________34 pts. b. Why do you think the Archaebacteria evolved to use F420H2 instead of NADPH as the electrondonor for the reduction of CH2-H4MPT?Because the CH2-H4MPT/CH3-H4MPT redox couple is so much more negative (by ~200 mV) than the CH2-H4folate/CH3-H4folate redox couple, NADPH is not astrong enough reductant to ensure the reduction goes significantly in the directionof CH3-H4MPT - need a stronger reductant = F420H22. You have been sent samples of two new metabolic inhibitors for analysis. Using a preparationof isolated liver mitochondria incubated with pyruvate, O2, ADP and Pi you find that addition ofinhibitor A blocks both electron transport and oxidative phosphorylation. When you addinhibitor B in addition to A, you find to your surprise that electron transport is restored, but notoxidative phosphorylation.5 pts. a. Classify these inhibitors in regard to their mode of action in electron transport and oxidativephosphorylation and briefly describe where they might be acting.Inhibitor A = phosphorylation inhibitorInhibitor B = uncouplerWhen A is added alone, both electron transport and oxidative phosphorylation are blocked. Aslong as the mitochondria are tightly coupled, inhibition of either process will inhibit the other, soit is not possible to distinguish the site of inhibition. Addition of B restores electron transport,but not phosphorylation, suggesting that B is an uncoupler (dissapates the proton gradient).Compound A must be an inhibitor of phosphorylation, at the ATP synthase.2 pts. b. Name a pair of known inhibitors that would give the same result.A = oligomycin or dicyclohexylcarbodiimide (DCCD)B = dinitrophenol (DNP)5 pts. 3. Nigericin is an ionophore that exchanges K+ for H+ across membranes. Predict the effect oftreatment of functioning mitochondria with nigericin on electron transport and ATP synthesis(oxidative phosphorylation). Explain your predictions. What effects would valinomycin, anionophore that transports K+, but not H+, have on functioning mitochondria?Ionophores are not pumps, but rather carriers or channels that allow selected ions to flow downtheir concentration gradients. Nigericin exchanges K+ for H+ ions, thereby discharging the protongradient across the inner mitochondrial membrane (allows protons to flow back intomitochondria in exchange for K+ flowing out). Since the proton gradient is generated by electrontransport and discharged by the ATP synthase-mediated formation of ATP, nigericin uncouplesthese two processes. I.e., electron transport will continue (or even increase) but ATP synthesiswill cease . Valinomycin cannot transport protons, so it will have little effect on either electrontransport or ATP synthesis . Its transport of K+ will decrease the membrane potential, but notchange the proton gradient, thereby leaving ATP synthesis substantially undisturbed.Name________KEY_______________________________________ SSN__________________________________________46 pts 4. a. Briefly summarize the mechanism by which light energy is converted to chemical energy in thelight reactions of photosynthesis. (Just outline the fundamental processes involved.) Light absorption by PSI and PSII causes excitation of an electron to its singlet state. Electron isejected and it enters electron transport chain . Protons are pumped during electron transportthrough the cytochrome bf complex to produce a proton gradient across the thylakoid membrane,This protonmotive force provides energy for ATP synthesis via ATP synthase. The electronseventually flow to reduce NADP + to NADPH .2 pts b. The chemical energy produced by the light reactions resides in two compounds needed to carryout the dark reactions. Name them.NADPH, ATP2 pts c. List the ultimate electron donor and final electron acceptor in non-cyclic electron transport ofthe light reactions.H2O = ultimate electron donorNADP = ultimate electron acceptor5 pts. d. Ordinary sunlight is very “dilute”. Briefly explain how the photosynthetic apparatus dealswith this problem.Each photosynthetic reaction center chlorophyll is associated with a large number of antenna chlorophylls which absorb photons, and pass the energy eventually to the reaction center chlorophyllby exciton or resonance transfer . Thus, the antenna chlorophylls capture photons from "dilute"sunlight and funnel their energy to the reaction center.5 pts e. Which is the rate-limiting step in the dark reactions? Briefly discuss the manner by whichthis step is regulated by light.ribulose-1,5-bisphosphate + CO2 + H20 ‡ 2 3-phosphoglycerate (RUBISCO reaction)Regulation is