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Oxidative Phosphorylation
Pratt and Cornely, Chapter 15
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Goal: ATP Synthesis
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Overview Redox reactions Electron transport chain Proton gradient
ATP synthesis Shuttles
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Standard Reduction Potential
“potential to be reduced” Listed according to oxidized compound being reduced High RP means that the compound is a strong oxidizer (ie O2) Its conjugate is a strong reducing agent (ie NADH) ½ O e + 2 H+ H2O
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Half Reactions Reduction potential written in terms of a reduction half reaction Aox Ared Example: Calculate DEo for reduction of FMN by NADH, given the Eo of FMN to be -0.30V Then calculate DGo’ NADH + FMN NAD+ + FMNH2
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Redox reactions: electricity
NAD+: Eo’ = -.32 FMN: Eo’=-.30 DEo’ = +0.02V Calculate DGo’:2 e- transfer DGo’ = -nFDEo’ = -2(96485 J/mol V)(0.02V) = -3.9 kJ/mol
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Numerous Redox Substrates
Chain of increasing reduction potential (potential to accept e-) Mobile carriers vs. within Complex Types of redox groups Organic cofactors Metals (iron/sulfur clusters) Cytochromes O2
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Overall chain from NADH
Complex I Q Complex III Cytochrome c Complex IV O2
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Within Complexes FAD/FMN Iron-sulfur clusters Prosthetic group
Bridge from 2 e- donors to 1 e- donors Iron-sulfur clusters
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Coenzyme Q: Mobile Carrier
1 or 2 e- carrier, 1 e- acceptor/donor Ubiquinone is a mobile carrier Can diffuse through nonpolar regions easily “Q pool” made by Complex I and Complex II (and others)
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Cytochrome c Mobile carrier Protein/heme 1 electron carrier
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Oxygen: the final electron acceptor
Water is produced—has very low reactivity, very stable Superoxide, peroxide as toxic intermediates Overall reaction NADH + H+ + ½ O2 NAD+ + H2O
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Flow Through Complexes
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Compartmentalization
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Protonmotive Force NADH + H+ + ½ O2 NAD+ + H2O + 10 H+ pumped
succinate + ½ O2 fumarate + H2O + 6 H+ pumped
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Complex I NADH Q through “Q pool” 4 protons pumped FMN
Iron-sulfur clusters “Q pool” 4 protons pumped Proton wire
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Complex III QH2 cytochromes 4 protons pumped Through Q cycle
Problem 10: An iron-sulfur protein in Complex III donates an electron to cytochrome c. Use the half reactions below to calculate the standard free energy change. How can you account for the fact that this process is spontaneous in the cell? FeS (ox) + e- FeS (red) Eo’ = V Cyt c (Fe3+) + e- cyt c (Fe2+) Eo’ = V
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Complex IV Cytochromes O2 Stoichiometry of half of an oxygen atom
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Complex II (and others)
Non-NADH sources Complex II (part of the citric acid cycle) Fatty acid oxidation Glycolysis NADH shuttle (Glycerol-3-phosphate) Bypasses Complex I Loss of 4 protons pumped
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Two paths of NADH into Matrix
NADH of glycolysis must get “into” matrix Not direct Needs either malate-aspartate shuttle (liver) Glycerol-3-phosphate shuttle (muscle) Costs 1 ATP worth of proton gradients, but allows for transport against NADH gradient
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Glycerol-3-phosphate Shuttle
Glycerol phosphate shuttle (1.5 ATP/NADH) Produces QH2 Operational in some tissues/circumstances
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Net ATP Harvest from Glucose
Glycolysis = 2 ATP Plus 3 or 5 ATP from NADH What leads to difference in this case? Pyruvate DH = 5 ATP Citric Acid Cycle = 20 ATP Total: ATP/glucose
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Overall Chemiosmosis 10 protons shuttled from matrix to intermembrane space Makes pH gradient and ion gradient
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Protonmotive Force and Oxidative Phosphorylation
Flow of electrons is useless if not coupled to a useful process Battery connected to wire Proton gradient across mitochondrial membrane
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Proton Gradient Gradient driven by concentration difference + charge difference Assume DpH 0.5 and 170mV membrane potential
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Using the Gradient Coupled to ATP synthesis
Uncouplers used to show link of oxygen uptake and ATP synthesis
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Uncouplers “Uncouple” protonmotive force from ATP synthase
DNP pKa / solubility perfectly suitable
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Respiratory Poisons Other respiration poisons
Cyanide—binds Complex IV in place of oxygen
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Complex V: ATP Synthase
Molecular motor Rotor: c, g, e Proton channel
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Proton Channel Protons enters channel between rotor and stator
Rotor rotates to release strain by allowing proton to enter matrix 8- 10 protons = full rotation Species dependent
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“Stalk” (g) moves inside the“knob”—hexameric ATP synthase
Knob held stationary by “b”
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Hexameric Knob
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Binding-Change Mechanism
Stalk causes ATP synthase to have three different conformations: open, loose, tight In “tight” conformation, energy has been used to cause an energy conformation that favors ATP formation
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Problem 39 How did these key experiments support the chemiosmotic theory of Peter Mitchell? The pH of the intermembrane space is lower than the pH of the mitochondrial matrix. Oxidative phosphorylation does not occur in mitochondrial preparations to which detergents have been added. Lipid-soluble compounds inhibit oxidative phosphorylation while allowing electron transport to continue.
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Energy Accounting ATP costs 2.7 protons
8 protons produces 3 ATP NADH pumps 10 protons when 2 e- reduce ½ O2 4 protons in Complex I, 4 protons in Complex III, and 2 protons in Complex IV P/O ratio--# of phosphorylation per oxygen atom 10H+/NADH (1 ATP/2.7 H+) = 3.7 ATP/NADH 6H+/QH2 (1 ATP/2.7 H+) = 2.3 ATP/QH2 In vivo, P/O ratio closer to 2.5 and 1.5 due to other proton “leaking” i.e. importing phosphate
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Active Transport of ATP
ATP must go out, ADP and Pi must go in Together, use about 1 proton of protonmotive force
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Regulation of Oxidative Phosphorylation
Electron transport is tightly coupled to ATP production Oxygen is not used unless ATP is being made Avoid waste of fuels Adding ADP causes oxygen utilization Respiratory control
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Problem 47 A culture of yeast grown under anaerobic conditions is exposed to oxygen, resulting in dramatic decrease in glucose consumption. This is called the Pasteur effect. Explain. The [NADH]/[NAD+] and [ATP]/[ADP] ratios also change when an anaerobic culture is exposed to oxygen. Explain how the ratios change and what effect this has on glycolysis and the citric acid cycle in yeast.
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