Oxidative Phosphorylation Pratt and Cornely, Chapter 15

Goal: ATP Synthesis

Overview Redox reactions Electron transport chain Proton gradient ATP synthesis Shuttles

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 O2 + 2 e + 2 H+ H2O

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

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

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

Overall chain from NADH Complex I Q Complex III Cytochrome c Complex IV O2

Within Complexes FAD/FMN Iron-sulfur clusters Prosthetic group Bridge from 2 e- donors to 1 e- donors Iron-sulfur clusters

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)

Cytochrome c Mobile carrier Protein/heme 1 electron carrier

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

Flow Through Complexes

Compartmentalization

Protonmotive Force NADH + H+ + ½ O2  NAD+ + H2O + 10 H+ pumped succinate + ½ O2  fumarate + H2O + 6 H+ pumped

Complex I NADH  Q through “Q pool” 4 protons pumped FMN Iron-sulfur clusters “Q pool” 4 protons pumped Proton wire

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’ = 0.280 V Cyt c (Fe3+) + e-  cyt c (Fe2+) Eo’ = 0.215 V

Complex IV Cytochromes  O2 Stoichiometry of half of an oxygen atom

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

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

Glycerol-3-phosphate Shuttle Glycerol phosphate shuttle (1.5 ATP/NADH) Produces QH2 Operational in some tissues/circumstances

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: 30-32 ATP/glucose

Overall Chemiosmosis 10 protons shuttled from matrix to intermembrane space Makes pH gradient and ion gradient

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

Proton Gradient Gradient driven by concentration difference + charge difference Assume DpH 0.5 and 170mV membrane potential

Using the Gradient Coupled to ATP synthesis Uncouplers used to show link of oxygen uptake and ATP synthesis

Uncouplers “Uncouple” protonmotive force from ATP synthase DNP pKa / solubility perfectly suitable

Respiratory Poisons Other respiration poisons Cyanide—binds Complex IV in place of oxygen

Complex V: ATP Synthase Molecular motor Rotor: c, g, e Proton channel

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

“Stalk” (g) moves inside the“knob”—hexameric ATP synthase Knob held stationary by “b”

Hexameric Knob

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

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.

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

Active Transport of ATP ATP must go out, ADP and Pi must go in Together, use about 1 proton of protonmotive force

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

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.