Oxidative Phosphorylation Pratt and Cornely, Chapter 15

Goal: ATP Synthesis

Overview Redox reactions Electron transport chain Proton gradient ATP synthesis Shuttles Analogy: How does burning coal put flour in the grocery store?

Standard Reduction Potential

Half Reactions Reduction potential written in terms of a reduction half reaction A ox  A red Example: NADH + FMN  NAD + + FMNH 2

Redox reactions: electricity 2 e- transfer Calculate  G by reduction potential NADH: E o ’ = -.32 FMN: E o ’=-.30  G o ’ = -nF  E o ’ = -2(96485)(0.02) = -3.9 kJ/mol

Numerous Redox Substrates O 2 : high “reduction potential” Substrates – Organic cofactors – Metals (iron/sulfur clusters) – cytochromes

Coenzyme Q: Mobile Carrier FADH 2 is a one e- donator Many reactions, including metals Ubiquinone is a key intermediate Can diffuse through nonpolar regions easily

Oxygen: the final electron acceptor Water is produced—has very low reactivity, very stable Superoxide, peroxide as toxic intermediates Overall reaction NADH + H + + ½ O 2  NAD + + H 2 O

Flow Through Complexes

Compartmentalization

Protonmotive Force NADH + H + + ½ O 2  NAD + + H 2 O + 10 H + pumped succinate + ½ O 2  fumarate + H 2 O + 6 H + pumped

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

Complex II Non-NADH sources – Complex II (citric acid cycle) – Fatty acid oxidation – Glycerol-3-phosphate shuttle (glycolysis) Bypasses Complex I – Loss of 4 protons pumped

Complex III QH 2  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) E o ’ = V Cyt c (Fe 3+ ) + e-  cyt c (Fe 2+ ) E o ’ = V

Complex IV Cytochromes  O 2 Stoichiometry of half of an oxygen atom

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 QH 2 Operational in some tissues/circumstances

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

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.

Proton Gradient Gradient driven by concentration difference + charge difference – Assume  pH 0.5 and 170mV membrane potential Free energy of ATP hydrolysis = -48 kJ/mole How many protons needed to fuel ATP formation? Minimum of 3

Protonmotive Force Flow of electrons is useless if not coupled to a useful process – Battery connected to wire Proton gradient across mitochondrial membrane

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

Complex V: ATP Synthase Molecular motor Rotor: c, ,  – Proton channel

Proton Channel Protons enters channel between rotor and stator Rotor rotates to release strain by allowing proton to enter matrix protons = full rotation – Species dependent

“Stalk” (  ) 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

Remember Analogy Fuel  electricity  water pumped uphill  flows down to grind flour But we don’t have bread until flour is transported to where it needs to go! Compartmentalization: ATP is in matrix, but must get to the rest of the cell

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

Energy Accounting ATP costs 2.7 protons – 8 protons produces 3 ATP NADH pumps 10 protons when 2 e - reduce ½ O 2 – 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 + /QH 2 (1 ATP/2.7 H + ) = 2.3 ATP/QH 2 In vivo, P/O ratio closer to 2.5 and 1.5 due to other proton “leaking” – i.e. importing phosphate

Uncouplers “Uncouple” protonmotive force from ATP synthase – DNP pKa / solubility perfectly suitable Other respiration poisons – Cyanide—binds Complex IV in place of oxygen

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

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]/A[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.