Lecture 10. Proton Gradient-dependent ATP Synthesis. Oxidative. Photo-Phosphorylation

Lecture 10 Proton Gradient-dependent ATP Synthesis Oxidative Phosphorylation Photo-Phosphorylation

Model of the Electron Transport Chain (ETC) Glycerol-3-P Shuttle Outer Mitochondrial Membrane G3P DHAP FADH 2 4H + 4H + 2H + + + + + + + + + + + + Cyt c Intermembrane space + + + + + + + + + + I Q F 0 II III IV - - - - - - - - - - - - - - - - FADH 2 Succinate Fumarate F 1 NADH + H + NAD + TCA-Cycle FADH 2 Fatty acid degradation (β-oxidation) ½O 2 + 2H + H 2 O ADP + Pi ATP H + Matrix p. 55

Proton Gradient-dependent dependent ATP Synthesis ( Oxidative Phosphorylation) Glycolysis, PDH, TCA-Cycle, degradation of fatty acids, amino acids, other sugars NADH FADH 2 Glycolysis, TCA-Cycle, degradation of fatty acids CoQ Cyt c Oxygen

Proton Gradient-dependent dependent ATP Synthesis ( Oxidative Phosphorylation) H + (out) ADP ATP ATP H + (in) Proton Motive Force (PMF) ATP Synthesis

Mechanism of ATP Synthase p. 57

L ADP + Pi γ O T γadp + Pi H + (P) H + (P) H + (P) ATP γ L O γ T ADP + Pi ATP L γ O T O L T H + (N) H + (N) H + (N) Proton movement through F O causes rotation of γ subunit and conformational changes of β subunits p. 57

Bioenergetics of the ETC and ATP Synthesis Do the numbers fit? (yes, more or less )

1. What is ΔG o of NADH oxidation? NADH + H + + 0.5 O 2 NAD + + H 2 O ΔE o = E o ox E o red ΔE o = 0.82 V ( 0.32 V) ΔE o = 1.14 V ΔG o = nfδe o ΔG o = 220 kj mol -1

2. What is ΔG of H + transport against H + gradient? Experimental data: ΔpH = 0.75 and ΔΨ = 150 mv ΔG = ΔG o + RT ln [H + ] P / [H + ] N + ZFΔΨ ΔG o = 0 ΔG = RT ln [H + ] P / [H + ] N + ZFΔΨ ln = 2.303 log ΔG = 2.303 RT log [H + ] P / [H + ] N + ZFΔΨ ΔG = 2.303 RT (log [H + ] P log[h + ] N ) + ZFΔΨ ph = log [H + ] ΔG = 2.303 RT (ph N ph P ) + ZFΔΨ ΔG = 2.303 RT ΔpH + ZFΔΨ ΔG = 2.303 RT 0.75 + ZF 0.15 V Z = charge of H + (+1) ΔG = ~ + 20 kj per mol H +

NADH + H + + 0.5 O 2 NAD + ETC 10 H + N 10 H + P + H 2 O 1. ΔG o of NADH oxidation ΔG o = 220 kj mol -1 Energy gained and coupled to proton transport 2. ΔG G of H + transport against electro-chemical chemical gradient ΔG = ~ + 20 kj per mol H + Energy gained is sufficient to pump 10 protons! ΔG = ~ + 200 kj per 10 mol H +

NADH + H + + 0.5 O 2 NAD + ETC 10 H + N 10 H + P + H 2 O 10 H + N ATP + H 2 O F o F 1 10 H + P ADP + Pi 3. How many ATP are generated per NADH? EMF (NADH) PMF (10 Protons)? ATP ( 220 kjmol - 1 ) ( 200 kjmol - 1 ) ΔG o (ATP Synthesis) = 30.5 kj mol -1 6-77 ATP ΔG (ATP Synthesis) ~ 50 kj mol -1 4 ATP In reality: 2-33 ATP

Inner Membrane Transporters Tap into the PMF H + H + H + H + Intermembrane Space Pyr H + H + H + H + Pi ADP 3- + + + + ATP 4- + + + + ----- ----- H + Pyr H + Pi ADP 3- ATP 4- OH - OH - OH - OH - OHŌH- OH - OH - Mitochondrial Matrix

4. How many ATP are generated per FADH 2? FADH 2 No complex I (only complex II-IV) + 0.5 O 2 FAD + H 2 O ETC 6 H + N 6 H + P 6 H + N ATP + H 2 O F o F 1 6 H + P ADP + Pi ΔE o = E o ox E o red = 0.82 V (+0.06 V) = 0.76 V ΔG o = nfδe o = 147 kj mol -1 EMF (FADH) PMF (6 Protons) ~2 2 ATP ( 140 kjmol - 1 ) ( 120 kjmol - 1 )

ATP Yield of Respiration Glucose 2 Pyruvate 2 ATP 2 NADH 2 + 6 (5%, anaerobic) 2 Pyruvate 2 Acetyl-CoA + 2 CO 2 2 NADH 6 2 Acetyl-CoA 4 CO 2 2 ATP 6 NADH 2 + 18 2 FADH 2 4 38 ATP 1 NADH = 3 ATP 1 FADH 2 = 2 ATP 36 ATP (if glycerol-3-p P shuttle) (~65% efficiency, ΔG of Glc Oxidation ATP)

Model of the Electron Transport Chain (ETC) Outer Mitochondrial Membrane G3P DHAP FADH 2 4H + 4H + 2H + + + + + + + + + + + + Cyt c Intermembrane space + + + + + + + + + + I Q F 0 II III IV - - - - - - - - - - - - - - - - FADH 2 Succinate Fumarate F 1 NADH + H + NAD + FADH 2 ½O 2 + 2H + H 2 O ADP + Pi ATP H + Amytal, Rotenone Antimycin A Cyanide Oligomycin p. 52

Isolated Mitochondria O 2 Consumption ATP Production No substrate, but ADP + Pi + Succinate + Cyanide Inhibition of Complex IV

O 2 Isolated Mitochondria ATP Succinate, but no (ADP, Pi) + (ADP, Pi) Inhibition of Fo-F1 F1 ATPase + Oligomycin + DNP Uncoupling of PMF and ATP synthesis Acceptor Control [ADP]

Dinitrophenol (DNP) Uncouplers O O N + O - N + - O G3P DHAP FADH 2 4H + 4H + 2H + + + + + + + + + + + + Cyt c HO Thermogenin (Proton Channel) + + + + + + + + + + I Q F 0 II III IV - - - - - - - - - - - - - - - - FADH 2 Succinate Fumarate F 1 NADH + H + NAD + FADH 2 ½O 2 + 2H + H 2 O ADP + Pi ATP H + Amytal, Rotenone Antimycin A Cyanide Oligomycin

Feedback Control ( Acceptor Control ) Glucose PFK-1 PK PDH CS IDH KGA-DH ETC ADP Glycogen Glucose-6-P Pyruvate Acetyl-CoA NADH ATP Activation Lactate Inhibition

Feedback Control by NADH Glucose Glycogen Glucose-6-P Pyruvate Acetyl-CoA NADH X ATP PK PDH CS IDH KGA-DH ETC Lactate

Allosteric Control of the TCA Cycle Pyruvate Pyr Carboxylase (+) Acetyl-CoA PDH Acetyl-CoA (+) AMP, NAD +, CoA (-)) ATP, NADH, Acetyl-CoA (+) Activation (-)) Inhibition CS (+) ADP (-)) ATP, NADH, Citrate, Succinyl-CoA Oxalaoacetate Citrate Malate Isocitrate IDH (+) ADP, NAD + (-)) ATP, NADH Fumarate α-ketoglutarate Succinyl-CoA α-kga (+) AMP (-)) ATP, NADH, Succinyl-CoA p. 49

Proton Gradient-dependent ATP Synthesis by Photo-phosphorylation

Plants and Photosynthetic Bacteria LIGHT O 2 ATP NADPH CO 2 + H 2 O ATP NADH Photosynthesis Respiration NADP + ADP + Pi H 2 O Autotrophic Metabolism Reduced Organic Compounds Heterotrophic Metabolism NAD + ADP + Pi p. 58

Plants and Photosynthetic Bacteria LIGHT O 2 ATP NADPH CO 2 + H 2 O ATP NADH Photosynthesis Respiration NADP + ADP + Pi H 2 O Reduced Organic Compounds NAD + ADP + Pi Light Reactions CO 2 Fixation Heterotrophic Metabolism p. 58

E o - 1.0 V Chl* e - CO 2-0.3 V L I G H T Chl o ΔE ATP NADP + NADPH NADH NAD + Food (Reduced Carbon) ΔE ATP O 2 H 2 O + 1.0 V Chl + p. 58

Proton Gradient-dependent dependent ATP Synthesis (1. Oxidative Phosphorylation) NADH/FADH 2 H + (out) ADP ATP ATP H + (in) O 2 H 2 O H + (in) Electron Motive Force (EMF) Proton Motive Force (PMF) ATP Synthesis

Proton Gradient-dependent dependent ATP Synthesis (2. Photo Phosphorylation) Chl* e - L I G H T H + (in) H + (out) NADP + NADPH ADP ATP ATP H + (in) Electron Motive Force (EMF) Proton Motive Force (PMF) ATP Synthesis

Oxidative Phosphorylation (per mole O 2 ) 2NAD H + 2H + + O 2 2NAD + ETC 20 H + N 20 H + P + 2H 2 O 20 H + N 6ATP + 6H 2 O F o F 1 20 H + P 6ADP + 6Pi

Photo-phosphorylation (per mole O 2 ) 2H 2 O + 2NADP + 8 Photons (~1,400-2,400 kj) PS 12 H + in 12 2NADP H + 2H + + O 2 12 H + out 12 H + in 3ATP + 3H 2 O F o F 1 12 H + out 3ADP + 3Pi 3ATP + 2NADPH + CO 2 + H 2 O 1/3 Triose-P + 3ADP + 2Pi + 2NADP + Carbon Assimilation (CO 2 Fixation)

Generation of ATP and NADPH in Photosynthesis p. 59

Photosynthetic Pigments Chlorophylls β-carotene H 3 C R 1 R 2 I II N N Mg N N R 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 CH 3 H 3 C IV III CH 3 Phycoerythrin H H CH 2 Protein CH 2 C O OR 4 H C O O CH 3 O O HOOC CH S COOH 3 CH 3 CH 3 CH 3 CH 3 N N N N O R 1, R 2, R 3 (short-chain substituents, e.g., -CH 3, -CH 2 -CH 3 ) R 4 (long-chain substituents, e.g., phytyl or geranylgeranyl side chain [C 20 ]) p. 60

Photosynthetic Pigments Blue Green Yellow Red p. 60