MitoSeminar II: Some calculations in bioenergetics

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1 MitoSeminar II: Some calculations in bioenergetics MUDr. Jan Pláteník, PhD. Ústav lékařské biochemie 1.LF UK Helpful comments of Prof. MUDr. Jiří Kraml, DrSc., are acknowledged. 1 Respiratory chain and oxidative phosphorylation: Summary * Transfer of electrons via electron carriers (respiratory chain) to oxygen in the inner membrane * Proton pumping from the matrix to the cytosolic site of the inner mitochondrial membrane (proton gradient). Protonmotive force = ph gradient + membrane potential * Protons flow through ATP synthase and power the synthesis of ATP. 2 1

2 Stoichiometry of oxidative phosphorylation If F 0 complex has 10 c subunits: one complete turn means flow of 10 protons. The F 1 part has three ATP-producing sites: one complete turn of the shaft means synthesis of three ATP from ADP + Pi At least 3 H + have to be pumped out of the matrix for production of 1 ATP One more H + is consumed for import of phosphate Transport of 2 electrons from NADH to oxygen (complexes I, III, IV) pumps 10 protons, from FADH 2 to oxygen (complexes III, IV) 6 protons. Oxidation of 1 NADH produces 2.5 ATP Oxidation of 1 FADH 2 produces 1.5 ATP 3 G G = H T S The chemical reaction can occur only if the G G is negative. It means: when the products have less free energy than the reactants have. 4 2

3 G G anda chemical equilibrium A + B C + D K eq = [C eq ][D eq ] [A eq ][B eq ] G = RT ln K eq + RT ln [C][D] [A][B] A + B C + D G negative A + B C + D G positive (negative for reverse reaction) A + B C + D G = 0 (equilibrium) 5 Standard free enthalpy G o = RT lnk eq 1.0M, 25 C ph 7.0 R... universal gas constant J mol -1 K -1 T... absolute temperature in Kelvins ( K = 25 o C) G = RT ln K eq + RT ln [C][D] [A][B] G = G o + RT ln [C][D] [A][B] 6 3

4 7 Redox potential X + Y X + Y X X + e Y + e Y Difference in affinities to electrons between two redox couples, in volts. 8 4

5 E o standard redox potential,, 1.0 M, ph 0 E o standard redox potential,, 1.0 M, ph 7 Redox potential Reference hydrogen electrode: E o = 0.0 V E o = 0.42 V 9 (from Harper s Illustrated Biochemistry, 27th edition, McGraw-Hill Co. 2006) 10 5

6 Relationship between free enthalpy and redox potential G G = nf E n number of transfered electrons F... Faraday constant = C /mol C (Coulomb) = J/ V 11 The Nernst equation Tells voltage of galvanic cell, or redox potential, for various concentrations of components. [C][D] G G = G o + RT ln [A][B] + G G = nf E RT [OX] E = E o + ln nf [RED] Walther Hermann Nernst ( ): Nobel Prize 1920 Equation also called Nernst-Peters (Peters applied Nernst equation for redox processes) 12 6

7 Example I: Transfer of electrons from NADH to oxygen: NADH + H + + 1/2O 2 NAD + + H 2 O Redox couples: NADH + H + NAD + + 2H + + 2e E o = 0.320V 1/2O 2 + 2H + + 2e H 2 O E o = V For the whole reaction: E o = 0.816V ( 0.320V )= = 1.136V G o = nf E o G o = 2(96.5 kj V 1 mol 1 )(1.136V) = kj mol 1 13 Example II: Succinate dehydrogenase Redox couples: succinate fumarate + 2H + + 2e E o = V FAD + 2H + + 2e FADH 2 E o = V G o = 2(96.5 kj V 1 mol 1 )( V) = kj mol 1 Reaction cannot proceed??? 14 7

8 Example II: Succinate dehydrogenase Redox couples: succinate fumarate + 2H + + 2e E o = V FAD + 2H + + 2e FADH 2 E o = V But, if fumarate is consumed in the next reaction, and FAD reoxidized, the actual ratios succinate/fumarate and FAD/FADH 2 would not be 1:1 If succinate : fumarate is 500:1, redox potential of the system using the Nernst equation would be: RT [OX] E = E o + ln = ( 0.08)( = 0.05 V nf [RED] And for FAD/FADH 2 500:1 E = = 0.04 V 15 Likewise malate dehydrogenase reaction: Redox couples: malate oxaloacetate + 2H + + 2e E o = V NAD + + 2H + + 2e NADH + H + E o = V 16 8

Ionic gradient on a membrane can also do work c( + ) 1 = 120 mmol/l c( + ) 2 = 12 mmol/l G G = c 1 RT ln c 2 = (8.

9 Ionic gradient on a membrane can also do work c( + ) 1 = 120 mmol/l c( + ) 2 = 12 mmol/l G G = c 1 RT ln c 2 = ( ) ln 10 = = 5.7 kj/mol Equation is valid if c 1 > c 2 (diffusion to c 2 ); for 37 C the coefficient is

10 Protonmotive force p = ph + ψ Difference in concentration of protons - about 1 ph unit from Nernst equation is 60 mv Electric potential on the inner membrane. Must be measured, value e.g. 160 mv p = 60 mv mv = 220 mv 19 Efficiency of mitochondrial production of ATP Oxidation of 1 mole of NADH leads to pumping of 10 mol protons and production of 2.5 mol ATP, protonmotive force is 220 mv Oxidation of 1 mol NADH: G o = kj/mol Protonmotive force p = 220 mv corresponds to: µ H + G = µ H + = F p = 21.2 kj/mol For pumping 10 mol protons: G = 10 x (21.2) = 212 kj is electrochemical proton gradient Most of the energy from oxidation is converted to proton gradient 20 10

11 Efficiency of mitochondrial production of ATP Oxidation of 1 mole of NADH leads to pumping of 10 mol protons and production of 2.5 mol ATP, protonmotive force is 220 mv Oxidation of 1 mol NADH: G o = kj/mol Protonmotive force p = 220 mv corresponds to: µ H + G = µ H + = F p = 21.2 kj/mol For pumping 10 mol protons: G = 10 x (21.2) = 212 kj For 1 ATP is needed: G o = 30.5 kj/mol, In the cell really (excess of ATP): G = asi 50 kj/mol 4 protons do work 84.9 kj/mol, which is certainly enough for 1 ATP is electrochemical proton gradient 21 Efficiency of mitochondrial production of ATP Oxidation of 1 mole of NADH leads to pumping of 10 mol protons and production of 2.5 mol ATP, protonmotive force is 220 mv Oxidation of 1 mol NADH: G o = kj/mol Protonmotive force p = 220 mv corresponds to: µ H + G = µ H + = F p = 21.2 kj/mol For pumping 10 mol protons: G = 10 x (21.2) = 212 kj For 1 ATP is needed: G o = 30.5 kj/mol, In the cell really (excess of ATP): G = asi 50 kj/mol is electrochemical proton gradient Oxidation of 1 mol NADH gives theoretically energy for making 4.4 mol ATP, practically 2.5 mol is produced efficiency cca 57 % (for( standard conditions cca 35 %) 22 11

BCH 4054 Spring 2001 Chapter 21 Lecture Notes

BCH 4054 Spring 2001 Chapter 21 Lecture Notes 1 Chapter 21 Electron Transport and Oxidative Phosphorylation 2 Overview Oxidation of NADH and CoQH 2 produced in TCA cycle by O 2 is very exergonic. Some