SBS 922 Membrane Biochemistry Mitochondria and chloroplasts John

SBS 922 Membrane Biochemistry Mitochondria and chloroplasts John F. Allen School of Biological and Chemical Sciences, Queen Mary, University of London 1

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Observed characteristics of oxidative phosphorylation By early 1960’s researchers had determined certain observed characteristics of oxidative phosphorylation linked to respiratory electron transfer in animal mitochondria, that had to be explained by any theory proposed for the mechanism of this energy transduction (transducing redox energy of electron transfer into chemical energy of ATP). Could measure two main parameters at that time a) rate or amount of electrons transferred along chain, by rate at which oxygen consumed (1 0 atom = 2 e-) b) amount of ATP synthesised (or amount of ADP or Pi converted into ATP)

From these sorts of measurements observed characteristics fall into 5 main categories 1] Energy coupling sites 2] Respiratory control 3] Uncoupling agents 4] Phosphorylation inhibitors 5] Reverse electron transfer You will observe 1 -4 in Practical 4.

1] Energy Coupling Sites Became apparent that amount of ATP formed (or ADP/Pi converted into ATP) was stoichiometric with amount of oxygen consumed stoichiometry called P/O or P/2 e- quotient ATP/O or ATP/2 e- quotient ADP/O or ADP/2 e- quotient value reflected several parameters 1) nature of substrate undergoing oxidation 2) integrity of coupling membrane 3) redox carrier composition of respiratory chain

In late 40’s and early 50’s several labs showed that oxidation of NADH (in fact oxidation of substrates like HOB that feed into resp. chain via NADH) lead to production of 3 molecules of ATP (ATP/O quotient of 3). Results with different electron donors and acceptors to isolate particular sections of respiratory chain (together with inhibitors to inhibit those parts of chain that are not being measured) showed varying ATP/O (or ATP/2 e-) quotients.

Reductant Oxidant Inhibitor ATP/O ATP/2 e- O 2 hydroxybutyrate (NADH) None 3 Succinate O 2 Rotenone 2 Ascorbate/ TMPD O 2 Rotenone + Antimycin A 1. 5 ferricyanide Cyanide hydroxybutyrate (NADH) 1. 5 Succinate 0. 5 ferricyanide Cyanide

Results showed three phosphorylation/coupling sites present Site I In complex 1 Site 2 In complex III Site 3 in complex IV (Shown subsequently if you generate high [NADPH][NAD+]/[NADP+][NADH] ratio can observe fourth coupling site in complex 0, but not of physiological importance) each of three sites exhibited different ATP/2 e- quotient site 1 2 3 ATP/2 e- quotient 1. 0 0. 5 1. 5 However these stoichiometries determined using added (exogeneous) ADP and Pi so include expenditure of energy required to transport ADP and Pi into matrix to site of ATP synthesis (ATP synthase). Get increased ATP/2 e- quotient if determined with ADP and Pi already inside (endogeneous) mitochondria. About 25% of energy released by electron transfer used for this active transport of ATP, ADP and Pi.

2] Respiratory Control In 1956 Chance and Williams observed that “as long as substrate (reductant, source electrons, electron donor), oxygen (oxidant, electron acceptor) and phosphate not limiting, rate of electron transfer effectively controlled by availability of ADP” i. e. whether ATP synthesis taking place or not. This phenomenon called respiratory control a) in absence of ADP, rate of electron transfer is low, reflects slow rate at which energy for ATP synthesis (provided by electron transfer) is dissipated in absence of ATP synthesis. Called “controlled state” or state IV b) if then add ADP the rate of electron transfer will increase dramatically until rate (state III) controlled by rate at which energy diissipated (actually limiting factor is activity of ADP/ATP translocase importing ADP and exporting ATP) c) rate then remains fast until almost all ADP phosphorylated to ATP. At this point rate declines to state IV again