Coupling of Electron Transport and ATP Synthesis

It is now generally accepted that the coupling of electron transport and ATP synthesis is brought about by the action of a proton electrochemical-potential gradient, denoted by the symbol A/uH+. This gradient arises as a consequence of electron transport and is dissipated by ATP synthase to generate ATP from ADP and P,. 

The most common way in which Aij is determined is from measurements of the concentrations inside and outside mitochondria, at equilibrium, of an ionizable compound that is permeable to the inner mitochondrial membrane. Aifi can then be calculated using the Nemst equation (14,1), written in the form  

The value of ApH across the inner mitochondrial membrane can be estimated from the equilibrium distribution of electroneutrally-permeant weak acids (or weak bases). The logic underlying such experiments is illustrated below. 

assuming that the equilibrium constant Ka is the same for the ionization of HA inside and outside the mitochondrion, and the value of Ka is sufficiently low that [HA]=0 and it will not contribute to the measurements, then 

5 THE RATIO OF PROTONS EXTRUDED FROM THE MITOCHONDRION TO ELECTRONS TRANSFERRED TO OXYGEN 

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Many models were proposed to account for the coupling of electron transport and ATP synthesis. A persuasive model, advanced by E. C. Slater in 1953, proposed that energy derived from electron transport was stored in a high-energy intermediate (symbolized as X P). This chemical species—in essence an activated form of phosphate—functioned according to certain relations according to Equations (21.22)-(21.25) (see below) to drive ATP synthesis. 

Proton transport via complexes I, III, and IV takes place vectorially from the matrix into the intermembrane space. When electrons are being transported through the respiratory chain, the concentration in this space increases—i. e., the pH value there is reduced by about one pH unit. For each H2O molecule formed, around 10 H ions are pumped into the intermembrane space. If the inner membrane is intact, then generally only ATP synthase (see p. 142) can allow protons to flow back into the matrix. This is the basis for the coupling of electron transport to ATP synthesis, which is important for regulation purposes (see p. 144). 

An overview of electron transport from water to NADP will be presented, and a discussion of photophosphorylation. This will include an appraisal of the recent observations and controversies about the localized versus delocalized nature of the proton pool(s) contributing to the proton electrochemical gradient involved in the mitchellian coupling of electron transport to ATP synthesis [5,6]. 

Oxidative phosphorylation is the name given to the synthesis of ATP (phosphorylation) that occurs when NADH and FADH2 are oxidized (hence oxidative) by electron transport through the respiratory chain. Unlike substrate level phosphorylation (see Topics J3 and LI), it does not involve phosphorylated chemical intermediates. Rather, a very different mechanism was proposed by Peter Mitchell in 1961, the chemiosmotic hypothesis. This proposes that energy liberated by electron transport is used to create a proton gradient across the mitochondrial inner membrane and that it is this that is used to drive ATP synthesis. Thus the proton gradient couples electron transport and ATP synthesis, not a chemical intermediate. The evidence is overwhelming that this is indeed the way that oxidative phosphorylation works. The actual synthesis of ATP is carried out by an enzyme called ATP synthase located in the inner mitochondrial membrane (Fig. 3). 

The result of such a process would be that two electrons are cycled twice through the PQ, and the ratio of H /e between PS II and PS I would be higher than one. This, if definitively confirmed, would be of great importance from the point of view of understanding the coupling of electron transport to the synthesis of ATP, and of the quantum yield of photosynthesis (see discussion under photophosphorylation). 

ATP synthase also can be inhibited. Oligomycin and dicyclohexylcarbodiimide (DCCD) prevent the influx of protons through ATP synthase. If actively respiring mitochondria are exposed to an inhibitor of ATP synthase, the electron-transport chain ceases to operate. Indeed, this observation clearly illustrates that electron transport and ATP synthesis are normally tightly coupled. 

Studies with beef-heart submitochondrial particles initiated in Green s laboratory in the mid-1950s resulted in the demonstration of ubiquinone and of non-heme iron proteins as components of the electron-transport system, and the separation, characterisation and reconstitution of the four oxidoreductase complexes of the respiratory chain. In 1960 Racker and his associates succeeded in isolating an ATPase from submitochondrial particles and demonstrated that this ATPase, called F, could serve as a coupling factor capable of restoring oxidative phosphorylation to F,-depleted particles. These preparations subsequently played an important role in elucidating the role of the membrane in energy transduction between electron transport and ATP synthesis. 

Mitchell suggested that the free energy release associated with electron transport and ATP synthesis is coupled by the protonmotive force created by the ETC. (The term chemiosmotic emphasizes that chemical reactions can be coupled to osmotic gradients.) An overview of the chemiosmotic model as it operates in the mitochrondion is illustrated in Figure 10.12. 

Control of oxidative phosphorylation allows a cell to produce only the amount of ATP immediately required to sustain its current activities. Recall that under normal circumstances, electron transport and ATP synthesis are tightly coupled. 

The value of the P/O ratio (the number of moles of Pi consumed for each oxygen atom reduced to HzO) reflects the degree of coupling observed between electron transport and ATP synthesis. The measured maximum ratio for the oxidation of NADH is 2.5. The maximum P/O ratio for FADH2 is 1.5. 

The oxidation of NADH and FADH2 by molecular oxygen is coupled in mitochondria to the endergonic synthesis of ATP from ADP and Pi. For many years the nature of the common intermediate between electron transport and ATP synthesis was elusive. Peter Mitchell, who received a Nobel Prize in chemistry in 1978 for his extraordinary insights, suggested that this common intermediate was the proton electrochemical potential. He proposed in the early... 

Engelhardt s experiments in 1930 led to the notion that ATP is synthesized as the result of electron transport, and, by 1940, Severo Ochoa had carried out a measurement of the P/O ratio, the number of molecules of ATP generated per atom of oxygen consumed in the electron transport chain. Because two electrons are transferred down the chain per oxygen atom reduced, the P/O ratio also reflects the ratio of ATPs synthesized per pair of electrons consumed. After many tedious and careful measurements, scientists decided that the P/O ratio was 3 for NADH oxidation and 2 for succinate (that is, [FADHg]) oxidation. Electron flow and ATP synthesis are very tightly coupled in the sense that, in normal mitochondria, neither occurs without the other. 

The uncouplers which abolish the coupling of respiratory rate to ATP synthesis act as proton translocators, inducing net proton translocation across the membranes. In this way the proton circuit can be short-circuited , allowing the protons translocated by the generator of to cross back across the membrane without passing through the ATP synthase and producing ATP. The majority of the uncouplers are protonatable, lipophilic compounds with an extensive pi-orbital system which allows the electron of the anionic, de-protonated form to be delocalized [11]. This enhances the permeability of the anionic form in the hydrophobic membrane, and allows the proton translocators to permeate in both their neutral (protonated) and anionic (deprotonated) forms. In this way they can catalyze the net transport of protons... 

Mitochondria produce most of the energy in cells by oxidative phosphorylation. This process combines two distinct but tightly coupled parts Electron transport and phosphorylation of ADP to ATP - as discussed in detail in Chapter 13.1. Most modem insecticides and acaricides that dismpt mitochondrial ATP synthesis [1] interfere with the electron transport (mainly at complex 1, less frequently at complex III) (see Chapter 28.3). 

This enzyme present in all animals and plants, aerobic yeast and some bacteria is involved in the next to the last step of oxidative phosphorylation, a process that couples electron transport to ATP synthesis cytochrome c (cytc) delivers electrons to the final component of the respiratory chain, cytochrome c oxidase which reduces O2 to water [39,40]. Crystal structures for both bacterial and mitochondrial multisubunit membrane-boimd COXs are available [41—45]. The enzyme contains two heme groups and three Cu ions. Of these, one heme and one Cu ion (called the heme site) constitute the catalytic center (Fig. 11.5), the remaining... 

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