Chemiosmotic proton gradient

In 1961, Peter Mitchell proposed a novel coupling mechanism involving a proton gradient across the inner mitochondrial membrane. In Mitchell s chemiosmotic hypothesis, protons are driven across the membrane from the matrix to the intermembrane... 

When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it, and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthasereconstituted in simple lipid vesicles with bac-teriorhodopsin, a light-driven proton pump from Halobaeterium halobium. As shown in Eigure 21.28, upon illumination, bacteriorhodopsin pumped protons... 

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a protonpump. Q, ubiquinone C, cytochrome c F Fq, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H" across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H" through Fq.

In Mitchell s earliest physicochemical formulations of the chemiosmotic theory, any involvement of the membrane across which the proton gradient was established received little attention. Between 1961... 

The ability to catalyse the evolution or oxidation of H2 may have been exploited by the earliest life forms as H2 would have been present in the early prebiotic environments. The origins of the proton-dependent chemiosmotic mechanism for ATP synthesis may also reflect the formation of proton gradients created by hydrogenases on either side of the cytoplasmic membrane. In addition, it has been speculated that the coupling of H2 and S metabolisms was also of fundamental importance in the origin of life. These two processes seem intimately coupled in the bifunctional sulfhydrogenase found in Pyrococcus furiosus (a combination of subunits for hydrogenase and sulfite reductase) which can dispose of excess reductant either by the reduction of protons to H2 or S° to H2S (Ma et al. 1993 Pedroni et al. 1995). 

Chemiosmotic theory readily explains the dependence of electron transfer on ATP synthesis in mitochondria. When the flow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping... 

A prediction of the chemiosmotic theory is that, because the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artificially created proton gradient should be able to replace electron transfer in driving ATP synthesis. This has been experimentally confirmed (Fig. 19-20). Mitochondria manipulated so as to impose a difference of proton concentration and a separation of charge across the inner membrane synthesize ATP in the absence of an oxidizable substrate the proton-motive force alone suffices to drive ATP synthesis. 

A fundamental postulate of the chemiosmotic theory is the presence of an oriented ATP synthase that utilizes the Gibbs energy difference of the proton gradient to drive the synthesis of ATP (Fig. 18-9). 

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). 

Figure 17.2 The chemiosmotic hypothesis. Electrons from NADH and/or FADH2 are passed to Oz via the electron transport chain. In the process, protons are extruded into the mitochondrial intermembrane space. The proton gradient thus created causes the movement of protons back into mitochondria through a channel in the F ATPase. In the process, one molecule of ATP is formed frm ADP and phosphate for every two to three protons channeled back into the mitochondria. ATP moves into the intermembrane space and cytosol in exchange for ADP moving in the opposite direction. Phosphate is taken up in exchange for OH".

Figure 1. The major transmembrane photosynthetic reaction centers (RC) (top) and respiratory complexes (bottom) are composed of light (zigzag) activated chains (dark gray) of redox centers (open polygons) that create a transmembrane electric field and move protons (double arrows) to create a transmembrane proton gradient, fulfilling the requirements of Mitchell s chemiosmotic hypothesis. Diffusing substrates include ubiquinone (hexagon) and other sources of oxidants and reductants. PSI and PSII, photosystems I and II, respectively.

Photosynthetic reaction centers plug into the chemiosmotic scheme by using light-excited states to create both an oxidant and a reductant. For the purple bacterial reaction centers, these oxidants and reductants are the redox carriers already described, oxidized cytochrome c and reduced ubiquinone QH2. Thus, in combination with Complex III, light drives a relatively straightforward cyclic electron transfer that generates a transmembrane electric field and proton gradient. 

The whole process of the light reactions generates a proton gradient across the membrane. This is used for the chemiosmotic production of ATP. This is called Z-scheme (or straight chain) photo-phosphorylation (Fig. 13.10). 

The anisotropic organization of electron carriers across the membrane accounts for the vectorial transport of protons from the inside to the outside of the membrane, which occurs with the passage of electrons. The coupling of this proton gradient to a proton-translocating ATP synthase (also known as ATP synthetase) accounts for the chemiosmotic coupling in oxidative phosphorylation. 

In a previous section, we have already seen two good examples of experimental results that are consistent with the chemiosmotic theory the difference in electron-transfer behavior between TMPD and DAD, two compounds with identical chemical composition but with different electrochemical behavior and the photophosphorylation coupled to a proton gradient formed in a minimum PS-I/membrane model system. In this section we present additional examples that render evidence that even more directly supports the chemiosmotic theory. 

According to Mitchell s chemiosmotic theory, photophosphorylation is driven by energy derived from electron transfer coupled to proton translocation. The results of postillumination discussed in the previous section further supports the notion that a proton gradient is the driving force for phosphorylation. It is therefore possible in principle that a similar proton gradient produced by artificial means might also be able to drive phosphorylation in a chloroplast membrane, entirely in the dark, i. e., without the aid of photo-induced electron transport. Such a scheme was indeed realized by the so-called acid-bath ATP-forma-tion demonstrated by Jagendorf and Uribe " in 1966. 

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