Chemiosmotic proton pumping

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... 

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

The chemiosmotic hypothesis had the great virtue of predicting the following consequences which could be tested (1) electron-transport driven proton pumps with defined stoichiometries and (2) a separate ATP synthase, which could be driven by a pH gradient or membrane potential. Mitchell s hypothesis was initially greeted with skepticism but it encouraged many people, including Mitchell and his associate Jennifer Moyle, to test these predictions, which were soon found to be correct.178... 

A consequence of the chemiosmotic theory is that there is no need for an integral stoichiometry between protons pumped and ATP formed or for an integral P / O ratio. There are bound to be inefficiencies in coupling, and Ap is also used in ways other than synthesis of ATP. 

Figure 18.26. Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated. The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion.

The ATP synthase is a reversible proton-translocating ATP ase The initial experiments which were important for the verification of the chemiosmotic hypothesis were those which showed that the complex was an autonomous proton pump when hydrolyzing ATP, and which showed that an artificial A/a could cause the ATP synthase to generate ATP. Thus, if a limiting amount of ATP is injected into an anaerobic mitochondrial incubation a net expulsion of protons is observed, followed by a decay which is accelerated by proton translocators [15]. Less complications arise if the experiment is repeated with inverted sub-mitochondrial... 

Fig. 2.2. The chemiosmotic proton circuit, a, during the synthesis of matrix ATP (State 3) b, during the synthesis of extra-mitochondrial ATP (State 3) c, during State 4 respiration d, in the presence of proton translocator. R, respiratory chain A, ATP synthase P, phosphate carrier U, uncoupler. The respiratory chain is simplified to a single proton pump.

Mitchell, P., Chemiosmotic coupling in oxidative and photosynthetic phosphorylation, Biol. Rev. Camb. Philos. Soc., 1966, 41, 445-502. WiKSTEOM, M.K., Proton pump coupled to cytochrome c oxidase in mitochondria. Nature, 1977, 266, 271-273. 

The chemiosmotic coupling mechanism explains how ATP is synthesized by mitochondria as a result of protons pumped during ETS. There is a considerable amount of evidence in support of this model. 

One of the tenets of the chemiosmotic theory is that energy from the oxidation-reduction reactions of the electron transport chain is used to transport protons from the matrix to the intermembrane space. This proton pumping is generally facilitated by the vectorial arrangement of the membrane spanning complexes. Their stracture allows them to pick up electrons and protons on one side of the membrane and release protons on the other side of the membrane as they transfer an electron to the next component of the chain. The direct physical link between proton movement and electron transfer can be illustrated by an examination of the Q cycle for the b-Ci complex (Fig. 21.9). The Q cycle involves a double cycle of CoQ reduction and oxidation. CoQ accepts two protons at the matrix side together with two electrons it then releases protons into the intermembrane space while donating one electron back to another component of the cytochrome b-Ci complex and one to cytochrome c. 

Dissimilatory nitrite reductase of denitrifying bacteria is usually a soluble enzyme and it has been difficult to ascribe a phosphorylative function associated with the conversion of nitrite to nitric oxide. However, the demonstration by Wood (1978) that the terminal reductase in nitrite respiration is located in the periplasm implies that electrons generated in the cytoplasm must traverse the cytoplasmic membrane to the periplasmic nitrite reduction site. This location would require proton pumping, thus facilitating phosphorylation by the chemiosmotic mechanism. 

The above is a very simplified account of the chemiosmotic theory and a number of the details of oxidative phosphorylation remain to be elucidated, in particular the molecular mechanism of proton pumping and the exact mechanism of action of the ATPase. However, the basic principles of the theory are now widely accepted. This theory is also able to account for photosynthetic phosphorylation in chloroplasts and for the synthesis of ATP by bacteria. 

A representation of the key experiment proving the chemiosmotic hypothesis. At the bottom is the lightactivated electrogenic proton pump, bacterial rhodopsin, and on the top the F,Fo ATP synthase. When light is shone on the vesicles coreconstituted with these two pumps in the presence of ADP and inorganic phosphate (Pi), ATP synthesis was observed. The only linkage between the two sets of structures is the electrochemical gradient of protons induced by illumination. 

Proton-coupled electron transfer (PCET) reactions play a fundamental role in the respiratory chain and in photosynthesis. In both membrane-bound systems, an electrochemical gradient is built up across the lipid bilayer by separating protons from electrons the resulting chemiosmotic proton potential serves to fuel a proton-driven pump synthesising the universal biological energy equivalent ATP (adenosine triphosphate). In addition to the relevance of PCET in these energy conversions, the coupled transfer of electrons and protons is an... 

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