Stoichiometry proton pumping

The Stoichiometry of Proton Pumping by the Mitochondrial Electron-Transport Chain 129... 

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 crucially important finding is that submitochon-drial particles or vesicles from broken chloroplasts will synthesize ATP from ADP and P , when an artificial pH gradient is imposed.172186 Isolated purified FjF0 ATPase from a thermophilic Bacillus has been coreconstituted into liposomes with the light-driven proton pump bacteiiorhodopsin (Chapter 23). Illumination induced ATP synthesis.187 These observations support Mitchell s proposal that the ATP synthase is both spatially separate from the electron carriers in the membrane and utilizes the protonmotive force to make ATP. Thus, the passage of protons from the outside of the mitochondria back in through the ATP synthase induces the formation of ATP. What is the stoichiometry of this process ... 

The stoichiometry of proton pumping was measured by Lehninger and associates using a fast-responding 02 electrode and a glass pH electrode.189 190 They observed an export of eight H+/ O for oxidation of succinate rat liver mitochondria in the presence of a permeant cation that would prevent the buildup of Em, and four H+/ O (2 H+/ e ) for the cytochrome oxidase system. These are equivalent to two H+/ e at each of sites II and III as is indicated in Fig. 18-4. 

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. 

Reynafarje, B., and A. L. Lehninger, The K+/site and H+/site stoichiometry of mitochondrial electron transport. J. Biol. Chem. 254 6331, 1978. Valinomycin is used to allow K+ to move inward across the inner membrane in response to the electric potential difference created by H+ efflux. The number of protons pumped out is found to be larger than previously estimated. 

Nitrate respiration can support the synthesis of ATP, while proton pumping has been quantified for several physiological substrates. Stoichiometries of about 4H+/NO, and 2H+/N03" have been found for L-malate and formate, and succinate, D-lactate and glycerol respectively. There is evidence that about one mole of ATP is synthesized by oxidative phosphorylation per mole of nitrate reduced.1440... 

Arai, H., Terres, G., Pink, S., and Forgac, M. (1988). Topography and subunit stoichiometry of the coated vesicle proton pump./. Biol. Chem. 263, 8796-8802. 

In these mechanisms, electron transport through the various components of the electron-transport chain leads to structural changes in the proteins of the chain, such that changes in their pKa values (Chap. 3) of ionizable amino acid residues occurs. For example, an increase in the pKa of a residue adjacent to the matrix side of the membrane would lead to proton uptake from the matrix, while a decrease in the pKa of a residue adjacent to the intermembranous side of the membrane could lead to release of a proton. The net effect of these processes is the transfer of protons from the matrix to the intermembranous side of the membrane. However, proton-pump mechanisms do not make strong predictions of the H+/e stoichiometries. 

No, it is inconsistent with this hypothesis, which predicts an H+/e value of 1.0. Such stoichiometries may, however, be explained by proton-pump mechanisms, in which electron transfer is coupled to changes in the pKa values of proteins within Complex I. 

Sacksteder, C.A., Kanazawa, A., Jacoby, M.E., and Kramer, D.M. 2000. The proton to electron stoichiometry of steady-state photosynthesis in living plants A proton-pumping Q cycle is continuously engaged. Proc. Natl. Acad. Sci. USA 97 14,283-14,288. 

Note The current value of 30 molecules of ATP per molecule of glucose supersedes the earlier one of 36 molecules of ATP. The stoichiometries of proton pumping, ATP synthesis, and metabolite transport should be regarded as estimates. About two more molecules of ATP are formed per molecule of glucose oxidized when the malate-aspartate shuttle rather than the glycerol 3-phosphate shuttle is used. 

Brzezinski, P., Johansson, A. L. (2010). Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase. Biochimica et Biophysica Acta, 1797, 710—713. 

We can now estimate how many molecules of ATP are formed when glucose is completely oxidized to GO2. The number of ATP (or GTP) molecules formed in glycolysis and the citric acid cycle is unequivocally known because it is determined by the stoichiometries of chemical reactions. In contrast, the AT P yield of oxidative phosphorylation is less certain because the stoi chiometries of proton pumping, ATP synthesis, and metabolite-transport processes need not be integer numbers or even have fixed values. As stated earlier, the best current estimates for the number of protons pumped out of the matrix by NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase per electron pair are four, two, and four, respectively. The synthesis of a molecule of ATP is driven by the flow of about three protons through ATP synthase. An additional proton is consumed in transporting ATP from the matrix to the cytoplasm. Hence, about 2.5 molecules of cytoplasmic ATP are generated as a result of the flow of a pair of electrons from NADH to O2. For electrons that enter at the level of Q-cytochrome c oxidoreductase, such as those from the oxidation of succinate or cytoplasmic NADH, the yield is about 1.5 molecules of ATP per electron pair, Hence, as tallied in Table 18.4, about 30 molecules of ATP are formed... 

Exact values for P/O ratios are difficult to determine because of the complexity of the systems that pump protons and phosphorylate ADP. The number of ADP molecules phosphorylated is direcdy related to the number of protons pumped across the membrane. This figure has been a matter of some controversy. It has been difficult for chemists and biochemists to accept uncertain stoichiometry. 

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