H+/site, charge/site, and ATP/site ratios in mitochondrial electron transport

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H+/site, charge/site, and ATP/site ratios have been determined at coupling sites I, II, and III. Three e- donors have been used for coupling site III: ferrocyanide, ascorbate + tetramethyl-p-phenylenediamine (TMPD), and succinate + TMPD. The H+/site ratios are 4.0 with ferrocyanide and 6.0 with succinate + TMPD (at pH <7.0); the charge/site ratios are 6.0 with ferrocyanide and with succinate + TMPD (at pH <7.0) and 4.0 with ascorbate + TMPD; the ATP/site ratio is 1.34 with ascorbate + ferrocyanide. These ratios have been obtained in the presence of amounts of antimycin A that provide full inhibition of site II. For coupling sites I and II, ferricyanide has been used as e- acceptor and succinate or NAD-linked substrates as e- donors. The H+/site ratios are 4.0 at sites I and II; the charge/site ratios are 4.0 at site I and 2.0 at site II; the ATP/site ratios are 1.0 at site I and 0.5 at site II. Two major factors affect the stoichiometries: (i) dimension of [unk]H and (ii) supply of H+ from the matrix. There is a correlation between collapse of [unk]H and increase of H+/site and charge/site ratios. This indicates that approximation of the phenomenologic stoichiometry of the H+ pump is obtained when flow ratios are measured at level flow. That charge/site and ATP/site ratios increase when ferrocyanide is e- donor and decrease when ferricyanide is e- acceptor is attributed to the localization of the redox couple. This leads to separation of 1 charge/e- when ferrocyanide is e- donor and to consumption of 1 charge/e- when ferricyanide is e- acceptor. To account for an extrusion of H+ in excess of that predicted by the loop model, it is proposed that each coupling site contains a channel acting as a H+ pump.

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