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

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.