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

Abstract

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.