Stoichiometry of H+ translocation coupled to electron flow from succinate to cytochrome c in mitochondria.

Measurements are reported of the number of H+ translocated from the matrix to the suspending medium as a pair of electrons passes through energy-conserving Site 2, as reflected by electron flow from succinate to the artificial electron acceptor ferricyanide in intact rat liver mitochondria. The reduction of ferricyanide was measured by dual wavelength spectrophotometry and the translocation of H+ with a glass electrode or spectrophotometrically with phenol red as indicator. Extraneous electron flow was blocked with rotenone and cyanide and interfering uptake of H+ on the H+H2P04symporter was prevented with N-ethylmaleimide. K+ (plus valinomycin) or Ca2+ were used as permeant charge-compensating cations. The number of H+ translocated per pair of electrons (the H+/2eratio) passing through Site 2 was found to be very close to 4, determined either from the ratio of the initial rates of electron flow and H+ translocation or from ferricyanide pulse experiments. In the absence of succinate or with antimycin A present, negligible electron flow and H+ translocation occurred. The translocation of 4 H+ into the medium was accompanied by uptake of either 2 K+ or 1 Ca’+, i.e. by uptake of two positive electric charges. In the presence of a proton-conducting uncoupler only 2 H+ appeared, derived from the dehydrogenation of succinate. The H+/2eratio of close to 4 for Site 2 of the respiratory chain is consistent with earlier reports from this laboratory showing that the average H+/site ratio for the three energy-conserving sites is close to 4.