H+ production by antimycin-inhibited mitochondria

1. Oxygen-pulse experiments on antimycin-treated liver mitochondria from young rats in the absence of added substrates indicate the presence of an endogenous hydrogenated reductant on the oxygen side of the antimycin inhibition site. This reductant has been estimated to be present in a 20--50-fold molar excess over cytochrome aa3. Oxidation of the reductant by small bursts of electron flow to oxygen gives a ratio H+ translocated:O atoms taken up of 2.1 +/- 0.2. 2. The endogenous hydrogenated reductant competes with added ferrocyanide and with ascorbate/NNN'N'-tetramethyl-p-phenylenediamine as a source of reducing equivalents to oxygen. 3. When the contribution of endogenous substrate to H+ production is taken into account, there still remains some respiration-dependent H+ translocation consistent with a H+ pumping activity of cytochrome oxidase. The stoichiometry of this 'extra' H+ translocation is variable and depends on the rate of electron flow.