Sidedness of e⁻ Donation and Stoichiometry of H⁺ Pumps at Sites II + III in Mitochondria from Rat Liver

The stoichiometry of the redox H⁺ pumps at site II and site II+III of the respiratory chain of rat‐liver mitochondria has been determined with three different e⁻ donors: succinate, duroquinol and NADH with duroquinone by‐pass of rotenone inhibition. Succinate provides reducing equivalents to the cytochrome b‐c₁ complex from the matrix side. Duroquinol, either added from the outside or obtained from the reduction of exogenous duroquinone by the NADH dehydrogenase, presumably is an intramembrane e⁻ donor to cytochrome b or to the endogenous ubiquinone. The stoichiometries were determined at steady‐state rates of ion translocation and e⁻ transfer. The ion translocation was followed on the basis of the rates of H⁺ extrusion and of K⁺ and Ca²⁺ uptake. The e⁻ transfer was followed either with the polarographic or with the spectrophotometric technique, i. e. absorbance changes during oxidation of duroquinol to duroquinone. In this latter case N‐ethylmaleimide was used to inhibit the NADH dehydrogenase activity responsible for the re‐reduction of the duroquinone formed during duroquinol oxidation. During operation of sites II+III, with oxygen as e⁻ acceptor, all three substrates tested gave H⁺/2e⁻ and charge/2e⁻ ratios close to 8. 0. During operation of site II, with ferricyanide as e⁻ acceptor, the H⁺/2e⁻ ratios were about 4. 0 and the charge/2e⁻ ratios close to 2. 0. The present results support previous reports from our laboratory on the stoichiometries of the redox H⁺ pumps at sites II and III. Furthermore the equality of the H⁺ and charge stoichiometry, with substrates having different sides of interaction with complex III suggests that e⁻ donors located on the matrix side or intramembrane (and possibly on the cytoplasmic side) share the same pathway in order to feed electrons into the H⁺ pump at site II.