Stoichiometry of mitochondrial H+ translocation coupled to succinate oxidation at level flow

The mechanistic stoichiometry of vectorial H⁺ translocation coupled to succinate oxidation by rat liver mitochondria in the presence of a permeant cation has been determined under level flow conditions with a membraneless fast responding O₂ electrode kinetically matched with a glass pH electrode. The reactions were initiated by rapid injection of O₂ into the anaerobically preincubated test system under conditions in which interfering H⁺ backflow was minimized. The rates of O₂ uptake and H⁺ ejection, obtained from computer-fitted regression lines, were monotonic and first order over 75% of the course of O₂ consumption. Extrapolarization of the observed rates to zero time, at which zero Δμ(H⁺) and thus level flow prevails, yielded vectorial H⁺/O flow ratios above 7 and closely approaching 8. The mitochondria undergo no irreversible change and give identical H⁺/O ratios on repeated tests. In a further refinement, the lower and upper limits of the mechanistic H⁺/O ratio were determined to be 7.55 and 8.56, respectively, from plots of the rates of O₂ uptake versus H⁺ ejection at increasing malonate and increasing valinomycin concentrations, respectively. It is therefore concluded that the mechanistic H⁺/O ratio for energy-conserving sites 2 + 3 is 8, in confirmation of earlier measurements. KCl concentration is critical for maximal observed H⁺/O ratios. Optimum conditions and possible errors in determination of mechanistic H⁺/O translocation ratios are discussed.