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. Extrapolation of the observed rates to zero time, at which zero delta mu 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.
D000818:Animals; D004566:Electrodes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D011189:Potassium Chloride / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D013386:Succinates / Q000378:metabolism*; D019802:Succinic Acid
D013386:Succinates; D011189:Potassium Chloride; D019802:Succinic Acid; D006180:Proton-Translocating ATPases