Dependence of the 3-OH-Butyrate Dehydrogenase and Cytochrome c Oxidase Reactions on Intramitchondrial pH

Summary

Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pH_e) with ATP, ADP, Pi, 3-OH-butyrate (3-OH-B), and acetoacetate (acac) (the last two were varied to maintain [3-OH-B]/[acac] constant), with or without sodium propionate to change the intramitochondrial pH. Measurements were made of the steady state water volume of the mitochondrial matrix, transmembrane pH difference, level of cytochrome c reduction, concentration of metabolites, and rate of oxygen consumption.

For each experiment, conditions were used for which PH_i was near maximal and minimal values and the measured extramitochondrial [ATP], [ADP] and [Pi] were used to calculate log [ATP]/[ADP] [Pi]. When [3-OH-B]/[acac] and [cyt c ⁺²]/[cyt c ⁺³] were constant, and PH_i was decreased from values near 7.7 to values near 7.2, log [ATP]/[ADP] [Pi] at high PH_i was significantly (P<0.02) greater than at low pH. The mean values for change in log [ATP]/[ADP] [Pi] divided by change in pH_i was 1.08 ± 0.15 (mean SEM). This agrees with the slope of 1.0 predicted if the energy available for ATP synthesis is dependent on the pH at which 3-OH-butyrate dehydrogenase operates, that is, the pH of the matrix space.

Plots of steady state respiratory rate vs. % cytochrome c reduction at different intra- and extramitochondrial pH values indicated that the rate of the cytochrome c oxidase reaction is dependent upon pH_i and not on pH_e. This implies that the matrix space is the source of protons consumed in the reduction of dioxygen to water in coupled mitochondria.