Dependence of the 3-OH-Butyrate Dehydrogenase and Cytochrome c Oxidase Reactions on Intramitchondrial pH
Suspensions of isolated rat liver mitochondria were incubated at constant extramitochondrial pH (pHe) 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 PHi 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 PHi was decreased from values near 7.7 to values near 7.2, log [ATP]/[ADP] [Pi] at high PHi was significantly (P<0.02) greater than at low pH. The mean values for change in log [ATP]/[ADP] [Pi] divided by change in pHi 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 pHi and not on pHe. This implies that the matrix space is the source of protons consumed in the reduction of dioxygen to water in coupled mitochondria.
KeywordsRespiratory Rate Oxidative Phosphorylation Free Energy Change Choline Chloride Matrix Space
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