Oxygen, pH, and mitochondrial oxidative phosphorylation

oxygen dependence of mitochondrial oxidative phosphorylation was measured in suspensions of isolated rat liver mitochondria using recently developed methods for measuring oxygen and cytochrome c reduction. Cytochrome-c oxidase (energy conservation site 3) activity of the mitochondrial respiratory chain was measured using an artificial electron donor (N,N,N=,N=-tetramethyl-p-phenylenediamine) and ascor-bate to directly reduce the cytochrome c, bypassing sites 1 and 2. For mitochondrial suspensions with added ATP, metabolic conditions approximating those in intact cells and decreasing oxygen pressure both increased reduction of cytochrome c and decreased respiratory rate. The kinetic parameters [KM and maximal rate (VM)] for oxygen were determined from the respiratory rates calculated for 100% reduction of cytochrome c. At 22°C, the KM for oxygen is near 3 Torr (5 M), 12 Torr (22 M), and 18 Torr (32 M) at pH 6.9, 7.4, and 7.9, respectively, and VM corresponds to a turnover number fo