Phosphorylation coupled with the oxidation of α-ketoglutarate by heart-muscle sarcosomes. 3. Experiments with ferricytochrome c as hydrogen acceptor

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Abstract
Oxidative phosphorylation was demon-strated with alpha-ketoglutarate as hydrogen donor and ferri-cytochrome c as acceptor. Since the ratio, phosphorus esterified: oxygen equivalent of ferricytochrome c reduced, was close to 2 and was sensitive to 2:4-dinitrophenol, respiratory chain phosphorylation must be involved. These experiments were carried out under completely anaerobic conditions in order to prevent reoxidation of ferrocytochrome c. A sensitive enzymic method of detecting residual O2 is described. Respiratory inhibitors could not be used satisfactorily. Cyanide inhibits phosphorylation and combines with alpha-ketoglutarate; azide also inhibits phosphorylation and is a relatively ineffective respiratory inhibitor with intact sarcosomes. Sulfide rapidly reduces ferricytochrome c. The combination between alpha-ketoglutarate and cyanide was a slow reaction. The yield of oxidative phosphorylation with ferricytochrome c as acceptor was compared with that obtained with O2. The phosphorus: oxygen ratios with ferricytochrome c varied between 1.5 and 1.9 (mean 1.72) in 9 experiments (phosphorus, alpha-keto-glutarate ratios, 1.6-2.0, mean 1.80), compared with just under 3 obtained with O2.Two possible explanations of the results are suggested. (1) Respiratory chain phosphorylation occurs between ferrocytochrome c and O2, as well as between substrate and ferricytochrome c. (2) All the phosphorylation occurs between substrate and the endogenous cytochrome c of the sarcosome, but the reaction with added ferricytochrome c by-passes a phosphorylating reaction.