Abstract
The spectrophotometric oxidation of horse heart ferrocytochrome c was examined by use of the particulate electron transport fraction (R3) of Azotobacter vinelandii strain O. Unlike cytochrome c, purified preparations of native Azotobacter cytochromes c4 + c5 were oxidized only slowly by the electron transport fraction. The oxidation of mammalian cytochrome c proceeded at an appreciable rate and displayed “apparent” first-order kinetics at a pH optimum of 9.0 with tris(hydroxymethyl)aminomethane-chloride buffer. The calculated Vmax value was 0.22 μmole of cytochrome c oxidized per min per mg of protein (25 C) and a Km value for cytochrome c of 2.3 × 10−5m was obtained. Ferricytochrome c was a “strict” competitive inhibitor for this oxidation. Cytochrome c oxidation by the Azotobacter electron transport system was markedly sensitive to cyanide, azide, and hydroxylamine, although carbon monoxide inhibition could not be demonstrated. It was sensitive also to high concentrations of phosphate, ethylenediaminetetraacetate, and some metal cations. “Aging” or prolonged storage of the Azotobacter R3 fraction, at 4 C for 10 days, resulted in a threefold increase in specific activity. The cytochrome c peroxidase type of reaction did not occur with the R3 electron transport fraction.
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