The Reaction of Reduced Cytochrome c Oxidase with Oxygen

The rate of reaction of reduced cytochrome oxidase with oxygen has been studied under a wide range of conditions by mixing the carbon monoxide compound of the reduced oxidase with oxygen-containing buffers and initiating the reaction by liberating free reduced oxidase photochemically. The method has been improved through the use of complementary filtration procedures and careful attention to electrical and magnetic shielding to a point at which the measurement of first order constants of up to 105 per sec has been possible. With the use of a wide range of oxygen concentrations, the rate of the initial portion of the biphasic record obtained at 445 mµ increases roughly in proportion to oxygen concentration until a rate of about 104 per sec is reached, beyond which it continues to increase but no longer does so in proportion to the oxygen concentration. Good agreement with the experimental findings has been obtained with a scheme in which 1 oxygen molecule diffuses into the oxidase unit (rate, 1 x 108 m-1sec-1) followed by the first order oxidation of cytochrome a3 (3 x 104 sec-1) and of a (7 x 102 sec-1). Previous work by these authors suggests that copper associated with the oxidase unit reacts at 7 x 103 sec-1 and the present publication describes the effect of temperature on these individual steps. In no case was the effect large; it was greatest on the cytochrome a oxidation step, corresponding to a Q10 of 1.4. The improved time resolution has permitted the spectro-kinetic separation of the cytochromes a3 and a oxidized-reduced difference spectra, which, although basically similar to those obtained in static experiments, show several important differences. The isosbestic points for the a3'' - a3''' and the a'' - a''' differences are 434 mµ and 430 mµ, respectively, the converse of the findings from static difference spectrophotometry and from previous work on the dissociation of CO in the presence of O2. Another important difference is that the ratio of the contribution of cytochrome a3 to that of a at 445 mµ was 2.3:1, compared to 1:1 by the static method.