Reactions of cytochrome oxidase with oxygen and carbon monoxide

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Abstract
The reaction of the Yonetani (1960a, 1961) preparation of cytochrome oxidase with carbon monoxide is accurately second-order with a rate constant 8 x 104 M -l sec.-l at 20[degree] and activation energy of 6.4 kcal. The dissociation velocity constant of carbon monoxide is 0.023 sec.-l at 20[degree] and pH 7.4. In the Yonetani (1960a, 1961) preparation, one-third of the total iron reacts with carbon monoxide to form the carbon monoxide compound as determined both by spectrophotometry and by gasometric methods. The reaction of oxygen with reduced cytochrome oxidase is rapid and the course of the reaction is complex. At low oxygen concentrations the approximate second-order rate constant is 6 x 107 M -1 sec.-1 for the Yonetani (1060a, 1961) preparation and 3 x 107 M -1 sec-1 for the Greenwood (1963) preparation. When the reaction between reduced cytochrome oxidase and oxygen is followed at different wavelengths, the course of the reaction changes. At low oxygen concentrations (less than 5[mu]M) the reaction proceeds faster when followed at 605 m[mu] than when followed at 445 m[mu]. At concentrations of oxygen above 10[mu]M, the observed changes at 605 m[mu] are slower than those at 445 m[mu]. The apparent second-order rate constant at 445 m[mu] and at low oxygen concentrations increases as the reaction proceeds; at high oxygen concentrations it decreases. These changes are attributed to consecutive reactions between oxygen and reduced cytochrome a3, and between reduced cytochrome a3 and oxidized cytochrome a. Satisfactory agreement between observation and calculation has been obtained by assuming that reduced cytochrome a3 can react with oxygen with a velocity constant of 1.5 x 108 M -1 sec.-1 and that the reaction between reduced cytochrome a and cytochrome a3 is first-order with a rate constant of 1.5 x 103 sec.-l. The spectrophotometric changes at different wave-lengths may be reproduced by using the extinction coefficients given by Yonetani (1960a).