The Reaction of Cytochrome Oxidase with Cytochrome C.

In recent work on the reaction between oxygen and reduced cytochrome oxidase made by the method of Yonetani (1)) Gibson and Greenwood (2) found that the reaction is complex and that at least two spectroscopically distinguishable reactions must take place. The kinetic changes were interpreted in terms of a scheme proposed by Keilin and Hartree (3), according to which cytochrome oxidase preparations contain two components: cytochrome u3, which reacts directly with oxygen, and cytochrome a, which reacts with cytochrome u3 and cytochrome c but not with oxygen. The experiments afforded a semiquantitative confirmation of the difference spectra between the oxidized and reduced forms of cytochromes a and a3 found by Yonetani (1) by static difference spectrophotometry, and led to the conclusions that the initial reaction between oxygen and reduced cytochrome a3 is very fast indeed, with an apparent second order rate constant of 5 x 107 M-I see+, and that the reaction between cytochromes a3 and a (presumed first order) has a rate constant of 750 see-l at 20”. Yonetani’s preparation is soluble only in the presence of the nonionic detergent Emasol 4130 (Kao Soap Company, Tokyo), and the results obtained with it, like those obtained with other soluble preparations, are subject to criticism as artifacts. The active, deoxycholate-stabilized preparation of Griffiths and Wharton (4) has now been compared with Yonetani’s preparation, and has been found to agree closely with it in the properties examined. This agreement between enzymes prepared by different procedures from different starting materials goes some way to meet the criticism of artificiality, since there is no a priori reason why such close parallelism between the preparations should have been found unless their behavior is a reflection of the underlying properties of the enzyme. In addition to the experiments on the oxidation of the enzyme, we also report on its reaction with cytochrome c, and on some properties of a more complex system with oxygen, oxidase, cytochrome c, and ascorbic acid.