The level of reduction of cytochrome a and cua during the oxidation of ferrocytochrome c has been determined in stopped‐flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, cua is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and cua to cytochrome a 3‐Cub is a two‐electron process and, in addition, that the binding of oxidized cytochrome c to the electron‐transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate‐limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.

The resting as well as the 420 nm and 428 nm forms of cytochrome oxidase have been studied in kinetic experiments with an excess of enzyme over reduced cytochrome e. No difference was found in the behavior of the two activated forms. With all three forms, a fraction of cytochrome a was reoxidized with a rate which was much lower than k,,,. This suggests that intramolecular transfer to the dioxygen-reducing site occurs only if both cytochrome u and CuA are reduced. An initial rapid phase in the oxidation of cytochrome u in the pulsed and oxygenated enzymes is related to the presence of a three-electron-reduced dioxygen intermediate. The increased catalytic activity of pulsed and oxygenated oxidase can be explained on the basis of a shift in the redox equilibrium between cytochrome a and Cu+,.

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