Characterization of steady-state activities of cytochrome c oxidase at alkaline pH: Mimicking the effect of K-channel mutations in the bovine enzyme

The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s ^-1 at pH 7.0 to less than 10 s ^-1 at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30-50 s ^-1) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the K _m values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a ₃ at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a ₃ is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O ₂ suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a ₃/Cu _B center prior to the reaction with O ₂. This step is not required for the reaction with H ₂O ₂, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O ₂, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.