Current understanding of the oxygen reduction and proton translocation processes in cytochrome c oxidase is largely derived from the data obtained by a nonphysiological method for initiating the catalytic reaction: photolyzing carbon monoxide (CO) from the CO-inhibited enzyme in the presence of oxygen (O2). However, considerable evidence suggests that the use of CO introduces artifacts into the reaction mechanism. We have therefore developed a rapid solution mixer with a mixing time of 20 μs to study the catalytic reaction by directly mixing the enzyme with O2 without using CO. Unexpectedly, the resonance Raman scattering detected for the first 120 μs after the mixing show that the CO influences neither the structure of the primary oxy-intermediate, its rate of decay, nor the rate of oxidation of cytochrome a. This implies that CO has an effect on the later stages of the catalytic process, which may involve the proton translocation steps, and calls for the re-examination of the catalytic process by using the direct mixing method. In addition, these results demonstrate the feasibility of using the rapid mixing device for the study of biological reactions in the microsecond time domain.
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