MICROSECOND GENERATION OF OXYGEN-BOUND CYTOCHROME-C-OXIDASE BY RAPID SOLUTION MIXING

Current understanding of the oxygen reduction and proton translocation processes in cytochrome c oxidase is largely derived from the data ob tained by a nonphysiological method for initiating the catalytic react ion: photolyzing carbon monoxide (CO) from the CO-inhibited enzyme in the presence of oxygen (O-2). However, considerable evidence suggests that the use of CO introduces artifacts into the reaction mechanism. W e have therefore developed a rapid solution mixer with a mixing time o f 20 mu s to study the catalytic reaction by directly mixing the enzym e with O-2 without using CO. Unexpectedly, the resonance Raman scatter ing detected for the first 120 mu s 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, w hich 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 micro second time domain.