S. Takahashi et al., MICROSECOND GENERATION OF OXYGEN-BOUND CYTOCHROME-C-OXIDASE BY RAPID SOLUTION MIXING, The Journal of biological chemistry, 270(15), 1995, pp. 8405-8407
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.