Single-electron photoreduction of the P-M intermediate of cytochrome c oxidase
by Siletsky, S. A.; Han, D.; Brand, S.; Morgan, J. E.; Fabian, M.; Geren, L.; Millett, F.; Durham, B.; Konstantinov, A. A.; Gennis, R. B.
The P-M -> F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P-M state of the oxidase was generated by exposing the oxidized enzyme to CO Plus O-2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a(3) with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is similar to 5-fold more rapid than the rate of electron transfer from heme a to heme a(3) in the F -> O transition, but is significantly slower than formation of the F state from the PR intermediate in the reaction of the fully reduced enzyme with O-2 to form state F (70-90 mu s). The similar to 0.3 ms P-M -> F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P-M -> F transition is generated much later, with a time constant of 1.3 ms. In addition, the P-M -> F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with r values of 0.18 and 0.85 ms.