Single-electron photoreduction of the P M intermediate of cytochrome c oxidase

Abstract

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 ∼ 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 P R intermediate in the reaction of the fully reduced enzyme with O 2 to form state F (70–90 μs). The ∼ 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 τ values of 0.18 and 0.85 ms.