Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

The PM → 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 PM state of the oxidase was generated by exposing the oxidized enzyme to CO plus O2. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a3 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 a3 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 O2 to form state F (70–90 μs). The ∼ 0.3 ms PM → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the PM → F transition is generated much later, with a time constant of 1.3 ms. In addition, the PM → 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.