Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b(558), b(595), and d as the red ox metal centers. Taking advantage of spectroscopic properties of three hem es which exhibit distinct absorption peaks, we investigated electron transf er within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in ma inly an oxygenated form and partially an oxoferryl and a ferric low-spin fo rms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b(558 ) and b(595) with a second-order rate constant of 3 x 10(8) M-1 s(-1), sugg esting that a rapid equilibrium occurs for electron transfer between two b- type hemes long before 10 mu s. In the slower phase, an intramolecular elec tron transfer from heme b to the oxoferryl and the ferric heme d occurred w ith the first-order rate constant of 4.2-5.6 x 10(2) s(-1). In contrast, th e oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated th at the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the f erric and oxoferryl forms of heme d to the ferrous and ferric forms, respec tively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s(-1), respectively.