Transient formation of ubisemiquinone radical and subsequent electron transfer process in the Escherichia coli cytochrome bo

To elucidate a unique mechanism for the quinol oxidation in the Escherichia coli cytochrome bo, we applied pulse radiolysis technique to the wild-type enzyme with or without a single bound ubiquinone-8 at the high-affinity qu inone binding site (Q(H)), using N-methylnicolinamide (NMA) as an electron mediator. With the ubiquinone bound enzyme, the reduction of the oxidase oc curred in two phases as judged from kinetic difference spectra. Tn the fast er phase, the transient species with an absorption maximum at 440 nm, a cha racteristic of the formation of ubisemiquinone anion radical, appeared with in 10 ps after pulse radiolysis. In the slower phase, a decrease of absorpt ion at 440 nm was accompanied by an increase of absorption at 428 and 561 n m, characteristic of the reduced form. In contrast, with the bound ubiquino ne-8-free wild-type enzyme, NMA radicals directly reduced hemes b and o, th ough the reduction yield was low. These results indicate that a pathway for an intramolecular electron transfer from ubisemiquinone anion radical at t he Q(H) Site to heme b exists in cytochrome bo. The first-order rate consta nt of this process was calculated to be 1.5 x 10(3) s(-1) and is comparable to a turnover rate for ubiquinol-1. The rate constant for the intramolecul ar electron transfer decreased considerably with increasing pH, though the yields of the formation of ubisemiquinone anion radical and the subsequent reduction of the hemes were not affected. The pH profile was tightly linked to the stability of the bound ubisemiquinone in cytochrome bo [Ingledew, W . J., Ohnishi, T., and Salerno, J. C. (1995) Eur. J. Biochem. 227, 903-908] , indicating that electron transfer from the bound ubisemiquinone at the Q( H) site to the hemes slows down at the alkaline pH where the hound ubisemiq uinone can be stabilized. These findings are consistent with our previous p roposal that the bound ubiquinone at the Q(H) Site mediates electron transf er from the low-affinity quinol oxidation site in subunit II to low-spin he me b in subunit I.