A new ruthenium complex to study single-electron reduction of the pulsed OH state of detergent-solubilized cytochrome oxidase

by Brand, Sue Ellen; Rajagukguk, Sany; Ganesan, Krithika; Geren, Lois; Fabian, Marian; Han, Dan; Gennis, Robert B.; Durham, Bill; Millett, Francis

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru-(bipyrazine)(2)](2)(quaterpyridine), (Ru(2)Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru(2)Z allows it to bind electrostatically near Cu-A in subunit 11 of cytochrome oxidase. The photoexcited state Ru(II*) of Ru(2)Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces Cu-A with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "O-H" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru(2)Z, the oxidized OH state was formed within 5 ms. Ru(2)Z was then excited with a laser flash to inject one electron into Cu-A. Electron transfer from Cu-A -> heme a -> heme a(3)/Cu-B was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from Cu-A to heme a occurred with a rate constant of 20 000 s(-1) with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a(3)/Cu-B center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s(-1) and 110 s(-1) and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a(3) is observed, indicating that Cu-B is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.

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1520-4995; 0006-2960