A New Ruthenium Complex To Study Single-Electron Reduction of the Pulsed O_HState of Detergent-Solubilized Cytochrome Oxidase

Abstract

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), (Ru2Z). 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 Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB 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 CuA 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 a3/CuB 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 a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.

Computational study of the activated O H state in the catalytic mechanism of cytochrome c oxidase

“…It is important to note that these calculated redox potentials differ substantially from the experimentally determined values (26) due to the fact that the latter are equilibrium potentials where charge compensation by protonation has occurred. The calculated O H /O H,R redox potentials are consistently ∼0.4 V higher than for the O/O R redox couples, which is in accord with the experimental observations of an elevated redox potential of Cu B in the O H state (29,52). The 0.4 V higher redox potential of the activated form solves the dilemma, at least in part, of an apparently far too low redox potential of the O→E transition to be consistent with proton pumping, and with the overall energetics of the catalytic cycle (2) (see The Metastable O H State).…”

“…However, in that study the observed rate of reduction of the BNC in the presumed O H state was much slower than reported in ref. 52 for the bovine and in ref. 29 for the Paracoccus enzyme.…”

The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres

“…When the partial reactions are examined the P → F and F → O rates are reportedly of the order of 10 000 s −1 [25,26], whereas the O → E and E → R rates are much lower (approx. 1000 s −1 ) [27,28]. The maximal levels of P and F will therefore be no more than 10 % those of E and O, and therefore undetectable with the present methodology.…”

Copper Active Sites in Biology

“…The reaction proceeds via a fast electron transfer between Cu A and cytochrome a with an apparent lifetime of F of ~50 µs (rate constant of 2×10 4 s −1 ), 953,954 to form intermediate F′. Earlier work by Gray and coworkers estimated a shorter lifetime for intermediate F of 1.2–25 µs.…”

“…949,954 The formation of O H is pH dependent, slower at higher pH, with a k H / k D of 2.5 measured at pH 7.4 952 and is rate-limited by proton uptake. The transmembrane voltage generated is biphasic with rate constants of ~830 s −1 (coincident with electron transfer coupled to proton transfer) and 220 s −1 (associated with proton transfer) with relative amplitudes of 1:3.…”