Single Electron Reduction of Cytochrome c Oxidase Compound F:  Resolution of Partial Steps by Transient Spectroscopy

Abstract

The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.

Direct measurement of proton release by cytochrome c oxidase in solution during the F→O transition

“…The monomeric enzyme was converted to state F by H 2 O 2 in the presence of the photoreductant Ru 2 C, which binds noncovalently to the oxidase (presumably to the binding site for Cyt c) under the conditions used (23,24). The yield of the F state was determined spectroscopically (as described in ref.…”

“…The concentration of CcO was measured by using the absorbance in Soret band as described in ref. 24. The pH of the sample was adjusted to 9.5 by titration with dilute KOH by using the absorbance of the dye at 550 nm.…”

“…The photoreduction of CcO was achieved as described in ref. 24 by a laser pulse in the presence of 30 M Ru2C, 10 mM aniline, and 1 mM 3CP (the sacrificial electron donor). Compound F was made by incubation with 6 mM H 2O2 for 5 min followed by titration of the sample to pH 9.5.…”

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

“…Electron [29][30][31]. The different KIEs indicate that different proton-transfer events are rate-limiting for these reactions.…”

A Physical Chemist's Expedition to Explore the World of Membrane Proteins

“…In this scenario, the reduction potential of Cu A would become higher than that of cytochrome a, forestalling the electron transfer from Cu A to cytochrome a. In the absence of redox linkage, the electron equilibration between Cu A and cytochrome a would be rapid, of the order of 20,000 s (64), and possibly even faster (113). Aside from the redox potential difference between these two redox centers, the kinetics of the electron transfer between Cu A and cytochrome a could also be tuned by subtle conformational changes in the enzyme.…”