Formation of the “Peroxy” Intermediate in Cytochrome c Oxidase Is Associated with Internal Proton/Hydrogen Transfer

Abstract

When dioxygen is reduced to water by cytochrome c oxidase a sequence of oxygen intermediates are formed at the reaction site. One of these intermediates is called the "peroxy" (P) intermediate. It can be formed by reacting the two-electron reduced (mixed-valence) cytochrome c oxidase with dioxygen (called P(m)), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen (called P(r)). In recent years, evidence has accumulated to suggest that the O-O bond is cleaved in the P intermediate and that the heme a(3) iron is in the oxo-ferryl state. In this study, we have investigated the kinetic and thermodynamic parameters for formation of P(m) and P(r), respectively, in the Rhodobacter sphaeroides enzyme. The rate constants and activation energies for the formation of the P(r) and P(m) intermediates were 1.4 x 10(4) s(-1) ( approximately 20 kJ/mol) and 3 x 10(3) s(-1) ( approximately 24 kJ/mol), respectively. The formation rates of both P intermediates were independent of pH in the range 6.5-9, and there was no proton uptake from solution during P formation. Nevertheless, formation of both P(m) and P(r) were slowed by a factor of 1.4-1.9 in D(2)O, which suggests that transfer of an internal proton or hydrogen atom is involved in the rate-limiting step of P formation. We discuss the origin of the difference in the formation rates of the P(m) and P(r) intermediates, the formation mechanisms of P(m)/P(r), and the involvement of these intermediates in proton pumping.

Discovery of the True Peroxy Intermediate in the Catalytic Cycle of Terminal Oxidases by Real-time Measurement

“…We cannot exclude, however, that at the P state the O-O bond is already broken and P could be similar to P M in CcO. The latter would be apparently inconsistent with the same reaction catalyzed by the R CcO where no P M -like intermediate preceding the formation of P R has been observed (7,16,17). The P intermediate is further converted into F with k ϭ 2.1 ϫ 10 4 s Ϫ1 .…”

Conformational coupling between the active site and residues within the K C -channel of the Vibrio cholerae cbb 3 -type (C-family) oxygen reductase

“…By analogy with the reaction in the A-family HCOs, oxidation of the active site heme b 3 /Cu B and heme b, forming intermediate P R , presumably can occur with no concomitant uptake of bulk protons (27). In the P R intermediate in A-family enzymes, the O-O bond is broken, and the proton required for this is supplied by the cross-linked tyrosine at the active site.…”

Interconversions of P and F intermediates of cytochrome c oxidase from Paracoccus denitrificans

“…This scheme had to be modified because a three-electron reduced P state was observed during the reaction of fully reduced four-electron CcO with molecular oxygen (33), and by the observation that the P H state is converted to the F • state upon lowering the pH (15). Here we provide additional evidence that the UV/ visible spectra do not reflect the overall redox state of the enzyme as a simple and facile correlation because the presence of a previously undescribed ammonia ligand, presumably at Cu B , can cause the difference in absorption properties of the F and P N states without comprising any redox reaction or radical formation.…”