Mass spectrometric determination of dioxygen bond splitting in the “peroxy” intermediate of cytochrome c oxidase

Abstract

The ''peroxy'' intermediate (P form) of bovine cytochrome c oxidase was prepared by reaction of the two-electron reduced mixedvalence CO complex with 18 O2 after photolytic removal of CO. The water present in the reaction mixture was recovered and analyzed for 18 O enrichment by mass spectrometry. It was found that approximately one oxygen atom ( 18 O) per one equivalent of the P form was present in the bulk water. The data show that the oxygen-oxygen dioxygen bond is already broken in the P intermediate and that one oxygen atom can be readily released or exchanged with the oxygen of the solvent water. Bovine cytochrome c oxidase (CcO) catalyzes the oxidation of ferrocytochrome c and the reduction of dioxygen to water in a process that conserves a fraction of the energy released in the redox process by translocating protons across the inner membrane of the mitochondrion. For this process, the enzyme depends on four transition metal centers: cytochrome a, Cu A , cytochrome a 3 , and Cu B . Electrons from ferrocytochrome c are transferred to Cu A and subsequently delivered to the cytochrome a 3 -Cu B binuclear center via cytochrome a; the conversion of O 2 to H 2 O is effected at this binuclear center.When the enzyme is fully reduced (Fe ϩ2 a Cu ϩ A Fe ϩ2 a3 Cu ϩ B ), a process requiring four electrons, the reaction with O 2 is fast, and detection of intermediates usually requires rapid kinetic techniques and transient spectroscopic methods. In contrast, when partially reduced oxidase reacts with O 2 , it is possible to obtain relatively stable oxygenated intermediates of the enzyme (1, 2). One of these intermediates, the ''peroxy'' or P form, is a product of the reaction of the two-electron reduced enzyme with O 2 . This P form is readily prepared on reaction of the two-electron reduced mixed-valence CO complex (MV-CO or Fe ϩ3 a Cu ϩ2 A Fe ϩ2 a3 CO Cu ϩ B ) with oxygen after photolytic removal of the bound CO (3-9). The same compound P can be observed after reaction of oxidized enzyme with hydrogen peroxide (10-14) and in mitochondria during reverse electron flow (15-17). Because the P form of the enzyme is formally at the two-electron reduced state, it was initially expected to have an intact oxygen-oxygen bond (e.g., Fe refs. 10, 12, 15, and 17-19). However, recent resonance Raman experiments (20-23) and data from reductive titrations of the P form (24) suggest that the O-O bond is broken, and the presence of H 2 O 2 in this species could not be detected (25).Cleavage of the oxygen-oxygen bond requires that an additional two reducing equivalents be available. These would need to be provided by one or more groups present in the enzyme. The reaction mechanism might then be analogous to the formation of compound I in heme peroxidases, during which reaction hydrogen peroxide is decomposed.Because the nature of the P intermediate of CcO is controversial and our understanding of it is based entirely on spectroscopic observations, we have addressed the question directly as to whether the dioxygen bond is broken in thi...

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

“…If heme a is oxidized, the electron along with the proton is provided by adjacent Y244 [4,23], and P m state is formed. The P m state is characterized by Cu B (2+), tyrosyl radical and oxoferryl state of iron of heme a 3 [24], Fe 4þ @O 2À . The protonation state of His291 in the P m however is unknown, as is the nature of the ligand to Cu B (2+) ion (it can be OH À or H 2 O).…”

The proton donor for OO bond scission by cytochrome c oxidase

“…The catalytic cycle of CcO starts with binding of dioxygen to heme a 3 [formation of a ferrous O 2 adduct, compound A (2)]. This step is followed by scission of the OOO bond, which requires delivery of four electrons and a proton to dioxygen and leaves the binuclear site in the highly oxidized P state (3)(4)(5). Three of the four required electrons are donated by heme a 3 (two electrons) and Cu B (one electron).…”

Direct Detection of Fe(IV)=O Intermediates in the Cytochrome aa3 Oxidase from Paracoccus denitrificans/H2O2 Reaction

“…CcO contains a homo-dinuclear Cu A center, one low spin heme a, and a high spin heme a 3 -Cu B binuclear center where the reduction of dioxygen to H 2 O takes place (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). One of the unique properties of the binuclear heme a 3 -Cu B center that were determined by the crystal structures of the bovine, the Paracoccus denitrificans, and the Thermus thermophilus heme copper oxidases is the Tyr-280 -His-276 (P. denitrificans numbering) cross-linked structure (7)(8)(9)(10)(11).…”