Dioxygen activation and bond cleavage by mixed-valence cytochrome c oxidase

Abstract

Elucidating the structures of intermediates in the reduction of O 2 to water by cytochrome c oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O 2 with the mixed-valence enzyme, in which only heme a 3 and Cu B in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that OAO bond cleavage occurs within the first 200 s after reaction initiation; the presence of a uniquely stable FeOOOO(H) peroxy species is not detected. The product of this rapid reaction is a heme a 3 oxoferryl (Fe IV AO) species, which requires that an electron donor in addition to heme a 3 and Cu B must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E. OOH؊ , and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed OOO bond forming chemistry that occurs during O 2 evolution in photosynthesis.The molecular mechanism of dioxygen activation and reduction by the terminal respiratory enzyme, cytochrome c oxidase (CcO), is accessible because of its unique kinetic properties. Elucidation of this mechanism is of fundamental importance in understanding O 2 chemistry in biological systems and necessary for insight into the function of the protein as a redox-linked proton pump. CcO uses four redox-active metal centers, Cu A , heme a, and the heme a 3 ͞Cu B binuclear center, to sustain mitochondrial electron transport by reducing molecular oxygen to water. This reaction ensures a constant flow of electrons through the respiratory chain and the coupled generation of a proton gradient across the mitochondrial membrane, which is required for ATP synthesis. The oxygen chemistry catalyzed by CcO contributes directly to the build-up of the proton gradient because of its redox-linked proton pumping function (1). Thus, the overall reaction catalyzed by CcO may be written aswhere H in ϩ and H out ϩ indicate protons on the matrix (in) and cytosolic (out) sides of the membrane. Because its reaction kinetics is controlled by its proton-pumping function, unique insights into oxygen activation mechanisms are possible (2, 3).A number of reaction intermediates in dioxygen reduction have been identified recently (3, 4). Nonetheless, the timing and mechanism of the critical OAO bond cleavage process in the heme a 3 ͞Cu B binuclear center and the structure of a key intermediate at the peroxy level are poorly understood. In one model, a heme-peroxy adduct [Fe a3 III OOOO(H)] is uniquely stable (2,3,(5)(6)(7)(8). This model contrasts with peroxidases (9) and catalases (10), in which the peroxy OOO bond is spontaneously cleaved to yield an oxoferryl (Fe IV AO) product and a radical. Recent Raman work on the reaction of CcO with H 2 O 2 provided an alternati...

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

“…The data of the Y280H/H 2 O 2 and Y280H MV/O 2 reactions demonstrate that the 607-nm form is not linked to the redox properties of Tyr-280 and can be produced without its involvement to form a Fe(IV)ϭO/Cu B 2ϩ -His-Y ⅐ species, as recently proposed (13,14,16). It should be noted that Fabian and Palmer (24,25) reported the formation of 7% of a radical species in the oxidized CcO/H 2 O 2 reaction, whereas MacMillan et al (27) reported a yield of 20% for a tyrosine radical species produced in the same 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).…”

“…Tyr-280 is located at the end of the proton K-channel and is highly conserved among the heme copper oxidases. The suggestion that the O-O bond cleavage proceeds by concerted hydrogen atom transfer from the cross-linked His-Tyr species to produce the Fe(IV)ϭO/Cu B 2ϩ -His-Y ⅐ species raises important issues as to the means by which the electron transfer to these transient species is regulated for conformational transitions that are required to occur in any pumping mechanism (13,16).…”

CO impedes superfast O 2 binding in ba 3 cytochrome oxidase from Thermus thermophilus

“…If simultaneous binding of CO to Cu B þ and O 2 to heme a 3 2þ does occur, the CO-bound Cu B may not be able to act as an electron donor during dioxygen reduction. Because the rate of P I formation is the same in the absence of CO (this study) and presence of CO (our assignment) (6), the donor of the fourth electron required for dioxygen bond cleavage would presumably be the same in both cases, perhaps the cross-linked tyrosine (31,32), as suggested for the reaction of O 2 with the mixed-valence aa 3 enzyme (32). Alternatively, the binding of O 2 to heme a 3 2þ may reduce the affinity of Cu B þ for CO, causing CO to dissociate, thus allowing Cu B þ to act as an electron donor to the bound dioxygen.…”

Entrance of the proton pathway in cbb 3 -type heme-copper oxidases

“…This reaction can be studied by time-resolved spectroscopy using various detection techniques, which has provided detailed information about the catalytic cycle of the A-type HCuOs (see, e.g., refs. [22][23][24][25][26].…”