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...

Cytochrome oxidase activates and reduces O(2) to water to sustain respiration and uses the energy released to drive proton translocation and adenosine 5'-triphosphate synthesis. A key intermediate in this process, P, lies at the junction of the O(2)-reducing and proton-pumping functions. We used radioactive iodide labeling followed by peptide mapping to gain insight into the structure of P. We show that the cross-linked histidine 240-tyrosine 244 (His240-Tyr244) species is redox active in P formation, which establishes its structure as Fe(IV) = O/Cu(B)2+-H240-Y244. Thus, energy transfer from O2 to the protein moiety is used as a strategy to avoid toxic intermediates and to control energy utilization in subsequent proton-pumping events.

No abstract

The aa3-type cytochrome c oxidase from Rhodobacter sphaeroides is closely related to eukaryotic cytochrome c oxidases. Analysis of site-directed mutants identified the ligands of heme a, heme a3, and CuB [Hosler et al. (1993) J. Bioenerg. Biomembr. 25, 121-133], which have been confirmed by high-resolution structures of homologous oxidases [Iwata et al. (1995) Nature 376, 660; Tsukihara et al. (1995) Science 269, 1069; (1996) 272, 1136]. Since the protons used to form water originate from the inner side of the membrane, and the heme a3-CuB center is located near the outer surface, the protein must convey these substrate protons to the oxygen reduction site. Transmembrane helix VIII in subunit I is close to this site and contains several conserved polar residues that could function in a rate-determining proton relay system. To test this role, apolar residues were substituted for T352, T359, and K362 in helix VIII and the mutants were characterized in terms of activity and structure. Mutation of T352, near CuB, strongly decreases enzyme activity and disrupts the spectral properties of the heme a3-CuB center. Mutation of T359, below heme a3, substantially reduces oxidase activity with only minor effects on metal center structure. Two mutations of K362, approximately 15 A below the axial ligand of heme a3, are inactive, make heme a3 difficult to reduce, and cause changes in the resonance Raman signal specific for the iron-histidine bond to heme a3. The results are consistent with a key role for T352, T359, and K362 in oxidase activity and with the involvement of T359 and K362 in proton transfer through a relay system now plausibly identified in the crystal structure. However, the characteristics of the K362 mutants raise some questions about the assignment of this as the substrate proton channel.