Biochemistry zyxwvu 1990, 29, zyxwvu 10135-10140 10135 B. J., zyxwv & Rokach, J. (1987) Ado. Prostaglandin, Thromb- oxane, Le vkotriene Res. 17, 55-59. Yokoyama, C., Shinjo, F., Yoshimoto, T., Yamamoto, S., Oates, J. A., & Brash, zyxw A. R. (1986) J. Biol. Chem. 261, Yoshimoto, T., Miyamoto, Y., Ochi, K., & Yamamoto, zy S. Wong, P. Y.-K., Westlund, P., Hamberg, M., Granstrom, E., Chao, P. Ha-W., & Samuelsson, B. (1984) J. Biol. Chem. 259, 2683-2686. Woollard, P. M. (1986) Biochem. Biophys. Res. Commun. Yamamoto, S., Ueda, N., Yokoyama, S., Kaneko, F., Shinjo, F., Yoshimoto, T., Oates, J. A., Brash, A. R., Fitzsimmons, 136, 169-176. 167 14-1 672 1. (1982) Biochim. Biophys. Acta 713, 638-646. Room Temperature Characterization of the Dioxygen Intermediates of Cytochrome c Oxidase by Resonance Raman Spectroscopy+ Randy W. Larsen,t Wei Li,' Robert A. Copeland,rvs Stephan N. Witt,tJ Bih-Show Lou," Sunney I. Chan,' and Mark R. Ondrias*J Department of Chemistry, University zyxwvuts of New Mexico, Albuquerque, New Mexico 87131, and Arthur Amos Noyes Laboratory of Chemical Physics, 127-72, California Institute of Technology, Pasadena, California 91 125 Received February 23, 1990; Revised Manuscript Received July 30, 1990 ABSTRACT: Resonance Raman spectroscopy was employed to investigate the heme structures of catalytic intermediates of cytochrome zyxwvutsr c oxidase at room temperature. The high-frequency resonance Raman spectra were obtained for compound C (the two-electron-reduced dioxygen intermediate), ferryl (the three-elec- tron-reduced dioxygen intermediate), and the fully oxidized enzyme. Compound C was formed by photolyzing CO mixed-valence enzyme in the presence of 02. The ferryl intermediate was formed by reoxidation of the fully reduced enzyme by an excess of H202. Two forms of the oxidized enzyme were prepared by reoxidizing the fully reduced enzyme with 02. Our data indicate that, in compound C, cyt a3 is either intermediate or low spin and is nonphotolabile and its oxidation state marker band, v4, appears at a higher frequency than that of the resting form of the enzyme. The ferryl intermediate also displays a low-spin cyt u3, which is nonphotolabile, and an even higher frequency for the oxidation state marker band, v4. The reoxidized form of cytochrome c oxidase with a Soret absorption maximum at 420 nm has an oxidation state marker band zyxwvutsr (u4) in a position similar to that of the resting form, while the spin-state region resembles that of compound C. This species subsequently decays to a second oxidized form of the enzyme, which displays a high-frequency resonance Raman spectrum identical with that of the original resting enzyme. Cytochrome c oxidase is a multisubunit, membrane-bound protein that catalyzes the four-electron reduction of dioxygen in mitochondria. The oxygen reduction activity of the enzyme is coupled to proton translocation across the inner mitochon- drial membrane during respiration. The enzyme utilizes four redox-active metal centers to perform its catalytic function. These centers include two heme A chromophores and two Cu ions. The dioxygen reduction site consists of a binuclear heme A/Cu cluster (designated cytochrome a3, CU,). The two remaining metal centers (designated cyt a and Cu,) mediate the electron transfer from ferrocytochrome c to cytochrome a3, CU, (Wikstrom et al., 1977, 1981; Palmer et al., 1979). Although the dioxygen reduction kinetics have been studied extensively by a variety of spectroscopic techniques, much less Research supported in part by Grants G M 22432 (to S.I.C.) and GM 33330 (to M.R.O.) from the National Institute of General Medical Sciences, US. Public Health Service, Grant RR 08139 (to M.R.O.) from the Division of Research Resources, National Institutes of Health, and the donors of the Petroleum Research Fund, administered by the Am- erican Chemical Society (to S.I.C.). Contribution No. 8103 from Di- vision of Chemistry and Chemical Engineering, California Institute of Technology. * To whom correspondence should be addressed. *California Institute of Technology. *Current address: Department of Biochemistry and Molecular Bi- ology, University of Chicago, Chicago, IL 60637. Current address: Department of Chemistry, Stanford University, Stanford, CA 94305. University of New Mexico. is known about the structure of the intermediates formed during the turnover of the enzyme by O2 [see Hill et al. (1986) and Chan et al. (1988) for reviews]. Because the turnover rate of the enzyme can be as high as 400 electrons transferred per second, spectroscopic studies of the intermediates have been difficult. Low-temperature transient absorption studies of the fully reduced CO photolyzed enzyme in the presence of O2 revealed the existence of at least three distinct species during turnover (Chance et al., 1975). Recent room temperature resonance Raman and transient absorption spectra indicate the possibility of four intermediates (Babcock et al., 1985; Hill & Greenwood, 1984). The first intermediate is believed to be an O2 adduct bound to the ferrous heme a3 similar to oxyhemoglobin (compound A) (Babcock et al., 1985; Han et al., 1990; Hill & Greenwood, 1984). Electron transfers from CU, and heme a3 to the bound 0, produce the second intermediate, generally assumed to be a peroxo heme a3-Cu, bridged species (com- pound C) (Hill & Greenwood, 1984). Blair et al. (1985), using EPR spectroscopy in conjunction with the low-temperature triple trapping technique pioneered by Chance et al. (1975), reported evidence for two distinct intermediates at the three-electron level of dioxygen reduction. It has been pro- posed that the first of these species is a cupric hydroperoxide coordinated to a ferrous heme a3 in the binuclear cluster, while the second is an Fe(IV) heme a3 species resulting from heterolytic cleavage of the 0-0 bond (Blair et al., 1984). 0006-2960/90/0429-10135$02.50/0 0 1990 American Chemical Society