Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen," Biochemistry

ABSTRACT: Electron transfer during the reaction of fully reduced bovine heart cytochrome oxidase with dioxygen has been studied at 24°C in the near-infrared region following photolysis of the fully reduced CO-bound complex. The transient spectral changes and kinetics were followed on microsecond to millisecond time scales at nine different wavelengths between 597 and 935 nm and were analyzed using singular value decomposition and global exponential fitting. Four apparent lifetimes, 14 µs, 40 µs, 86 µs, and 1.1 ms, were resolved. The near-infrared spectra of the intermediates are extracted on the basis of a previously proposed mechanism [Sucheta et al. (1998) Biochemistry 37, 17905-17914] and compared to model spectra of the postulated intermediates. The data provide a comprehensive picture of the spectral contributions of the different redox centers in their respective oxidation or ligation states in the nearinfrared region and strongly support that Cu A is partially (2/3), but not fully, oxidized in the 3-electronreduced ferryl intermediate. The reduction of dioxygen to water by cytochrome oxidase has been extensively studied in the Soret and visible regions by single-wavelength detection (445, 605 nm) (1-4), and more recently in our laboratory, using a gated multichannel analyzer (5-7). These studies, along with time-resolved resonance Raman (TR 3 ) studies (8-14), have indicated that the reaction involves the formation of compound A, followed by the putative P 1 intermediate (607 nm form), and the subsequent generation of a ferryl, F (580 nm), form. The generation of the ferryl is followed by a more rapid electron transfer between Cu A and heme a (7). The nature of P has been the subject of intensive discussion, and recent investigations have suggested that it is a ferryl (a 3 4+ dO) with a radical on tyrosine 244 The formation of P has not been detected by TR 3 during the reaction of the fully reduced enzyme with dioxygen, but recent time-resolved optical absorption studies in our laboratory using a gated multichannel analyzer have suggested that P is transiently formed (6). The reduction of dioxygen to water by cytochrome oxidase has also been studied at 830 nm, the putative absorbance maximum of the oxidized Cu A In this study, we have investigated the reaction of the fully reduced cytochrome oxidase with dioxygen at selected wavelengths in the near-infrared region at room temperature. Using singular value decomposition (SVD) and global exponential fitting, we have extracted the near-infrared spectra of the intermediates, including that of compound A, the putative P, and F. MATERIALS AND METHODS Cytochrome oxidase was isolated from bovine hearts according to the method of Yoshikawa et al. (24), which involves sequential ammonium sulfate fractionations in cholate and Tween. The final precipitate was dialyzed against 0.1 M sodium phosphate buffer, pH 7.4. The fully reduced form was prepared by adding sodium ascorbate and ruthenium hexammine chloride to an anaerobic oxidized enzyme solution (5, 6). The mixed-valence CO-bound enzyme was made by deoxygenating the oxidized enzyme solution with purified N 2 , followed by exposure to CO until no further change a