RESONANCE RAMAN STUDIES OF ESCHERICHIA-COLI CYTOCHROME-BD OXIDASE - SELECTIVE ENHANCEMENT OF THE 3 HEME CHROMOPHORES OF THE AS-ISOLATED ENZYME AND CHARACTERIZATION OF THE CYANIDE ADDUCT

Cytochrome bd oxidase is a terminal bacterial oxidase containing three cofactors: a low-spin heme (b(558)), a high-spin heme (b(595)), and a chlorin d. The center of dioxygen reduction has been proposed to be at a dinuclear b(595)/d site, whereas b(558) is mainly involved in transferring electrons from ubiquinone. One of the unique functional features of this enzyme is its resistance to high concentrations of cyanide (K-i in the millimolar range). With the appropriate selection of laser lines, the ligation and spin states of the b(558), b(595), and d hemes can be probed selectively by resonance Raman (rR) spectroscopy. Wavelengths between 400 and 500 nm predominantly excite the rR spectra of the b(558) and b(595) chromophores. Spectra obtained within this interval show a mixed population of spin and ligation states arising from b(558) and b(595), With the former more strongly enhanced at higher energy, Red excitation wavelengths (590-650 nm) generate rR spectra characteristic of chlorins, indicating the selective enhancement of the d heme, These rR results reveal that cytochrome bd oxidase ''as isolated'' contains the b(558) heme in a six-coordinate low-spin ferric state, the b(595) heme in a five-coordinate high-spin (5cHS) ferric state, and the d heme in a mixture of oxygenated ((FeO2)-O-II paired left right arrows (FeO2-)-O-III; d(650)) and ferryl-oxo (Fe-IV=O; d(680)) states, However, the rR spectra of these two chlorin species indicate that they are both in the 5cHS state, suggesting that the d heme is lacking a strongly coordinated sixth ligand. Addition of inhibitory concentrations of CN- (10-40 mM) to the as-isolated enzyme leads to loss of the d(650) absorbance of the oxygenated species. Resonance Raman spectra of these cyanide-containing samples have been monitored by 413.1-, 514.5-, and 647.1-nm laser excitation. The data show that b(558) experiences little or no change, whereas b(595) is partially autoreduced to a 5cHS ferrous species. However, neither the remainder of the 5cHS ferric b(595) nor the autoreduced b(595) gives any indication of cyanide binding as judged by rR spectroscopy, even in the presence of 40 mM KCN. Loss of O-2 and electron transfer from the resultant heme d(2+) to b(595)(3+) would generate oxidized heme d and autoreduced b(595) We propose that the resultant heme d is present in a mixture of states, including residual oxygenated (Fe-O-2 stretch at 568 cm(-1)) and ferryl-oxo (Fe=O stretch at 815 cm(-1)) states as well as a cyanide-reacted, oxidized state. The combined spectra, including that of oxidized heme d, indicate the presence of predominantly high-spin, pentacoordinate species. The observation of a cyanide complex that is only five coordinate is consistent with the proposed absence or loss of an endogenous protein ligand to the chlorin cofactor.