Resonance Raman spectroscopic identification of a histidine ligand of b₅₉₅ and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b₅₅₈), a high-spin heme (b₅₉₅), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b₅₉₅/d site, whereas b₅₅₈ is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b₅₅₈ (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b₅₉₅ and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b₅₉₅²⁺ cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b₅₅₈²⁺ heme. Wavelengths between these values enhance contributions from both b₅₉₅²⁺ and b₅₅₈²⁺ chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d²⁺ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm^-1 to the (b₅₉₅)Fe^II-N(His) stretching vibration, based on its 1.2-cm^-1 upshift in the ⁵⁴Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b₅₉₅ is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b₅₉₅ or d (Fang, H., Lin, R.-J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b₅₉₅ is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In ∼700-fold excess cyanide (∼35 mM), the 629-nm UV/vis band of d²⁺ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different λ₀ (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b₅₉₅ and d remain as 5cHS species in the presence of CN^-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN^- to form the 5cHS d²⁺ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d³⁺ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d³⁺ to yield entirely the high-spin form of the cofactor.