Resonance Raman Spectroscopic Identification of a Histidine Ligand of b 595 and the Nature of the Ligation of Chlorin d in the Fully Reduced Escherichia coli Cytochrome bd Oxidase

• Published in: Biochemistry (Easton) Vol. 35; no. 7; pp. 2403 - 2412
• Main Authors: Sun, Jie, Kahlow, Michael A, Kaysser, Tamma M, Osborne, Jeffrey P, Hill, John J, Rohlfs, Ronald J, Hille, Russ, Gennis, Robert B, Loehr, Thomas M
• Format: Journal Article
• Language: English
• Published: American Chemical Society 20.02.1996
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9518252

Cytochrome bd oxidase is a bacterial terminal oxidase that contains 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 a binuclear b 595/d site, whereas b 558 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 b 558 (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 b 595 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 b 595 2+ cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b 558 2+ heme. Wavelengths between these values enhance contributions from both b 595 2+ and b 558 2+ chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595−650 nm). The rR data indicate that d 2+ 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 (b 595)FeII−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 b 595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b 595 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 b 595 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 d 2+ 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 b 595 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 d 2+ cyano adduct. These findings corroborate our companion study of the “as-isolated” enzyme in which we proposed a 5cHS d 3+ 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 d 3+ to yield entirely the high-spin form of the cofactor.