RESONANCE RAMAN-SPECTRA OF THE NITRIC-OXIDE ADDUCTS OF FERROUS CYTOCHROME P450CAM IN THE PRESENCE OF VARIOUS SUBSTRATES

Resonance Raman spectra of the nitric oxide adducts of ferrous cytochrome P450cam (from Pseudomonas putida) in the adamantanone-, camphor-, and norcamphor-bound forms, as well as in the substrate-free form, are reported. The lowered frequency of the nu-4 (1372 cm-1) and nu-3 (1499 cm-1) modes is consistent with the presence of endogenous thiolate ligation. The three expected normal modes of the natural abundance nitric oxide adduct of ferrous cytochrome P450cam are detected at 1591, 554, and 446 cm-1 and are assigned to nu(N-O), nu(Fe(II)-NO), and delta(Fe(II)-NO), respectively, based on a normal mode analysis, although extensive mixing of the latter two modes is indicated. The very strong band at 554 cm-1 shifts to 539 ((NO)-N-15-O-16), 552 ((NO)-N-14-O-18), and 538 cm-1 ((NO)-N-15-O-18) as the mass of NO increases incrementally by one atomic unit, while the 446-cm-1 feature exhibits a monotonous downshift to 442 ((NO)-N-15-O-16), and 440 ((NO)-N-14-O-18, and 437 cm-1 ((NO)-N-15-O-18). The substrate sensitivity of these two bands, distinctly different from those observed for the intrinsically linear Fe(II)-CO (Uno, T., et al. J. Biol. Chem. 1985, 260, 2023-2026) and Fe(III)-NO (Hu, S.; Kincaid, J. J. Am. Chem. Soc. 1991, 113, 2843-2850) adducts, is discussed in relation to the enzyme-catalyzed regio- and stereospecific hydroxylation of camphor.