Resonance Raman detection of bound dioxygen in cytochrome P-450cam.

We have used resonance Raman spectroscopy and isotopic labeling techniques to unambiguously assign the dioxygen stretching frequency (vo-o) in the substrate-bound oxygenated complex of cytochrome P-450cam. The frequency found for Vo-o in the P-450cam system (1140 cm-1) is in remarkable agreement with recent studies of thiolate heme model compounds. The general features of the oxy-P-450cam Raman spectra are tabulated and comparisons are made with the oxy complexes of hemoglobin, myoglobin, and various model compounds. Most of the results are qualitatively explained by consideration of electron donation into the pi g (O2)/d pi (M) orbitals of the oxygenated complex (M = Fe or Co). It is also noted that the effect of the "extra" electron in the nitrogen base Co(II) oxy complexes, in some ways, parallels the effect of the lone pair electrons of thiolate in the oxy-P-450cam complex. This is evidenced by the enhanced resonance Raman activity of vo-o in both the Co(II) and P-450 systems as well as by the similarity of the vo-o frequencies.

Resonance Raman studies of isotopically labeled chloroperoxidase.

Chloroperoxidase (CPO) and cytochrome P450cam have been shown by several techniques to have similar active site properties. Recent resonance Raman investigations using isotopically enriched 34S-labeled samples have demonstrated thiolate ligation in the P450cam system. We report here on a number of parallel studies involving CPO. On the basis of isotopic labeling (34S, 13CO), we assign the Fe-S and Fe-CO stretching frequencies of CPO at 347 (-vFe-S) and 488 cm-1 (-vFe-CO). The differences of the -vFe-S and -vFe-CO in CPO and P450cam may suggest subtle differences in the thiolate binding in the two systems.