Resonance Raman studies of cytochrome P450BM3 and its complexes with exogenous ligands

Resonance Raman spectra are reported for both the heme domain and holoenzym e of cytochrome P450BM3 in the resting state and for the ferric NO, ferrous CO, and ferrous NO adducts in the absence and presence of the substrate, p almitate. Comparison of the spectrum of the palmitate-bound form of the hem e domain with that of the holoenzyme indicates that the presence of the fla vin reductase domain alters the structure of the heme domain in such a way that water accessibility to the distal pocket is greater for the holoenzyme , a result that is consistent with analogous studies of cytochrome P450cam. The data for the exogenous ligand adducts are compared to those previously reported for corresponding derivatives of cytochrome P450cam and document significant and important differences for the two proteins. Specifically, w hile the binding of substrate induces relatively dramatic changes in the nu (Fe-XY) modes of the ferrous CO, ferric NO, and ferrous NO derivatives of c ytochrome P450cam, no significant changes are observed for the correspondin g derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for d istortion of the Fe-XY fragment, even in the absence of substrate. This app arent distortion, which is nonexistent in the case of substrate-free cytoch rome P450cam, is most reasonably attributed to interaction of the Fe-XY fra gment with the F87 phenylalanine side chain. This residue is known to lie v ery close to the heme iron in the substrate-free derivative of cytochrome P 450BM3 and has been suggested to prevent hydroxylation of the terminal, ome ga, position of long-chain fatty acids.