Functional and structural studies of cytochrome P450 by resonance Raman spectroscopy
Resonance Raman spectra are reported for both the heme domain and holoenzyme 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, palmitate. Comparison of the spectra of the palmitate-bound form of the heme domain with that of the holoenzyme indicates that the presence of the flavin 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 which is consistent with similar 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, while the binding of substrate induces relatively dramatic changes in the ν(Fe-XY) modes of the ferrous CO, ferric NO and ferrous NO derivatives of cytochrome P450cam, no significant changes are observed for the corresponding derivatives of cytochrome P450BM3 upon binding of palmitate. In fact, the spectral data for substrate-free cytochrome P450BM3 provide evidence for distortion of the Fe-XY fragment, even in the absence of substrate. This apparent distortion, which is non-existent in the case of substrate-free cytochrome P450cam, is most reasonably attributed to interaction of the Fe-XY fragment with the F87 phenylalanine side chain. This residue is known to lie very close to the heme iron in the substrate-free derivative of cytochrome P450 BM3 and has been suggested to prevent hydroxylation of the terminal, ω, position of long chain fatty acids. Resonance Raman spectroscopy is also applied to the cyanide adducts of cytochrome P450cam and its T252A and D251N site directed mutants, both in their substrate-free and camphor-bound forms, in order to probe active site heme structure and, in particular, interactions of the FeCN fragment with potential active H-bond donors. In contrast to the ferrous CO and ferric NO adducts, which form only essentially linear (very slightly distorted) FeXY fragment, clear evidence is obtained in the case of the ferric CN- adducts for existence of an additional, rather highly bent, conformer; i.e., the cyanide complexes form both linear and bent conformers in both the substrate-free and substrate-bound forms. Formation of this bent conformer in the case of the cyanide complexes is most reasonably attributed to the presence of off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments which are poorer H-bond acceptors. Interestingly, for all there proteins, comparison of the observed vibrational spectra indicate that the bent conformer is oriented towards the water filled substrate-binding site in the substrate-free form, but oppositely, toward the proposed proton delivery shuttle, in the substrate-bound form. Sensitivity of the FeCN modes to H2O/D2O exchange in the two camphor-bound mutants, which is apparently absent for the camphor-bound native protein, is attributable to the known presence of extra water in the active sites of these mutants. Finally, the O-O stretching mode of oxy intermediate of D251N cytochrome P450cam was studied. The ν(O-O) was found at 1137 cm-1 , a 2 cm-1 lower than that of wild type oxy complex. In the presence of its redox partner, putidaredoxin, the P(O-O) shifts to 1130 cm-1 . Such down-shift of O-O stretching mode is obtained by previous studies of putidaredoxin-bound CO adduct of cytochrome P450cam.
"Functional and structural studies of cytochrome P450 by resonance Raman spectroscopy"
(January 1, 1999).
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