Hydrogen-bonding interactions in the active sites of cytochrome P450cam and its site-directed mutants

Resonance Raman spectroscopy is applied to the cyanide adducts of cytochrom e P450cam and its T252A and D251N site-directed mutants, both in their subs trate-free and camphor-bound forms, to probe active-site heme structure and , in particular, interactions of the FeCN fragment with tial active-site H- bond donors. In contrast to the ferrous CO and ferric NO adducts, which fro m only essentially linear (slightly distorted) FeXY fragments, the spectra of the ferric CN- adducts provide clear evidence the for the existence of a n additional, rather highly bent, conformer; that is, the cyanide complexes form both linear and bent conformers in both the substrate-free and substr ate-bound forms. Formation of this bent conformer is most reasonably attrib uted to the presence df off-axis H-bond donors, which induce distortion on the FeCN fragment but not the FeCO and FeNO fragments, which are poorer H-b ond acceptors. For all three proteins, the substrate-free form exhibits a c omplex spectral pattern which arises because one of the modes associated wi th the FeCN fragment is coupled with two heme macrocycle deformation modes. Significantly, no evidence for such coupling is observed in the spectra of the camphor-bound forms. While various unknown factors may possibly give r ise to selective activation of such coupling in the substrate-free derivati ve, given the known facts about the active-site architecture of this enzyme , a plausible explanation is that the bent conformer is oriented toward the water-filled substrate-binding site in the substrate-free form, but opposi tely, toward the proposed proton delivery shuttle, in the substrate-bound f orm. Sensitivity of the FeCN modes to H2O/D2O exchange in the two camphor-b ound mutants, which is apparently absent for the camphor-bound native prote in, is most reasonably attributed to the known presence of extra water in t he active sites of these mutants.