Probing the heme iron coordination structure of pressure-induced cytochrome P420_cam

Cytochrome P450_cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form, P420_cam. The resultant protein was characterized by electron paramagnetic resonance, magnetic circular dichroism, circular dichroism, and electronic absorption spectroscopy. A range of exogenous ligands has been employed to probe the coordination structure of P420_cam. The results suggest that conversion to P420_cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420_cam is essentially the same in the presence or absence of camphor (-211 ±10 and -210 ±15 mV, respectively). Thus, the well-documented thermodynamic regulation of enzymatic activity for P450_cam in which the reduction potential is coupled to camphor binding is not found with P420_cam. Further, cyanide binds more tightly to P420_cam (k_d = 1.1 ± 0.1 mM) than to P450_cam (k_d = 4.6 ±0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420_cam as well as results from a parallel investigation of the spectroscopically related inactive form of chloroperoxidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420_cam.