Cytochrome P450cam was subjected to high pressures of 2.2 kbar, converting the enzyme to its inactive form, P420cam. 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 P420cam. The results suggest that conversion to P420cam involves a conformational change which restricts the substrate binding site and/or alters the ligand access channel. The reduction potential of P420cam 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 P450cam in which the reduction potential is coupled to camphor binding is not found with P420cam. Further, cyanide binds more tightly to P420cam (kd = 1.1 ± 0.1 mM) than to P450cam (kd = 4.6 ±0.2 mM), reflecting a weakened iron-sulfur ligation. Spectral evidence reported herein for P420cam 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 P420cam.
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