PROBING NG THE HEME IRON COORDINATION STRUCTURE OF PRESSURE-INDUCED CYTOCHROME P420(CAM)

Cytochrome P450(cam) was subjected to high pressures of 2.2 kbar, conv erting the enzyme to its inactive form. P420(cam). The resultant prote in was characterized by electron paramagnetic resonance, magnetic circ ular dichroism, circular dichroism, and electronic absorption spectros copy, A range of exogenous ligands has been employed to probe the coor dination structure of P420(cam). The results suggest that conversion t o P420(cam) involves a conformational change which restricts the subst rate binding site and/or alters the ligand access channel, The reducti on potential of P420(cam) is essentially the same in the presence or a bsence of camphor (-211 +/- 10 and -210 +/- 15 mV, respectively), Thus , the well-documented thermodynamic regulation of enzymatic activity f or P450(cam) in which thz reduction potential is coupled to camphor bi nding 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 inve stigation of the spectroscopically related inactive form of chloropero xidase lead to the conclusion that a sulfur-derived proximal ligand is coordinated to the heme of ferric cytochrome P420(cam).