Formation, crystal structure, and rearrangement of a cytochrome P-450cam iron-phenyl complex

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Abstract
Cytochrome P-450cam reacts with phenyldiazene (PhN .dbd. NH), or less efficiently with phenylhydrazine, to give a catalytically inactive complex with an absorption maximum at 474 nm. The prosthetic group extracted anaerobically from the inactivated protein has the spectroscopic properties of a .sigma. phenyl-iron complex and rearranges, on exposure to air and acid, to an approximately equal mixture of the four N-phenylprotoporphyrin IX regioisomers. The crystal structure of the intact protein complex, refined at 1.9-.ANG. resolution to an R factor of 20%, confirms that the phenyl group is directly bonded through one of its carbons to the iron atom. The phenyl ring is tilted from the heme normal by about 10.degree. in the opposite direction from that in which carbon monoxide tilts when bound to P-450cam. Camphor, the natural substrate for P-450cam, is larger than a phenyl group and hydrogen bonds to Tyr 96, the only hydrophilic residue near the active site. Electron density in the active site in addition to that contributed by the phenyl group suggests that water molecules occupy part of the camphor binding site but are not within hydrogen-bonding distance of Tyr 96. As observed in a previous crystallographic study of inhibitor-P-450cam complexes [Poulos, T. L., and Howard, A. J. (1987) Biochemistry 26, 8165-8174], there are large changes in both the atomic positions and mobilities of the residues in the proposed substrate access channel region of the protein. Furthermore, both the distal and proximal helix regions appear to be perturbed in the .sigma.-bonded phenyl-iron complex. Unambiguous characterization of the phenyldiazene complex of cytochrome P-450cam makes possible the use of such complexes to probe the active site topologies of membrane-bound cytochrome P-450 enzymes.