ACTIVE-SITE TOPOLOGY OF SACCHAROMYCES-CEREVISIAE LANOSTEROL 14-ALPHA-DEMETHYLASE (CYP51) AND ITS G310D MUTANT (CYTOCHROME-P-450SG1)

Incubation of phenyldiazene (PhN = NH) with lanosterol 14-alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14-alpha-methyl group of lanosterol, results in the appearance of a 478nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (N(C)). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the Nc regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 angstrom high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14-alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the N(A) and N(C) regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.