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Combining site-directed mutagenesis with analysis of the active-site topology of bovine cholesterol side-chain cleavage cytochrome P450scc (P450scc), we have investigated the roles of tyrosine residues 93 and 94 on substrate binding. Four single mutants (Y93A, Y93S, Y94A, and Y94S) and one double mutant (Y93S/Y94S) were examined. The largest increase in Ks was observed for binding of cholesterol and 25-hydroxycholesterol to the Y94S mutant (approximately 5.5-fold), with a smaller increase (< 2.5-fold) for binding of 22-hydroxycholesterol. Mutation of Y94 thus appears to influence the interaction with cholesterol, 25-hydroxycholesterol, and possibly 22-hydroxycholesterol. Y93 is not involved in binding of 22- and 25-hydroxycholesterol but may interact with cholesterol. The active-site topologies of P450scc and its mutants were probed by reaction with three arylhydrazines. The N-arylprotoporphyrin IX regioisomer patterns obtained with phenyl- and 2-naphthylhydrazine indicate that the active site is primarily open above pyrrole ring A and suggest that a region some distance above pyrrole ring D is also open. The single mutations Y93S, Y93A, Y94A, and Y94S do not detectably alter the regioisomer patterns obtained with the phenyl- and 2-naphthyl probes, but a small, reproducible change is observed with the 2-naphthyl probe for the Y93S/Y94S double mutant. The conformational alteration implied by this change could not be detected by titration with 22- and 25-hydroxycholesterol but is detectable by titration with cholesterol. The results indicate that cholesterol binds over pyrrole ring D of the heme in bovine P450scc, strongly suggest that Y94 interacts with the side chain of cholesterol, and provide evidence that the side chains of 22- and 25-hydroxycholesterol bind to a different region of the active site than the side chain of cholesterol.