Abstract
Ferricyanide oxidation of the aryl-iron complexes formed by the reaction of cytochrome P450 enzymes with arylhydrazines causes in situ migration of the aryl group from the iron to the porphyrin nitrogen atoms. The regiochemistry of this migration, defined by the ratio of the four possible N-arylprotoporphyrin IX isomers, provides a method for mapping the topologies of cytochrome P450 active sites. The method has been validated by using it to examine the active site of cytochrome P450cam (CYP101), for which a crystal structure is available. In agreement with the crystal structure, reaction with phenylhydrazine gives a 5:25:70 ratio of the NA:NC:ND (subscript indicates pyrrole ring) N-phenylprotoporphyrin IX isomers. Naphthylhydrazine, however, yields exclusively the NC regioisomer and 4-(phenyl)phenylhydrazine the NA:NC:ND isomers in a 14:40:46 ratio. These isomer ratio differences are readily explained by topological differences between the upper and lower reaches of the active site. Having validated the aryl-iron shift as a topological probe, we used it to investigate the structural changes caused by mutation of Phe-87, a residue that provides the ceiling over pyrrole ring D in the crystal structure of cytochrome P450cam. Mutation of Phe-87 to a tryptophan causes no detectable change in the regiochemistry of camphor hydroxylation and only minor changes in the N-aryl isomer ratios. However, mutation of Phe-87 to an alanine, which was expected to open up the region above pyrrole ring D, severely decreased the proportion of the ND in favor of the NA isomer. Less rather than more space is therefore available over pyrrole ring D in the F87A mutant despite the fact that the regiochemistry of camphor hydroxylation remains unchanged. These results provide evidence for significant structural reorganization in the upper regions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant.
Highlights
From the $Department of Pharmaceutical Chemistry, Universityof California, S u n Francisco, California 94143-0446 and the §Department of Biochemistry, University of Texas Southwestern Medical Center,Dallas, Texas 75235-9038
Ferricyanide oxidation of the aryl-iron complexes Membrane-bound cytochrome P450 enzymes play crucial formed by the reaction of cytochrome P450 enzymes rolesinavariety of biochemical processes, including the with arylhydrazines causes in situ migration of the biosynthesis of moststerols and the catabolismof fatty acids and virtually all atoms
The purpose of the present study was to determine ( a ) whether the topological information deduced from the isomer distributions obtained with a range of arylhydrazines reflects the topology of a known cytochrome P450 active site; [6] whether the probes could be used to examine the active site structural changes caused by site-specific mutagenesis of active site residues; and (c) the topological sensitivity of the active site of cytochrome P450, to mutations of Phe-87, one of the residues that define the active site
Summary
From the $Department of Pharmaceutical Chemistry, Universityof California, S u n Francisco, California 94143-0446 and the §Department of Biochemistry, University of Texas Southwestern Medical Center,Dallas, Texas 75235-9038. Lessrather thanmore spaceis avail- 11).This method involves reaction of the cytochrome P450 able over pyrrole ring D in the F87A mutant despite enzymeswith arylhydrazinesto give aryl-iron complexes, the fact that the regiochemistorfycamphor hydroxyl- followed by ferricyanide-induced in situ migration of the aryl ation remains unchanged. These results provide evidence for significant structural reorganization in the upperregions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant
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