Active site characteristics of CYP4B1 probed with aromatic ligands

The active site topography of rabbit CYP4B1 has been studied relative to CY P2B1 and CYP102 using a variety of aromatic probe substrates. Oxidation of the prochiral substrate cumene by CYP4B1, but not CYP2B1 or CYP102, resulte d in the formation of the thermodynamically disfavored omega -hydroxy metab olite, 2-phenyl-1-propanol, with product stereoselectivity for the (S)-enan tiomer. Reaction of CYP4B1, CYP2B1, and CYP102 with phenyldiazene produced spectroscopically observable sigma -complexes for each enzyme. Subsequent o xidation of the CYP2B1 and CYP102 complexes followed by LC/ESI-MS analysis yielded heme pyrrole migration patterns similar to those in previous litera ture reports. Upon identical treatment, no migration products were detected for CYP4B1. Intramolecular deuterium isotope effects for the benzylic hydr oxylation of o-xylene-alpha-H-2(3), p-xylene-alpha-H-2(3), 2-H-2(3),6-dimet hylnaphthalene, and 4-H-2(3),4'-dimethylbiphenyl were determined for CYP4B1 and CYP2B1 to further map their active site dimensions. These probes permi t assessment of the ease of equilibration, within P450 active sites, of oxi dizable methyl groups located between 3 and 10 Angstrom apart [Iyer et al. (1997) Biochemistry 36, 7136-7143]. Isotope effects for the CYP4B1-mediated benzylic hydroxylation of o- and p-xylenes were fully expressed (k(H)/k(D) = 9.7 and 6.8, respectively), whereas deuterium isotope effects for the na phthyl and biphenyl derivatives were both substantially masked (k(H)/k(D) a pproximate to 1) In contrast, significant suppression of the deuterium isot ope effects for CYP2B1 occurred only with the biphenyl substrate. Therefore , rapid equilibration between two methyl groups more than 6 Angstrom apart is impeded within the active site of CYP4B1, whereas for CYP2B1, equilibrat ion is facile for methyl groups distanced by more than 8 Angstrom. Collecti vely, all data are consistent with the conclusion that the active site of C YP4B1 is considerably restricted relative to CYP2B1.