Heterobifunctional photoaffinity probes for cytochrome P450 2B

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p-aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylamine (II), and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synthesized and characterized. These probes, containing a photolabile azido-group and an amino-group on opposite sides of the molecule, were designed for photoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity differing in distance from the heme iron. Spectroscopic studies proved that probes I and II coordinated with the heme iron via their amino-group in the enzyme active center, whereas probe III did not. This result in conjunction with data from kinetic studies suggests probes I and II are appropriate for photoaffinity labeling of the CYP 2B active center. Thus, probe II was used to identify amino acid residues within a distance of the probe length (about 16.5 A) from the heme. Analysis of a Lys-C digest of the probe II-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to position 192-197 of the CYP 2B4 sequence. Using postsource decay/matrix-assisted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional structures of bacterial CYPs and in CYP 2B homology models is discussed.