Heterobifunctional photoaffinity probes for cytochrome P4502B

Three heterobifunctional photoaffinity probes, N-(p-azidobenzyl)-N-methyl-p -aminobenzylamine (I), N-(p-azidobenzyl)-N-methyl-p-aminophenethylaine (II) , and N-(p-azidophenethyl)-N-methyl-p-aminophenethylamine (III), were synth esized and characterized. These probes, containing a photolabile azido-grou p and an amino-group on opposite sides of the molecule, were designed for p hotoaffinty labeling of the cytochrome P450 (CYP) 2B active site cavity dif fering in distance from the heme iron. Spectroscopic studies proved that pr obes I and II coordinated with the heme iron via their amino-group in the e nzyme 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 use d to identify amino acid residues within a distance of the probe length (ab out 16.5 Angstrom) from the heme. Analysis of a Lys-C-digest of the probe I I-labeled CYP 2B4 revealed a single labeled hexapeptide corresponding to po sition 192-197 of the CYP 2B4 sequence. Using post-source decay/matrix-assi sted laser desorption ionization-time of flight, Arg197 was identified as a probe II target. The location of the labeled site in three-dimensional str uctures of bacterial CYPs and in CYP 2B homology models is discussed. (C) 1 999 Academic Press.