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.