Identification and Characterization of Cytochrome P4501A1 Amino Acid Residues Interacting with a Radiolabeled Photoaffinity Diazido-benzphetamine Analogue

The photolabile benzphetamine analogueN-(p- azidobenzyl)-N-methyl-p-azidophenetylamine (N3-BP-N3) and its tritiated derivative were synthesized and used as photoaffinity ligands for P4501A1 substrate binding. The enzymatic activity of P4501A1 toward ethoxycoumarin was competitively inhibited by N3-BP-N3. After irradiation with UV light a radioactive photolysis product remained bound to P4501A1. After large scale labeling in the absence and in the presence of α-naphthoflavone, P-450 was digested with 1-p-tosylamino-2-phenylethyl chloromethyl ketone-treated trypsin and the resultant peptide fragments were separated with HPLC on a reverse-phase column. Six peptides with increased levels of incorporated radioactivity were detected and from a competition experiment in the presence of the inhibitor, four of them could be tentatively assigned as involved in substrate interaction. Amino acid sequences were determined and compared with the primary P-4501A1 sequence. N3-BP-N3 can bind amino acid residues through both ends of the molecule and, therefore, crosslinked peptides could be identified. Alignment of the primary structure of cytochrome P4501A1 with that of cytochrome P450102 revealed that two of the isolated crosslinked peptides can be placed in the vicinity of heme (in the L helix region and β10–β11 sheet region of cytochrome P450102) and could be involved in substrate binding. The other two peptides were located on the surface of the protein with the label bound specifically to Lys residues that were predicted to be involved in reductase–P450 interaction.