Heme binding and substrate-protected cysteine residues in P-450cam

Reactions of the Pseudomonas putida cytochrome P-450-substrate complex or enzyme alone with 14C-labeled iodoacetic acid have been investigated at pH 7.0. After subsequent conversion of all of the cysteine residues to S-beta-carboxymethylcysteinyl residues, tryptic peptides of the derivative were separated by either high performance liquid chromatography or two dimensional electrophoresis, and their amino acid compositions and partial sequences were determined. All but cysteine residue-134 reacted to some extent. This result implicated residue-134 as the thiol group which is the axial heme ligand since the heme was intact in all of the derivatives made. Reaction of the enzyme-substrate complex with "cold" iodoacetic acid followed by substrate removal and reaction with 14C-labeled iodoacetic acid resulted in radiolabeling of mainly cysteine-240. This suggested cysteine-240 to be an active site cysteine residue.