Insights into the active site of the cytochrome P-450 haemoprotein family--a unifying concept based on structural considerations.

Abstract

1. Cytochromes P-450CAM of Pseudomonas putida and P-450LM-2 of rabbit liver, and to a lesser extent also adrenocortical mitochondrial cytochromes P-450(11) beta and P-450SCC, were investigated by active site-targeted reagents and by immunochemical techniques. The results of these studies and of the alignment of the amino acid sequences of cytochromes P-450CAM and of phenobarbital-induced rat-liver P-450LM support the following conclusions. 2. Cytochrome P-450 haemoproteins follow a common architectural design, most readily apparent in the surroundings of the haem group. Sequence homology and associated folding leads to immunochemical similarities revealed by radioimmune assay. 3. The haem domain is partially exposed on the surface of the protein. This part appears to be tightly structured from several distinct portions of the polypeptide chain which carry the haem catalytic site and the substrate binding site, respectively. 4. After photocovalent labelling with substrate- or inhibitor-derived arylazido reagents, a substantial portion of the haem domain may be excised by BrCN or formic acid and purified without loss of haem ('haemopeptide'). A major antibody-binding site is associated with this fragment. 5. Exploration of the haem pocket of cytochrome P-450CAM, using bromoacetyl and mercapto derivatives of its substrate, camphor, revealed an activated SH group responsible for binding and precision alignment of camphor toward the haem iron, presumably by forming a transient thiohemiketal bond. This cysteine may also function in translocation of nascent product for facilitated release. Specific terpenoid substrates of other cytochrome P-450 haemoproteins may be bound in a similar manner. 6. From radiolabelled peptide studies the substrate-binding cysteine was identified as residue 56 in the amino acid sequence of cytochrome P-450CAM. The haemchelating cysteine occupies position 134 or 146 in cytochrome P-450CAM and position 152 or 173 in cytochrome P-450LM, respectively.