Structural and Functional Diversity of Cytochrome P450

Abstract

The elucidation of the crystal structure of P450cam has greatly advanced site-directed mutagenesis and other biochemical studies of P450. However, the proton supply system in the P450 active site was elusive for many years. In 1997, the crystal structure of the ferric substrate (6-deoxyerythronolide B)-bound P450eryF that is involved in erythromycin biosynthesis was solved. A water molecule that was presumed to be a proton donor to the heme-bound dioxygen molecule was found in the active site of P450eryF. Two water molecules were also found in the first crystal structure of P450cam-O₂. Two mutant P450cam-O₂ structures and other studies revealed that the I-helix Thr, which is a crucial structural feature for dioxygen activation, is not a proton donor, although it is a key residue for cleaving the O–O bond. No water molecules were found around the heme-bound O₂ in the P450eryF-O₂ structure. Therefore, the proton supply systems for P450eryF and P450cam are different and depend on the substrate and substrate cavity.

Although most P450s catalyze monooxygenation reactions, there are some exceptions. One example is P450 StaP, which is a key enzyme for forming the core structure of an anticancer agent, indolocarbazole, which catalyzes the intramolecular C–C bond formation in chromopyrrolic acid (CPA). The crystal structure of CPA-bound P450 StaP and theoretical calculations of the active species of this enzyme suggest that C–C bond formation occurs via an indole cation radical intermediate that is equivalent to cytochrome c peroxidase compound I. In addition to the indole ring of CPA, water molecules and His250 are also important for the P450 StaP C–C coupling reaction.