Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450(cam)

To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450(cam), we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new prepara tion procedure involving in vitro folding of P450(cam) from the inclusion b odies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm(-1), indicating a neutral hi stidine residue as the axial ligand. However, another internal ligand is co ordinated to the heme iron as the sixth ligand in the ferric and ferrous fo rms of the C357H mutant, suggesting the collapse of the substrate-binding s ite. The C357H mutant showed no catalytic activity for camphor hydroxylatio n and the reduced heterolytic/homolytic ratio of the O-O bond scission in t he reaction with cumene hydroperoxide. The present observations indicate th at the thiolate coordination in P450(cam) is important for the construction of the heme pocket and the heterolysis of the O-O bond.