Roles of the proximal heme thiolate ligand in cytochrome P450(cam)

To examine the roles of the proximal thiolate iron ligand, the C357H mutant of P450(cam) (CYP101) was characterized by resonance Raman, UV, circular d ichroism, and activity measurements. The C357H mutant must be reconstituted with hemin for activity to be observed. The reconstituted enzyme is a mixt ure of high and low spin species. Low temperature (10 degreesC). low enzyme concentration (1 muM), high camphor concentration (1 mM). and 5-50 mM buff er concentrations increase the high to low spin ratio, but under no conditi ons examined was the protein more than 60% high spin. The C357H mutant has a poorer K-m for camphor (23 vs 2 muM) and a poorer K-d for putidaredoxin ( 50 vs 20 muM) than wild-type P450(cam). The mutant also exhibits a greatly decreased camphor oxidation rate, elevated uncoupling rate, and much greate r peroxidase activity. Electron transfer from putidaredoxin to the mutant i s much slower than to the wild-type even though redox potential measurement s show that the electron transfer remains thermodynamically favored. These experiments confirm that the thiolate ligand facilitates the O-O bond cleav age by P450 enzymes and also demonstrate that this ligand satisfies importa nt roles in protein folding, substrate binding, and electron transfer.