Roles of the Proximal Heme Thiolate Ligand in Cytochrome P450cam

• DOI: 10.1021/ja0040262
• Publication Date: 2002-07-26T04:58:40Z
• Authors: Karine Auclair; Pierre Moënne-Loccoz; Paul R. Ortiz de ...
• Keywords: Hemeprotein

To examine the roles of proximal thiolate iron ligand, C357H mutant P450cam (CYP101) was characterized by resonance Raman, UV, circular dichroism, and activity measurements. The must be reconstituted with hemin for to observed. enzyme is a mixture high low spin species. Low temperature (10 °C), concentration (1 μM), camphor mM), 5−50 mM buffer concentrations increase ratio, but under no conditions examined protein more than 60% spin. has poorer Km (23 vs 2 μM) Kd putidaredoxin (50 20 wild-type P450cam. also exhibits greatly decreased oxidation rate, elevated uncoupling much greater peroxidase activity. Electron transfer from slower even though redox potential measurements show that electron remains thermodynamically favored. These experiments confirm ligand facilitates O−O bond cleavage P450 enzymes demonstrate this satisfies important in folding, substrate binding, transfer.