Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity.
The function of the unique axial thiolate ligand of cytochrome P450 has been investigated by mutagenesis of the active-site cysteine with other amino acids in NH(2)-truncated P450s 2B4 and 2E1. The expressed Ser-436 variant of P450 2B4 was highly purified but incurred considerable heme loss. The pyridine hemochrome spectrum of C436S is characteristic of protoporphyrin IX, and the absolute spectra display Soret maxima at 405 nm (ferric), 422 nm (ferrous), and 413 nm (ferrous CO). 2B4:C436S catalyzes the NADPH- and time-dependent formation of H(2)O(2) in the reconstituted enzyme system, with maximal rates at approximately equimolar amounts of P450 reductase and C436S hemeprotein. The 2-electron oxidase activity with saturating reductase is directly proportional to the concentration of 2B4:C436S, and the turnover is 60-70% of that of the wild-type enzyme. In contrast, the C436S variant is devoid of oxygenase activity with typical substrates such as d-benzphetamine, 1-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H(2)O(2)-supported peroxidation of guaiacol and pyrogallol is comparable with 2B4 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2B4 nor 2B4:C436S catalyzes H(2)O(2) decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2B4 mainly into a 2-electron oxidase.
Vatsis
Kostas P
KP
Department of Biological Chemistry, 5440 Medical Science Building I, Medical School, The University of Michigan, Ann Arbor 48109-0606, USA. kvatsis@umich.edu
Peng
Hwei-Ming
HM
Coon
Minor J
MJ
D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine; D000072467:Cytochrome P450 Family 2; D006861:Hydrogen Peroxide / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012694:Serine; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D006861:Hydrogen Peroxide; D010544:Peroxidases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine