Replacement of active-site cysteine-436 by serine converts cytochrome P4502B4 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 NH2-truncated P450s 2114 and 2E1. The expressed Ser-436 variant of P450 2134 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 H2O2 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 2134: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, I-phenylethanol, and 4-fluorophenol, and has only marginal 4-nitrophenol aromatic hydroxylation activity. H2O2-supported peroxidation of guaiacol and pyrogallol is comparable with 2134 and mutant C436S and negligible relative to the turnover of peroxidases with these substrates. Neither 2134 nor 2134:C436S catalyzes H2O2 decomposition. It is concluded that replacement of active-site Cys-436 by Ser converts P450 2114 mainly into a 2-electron oxidase. (C) 2002 Published by Elsevier Science Inc.