FUNGAL P450NOR - EXPRESSION IN ESCHERICHIA-COLI AND SITE-DIRECTED MUTAGENESES AT THE PUTATIVE DISTAL REGION

P450nor, nitric oxide reductase from Fusarium oxysporum, was expressed in the soluble fraction of Escherichia coli cells by modifying the N- terminal codons and utilizing the pCW vector. The modified P450nor pur ified to electrophoretic homogeneity had spectral and enzymatic proper ties identical to those of native P450nor obtained from the fungi. Res idues 239 to 247 of the modified P450nor were replaced with Lys by sit e-directed mutagenesis. Thr-243 to His- and Arg-mutants were also crea ted. Among II mutants, only the Thr-243 to Lys-mutant exhibited an abs orption spectrum characteristic of a nitrogenous ligand-bound form of P450 at pH 8.0 in the ferric state, but the spectrum was altered to th at of the wild-type P450nor as the pH was lowered Other mutants had sp ectra typical of the low- and high-spin mixed form of P450 in the ferr ic state. In the ferrous state, all mutants showed the same spectrum a s the wild-type P450nor. Nitric oxide reductase activity was considera bly decreased by the replacement of Thr-243 with Lys, His, or Arg or A la-239 with Lys. These findings indicate that Thr-243 is located more closely to the heme iron than other residues in the putative distal he lix of P450nor and plays an important role in the catalytic activity, but a specific difference in the structure of the heme pocket from oth er P450s is suggested. (C) 1997 Academic Press