NEGATIVELY CHARGED ANABAENA FLAVODOXIN RESIDUES (ASP(144) AND GLU(145)) ARE IMPORTANT FOR RECONSTITUTION OF CYTOCHROME-P450 17-ALPHA-HYDROXYLASE ACTIVITY

Catalysis by microsomal cytochromes P450 requires the membrane-bound e nzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfer s electrons to the P450 heme via a flavodoxin-like domain, Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electro n transfer protein, directly interacts with bovine cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C. M., and Waterman, M. R. (1994) J. Biol. Chem. 269, 27401-27408). To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena, Mutagenesis of two a cidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17 alpha-hydroxylase activit y, Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activ ity. P450-dependent hydrogen peroxide/superoxide production by wild-ty pe FNR-Fld was measurably higher than that generated by FNR-D144A or F NR E145A, indicating that the mutations do not lead to P450 heme-media ted electron uncoupling, Interestingly, the D144A and E145A mutants bi nd with equal or even greater affinity to P450c17 than wild-type Fld, Furthermore, these mutations (D144A and E145A) actually increased cyto chrome c reductase activity (35 and 100% higher than wild type), Anaba ena Fld residues Asp(144) and Glu(145) align closely with rat P450 red uctase residue Asp(208), which has been shown by mutagenesis to be imp ortant in electron transfer to P4502B1 but not to cytochrome c (Shen, A. L., and Rasper, C. B. (1995) J. Biol. Chem. 270, 27475-27480). Thus , these residues in flavodoxins and P450 reductase appear to have simi lar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-bi nding domain of P450 reductase.