FLAVODOXIN AND NADPH-FLAVODOXIN REDUCTASE FROM ESCHERICHIA-COLI SUPPORT BOVINE CYTOCHROME P450C17 HYDROXYLASE-ACTIVITIES

Two soluble flavoproteins, purified from Escherichia coli cytosol and identified as flavodoxin and NADPH flavodoxin (ferredoxin) reductase ( flavodoxin reductase), have been found in combination to support the 1 7 alpha-hydroxylase activities of heterologously expressed bovine 17 a lpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics o f the two flavoproteins including absorbance spectra, molecular weight s, and amino terminal sequences are identical with those reported prev iously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin red uctase, possessing FAD as a cofactor, is able to reconstitute P450c17 activities only in the presence of flavodoxin, an FMN containing prote in, and NAD(P)H. Reducing equivalents are utilized more effectively fr om NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P 450c17 directly and with relatively high affinity (apparent K-s simila r to 0.2 mu M) at low ionic strength, as evidenced by a change in spin state of the P450c17 heme iron upon titration with flavodoxin. This a pparent spin shift is attenuated at moderate ionic strengths (100-200 mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Se pharose in an ionic strength dependent manner. These data implicate ch arge pairing as being important for the interaction between flavodoxin and P450c17. We propose that the amino acid sequence similarity betwe en E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD, and NAD(P)H binding regions of cytochrome P450 reductase provides the basis for the reconstitution of P450c17 activities by this bacterial s ystem.