NADPH-Flavodoxin reductase and flavodoxin from Escherichia coli:: Characteristics as a soluble microsomal P450 reductase

In addition to their endogenous roles as an activation system for various Escherichia coli metabolic pathways, the soluble flavoproteins flavodoxin (Fld) and NADPH-flavodoxin (ferredoxin) reductase (Fpr) can serve as an electron-transfer system for microsomal cytochrome P450s. Furthermore, since Fld and Fpr are structurally similar to the functional domains (FMN binding and NADPH/FAD binding domains, respectively) of NADPH-cytochrome P450 reductases (P450 reductases), these bacterial proteins represent a potentially useful model system for eukaryotic P450 reductases. Here we delineate similarities and differences between the E. coli Fpr-Fld system and rat P450 reductase as electron donors to bovine 17 alpha-hydroxylase/17,20-lyase P450 (P450c17). Importantly, recombinant Fpr, in combination with recombinant Fld, supports both the hydroxylase and lyase activities of P450c17 to the same proportional extent (hydroxylase-to-lyase ratio) as does P450 reductase. Maximum P450c17 turnover [5-6 mol of 17 alpha-OH-progesterone (mol of P450c17)(-1) min(-1)] was achieved using a large molar excess (50-100-fold over P450c17) of a 1:1 ratio of Fpr-Fld, although this rate was an order of magnitude less than the maximal P350 reductase-supported activity. Using these conditions, we have examined the effects of increasing ionic strength and the presence of cytochrome b(5) (b(5)) on these two systems. Critical Fld-P450c17 electrostatic interactions are disrupted at moderate ionic strength (>100 mM NaCl) as evidenced by significant inhibition (>50%) of Fpr-Fld-supported P450c17 activity while much higher ionic strength (300 mM NaCl) is required to disrupt P450 reductase-P450c17 interactions to the same extent. Interestingly, cytochrome b(5) was found to dramatically inhibit both P450 reductase-and Fpr-Fld-supported P450c17 progesterone 17 alpha-hydroxylase activity while in contrast 17 alpha-OH-pregnenolone lyase activity was stimulated by b(5). Investigation of the fate of reducing equivalents from NADPH added to Fpr under aerobic conditions revealed that the majority of the protein-bound FAD of Fpr is converted to the hydroquinone form. In constrast, the FMN of Fld is reduced by Fpr to a stable blue, neutral semiquinone which serves as the predominant electron donor to P450c17 in reconstitution assays. Thus, while the Fpr-Fld system and P450 reductase are fundamentally different with respect to their electrostatic interactions with P450c17, their ability to support maximal P450c17 turnover, and the FMN redox states (one-electron-reduced for Fld and two-electron-reduced for P450 reductase) capable of transferring electrons to microsomal cytochrome P450s, these differences do not appear to influence the relative catalytic efficiency of the P450c17 hydroxylase and lyase reactions.