The interaction of NADPH-P450 reductase with P450: An electrochemical study of the role of the flavin mononucleotide-binding domain

The electrochemically reduced mediator cobalt sepulchrate requires the presence of a flavoprotein for the rapid transfer of electrons to cytochrome P450;. This electrochemical method has been used here to show the interaction of NADPH-P450 reductase (either the detergent-solubilized form, d-OR, or the proteolytic-cleaved truncated form, t-OR), as well as Escherichia coli flavodoxin (FLD), with P450c17 by measuring the rate of 17 alpha-hydroxylation of progesterone, When NADPH is used as electron donor with a reconstituted system composed of d-OR and P450c17, the? addition of t-OR, flavodoxin, or cytochrome c inhibited the rate of formation of 17 alpha-hydroxyprotesterone, These results suggest the presence of a common protein binding site on the surface of d-OR, t-OR, and flavodoxin which plays a role in the interaction of the flavoproteins with the P450, It is speculated that a do main composed of acidic amino acids, located near the, flavin mononucleotide-binding region of the flavoproteins, may serve as this site, No inhibition by t-OR, flavodoxin, or cytochrome c is observed when comparable experiments are carried out using the artificial recombinant fusion protein rF450[mBov17A/mRa tOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase, (C) 1996 Academic Press, Inc.