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-hydroxyprogesterone. 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 domain 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/mRatOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase.
Estabrook
R W
RW
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038, USA.
Shet
M S
MS
Fisher
C W
CW
Jenkins
C M
CM
Waterman
M R
MR
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D004563:Electrochemistry; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005418:Flavodoxin / Q000494:pharmacology; D006900:Hydroxylation; D007700:Kinetics; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011374:Progesterone / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003574:Cytochrome c Group; D005418:Flavodoxin; D011993:Recombinant Fusion Proteins; D011374:Progesterone; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase