PMID:8806785
| Citation |
Estabrook, RW, Shet, MS, Fisher, CW, Jenkins, CM and Waterman, MR (1996) The interaction of NADPH-P450 reductase with P450: an electrochemical study of the role of the flavin mononucleotide-binding domain. Arch. Biochem. Biophys. 333:308-15 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1006/abbi.1996.0395 |
| Keywords |
Amino Acid Sequence; Binding Sites/genetics; Cytochrome P-450 Enzyme System/chemistry; Cytochrome P-450 Enzyme System/metabolism; Cytochrome c Group/metabolism; Electrochemistry; Electron Transport; Escherichia coli/genetics; Escherichia coli/metabolism; Flavin Mononucleotide/chemistry; Flavin Mononucleotide/metabolism; Flavodoxin/pharmacology; Hydroxylation; Kinetics; Molecular Sequence Data; NADPH-Ferrihemoprotein Reductase/chemistry; NADPH-Ferrihemoprotein Reductase/genetics; NADPH-Ferrihemoprotein Reductase/metabolism; Progesterone/chemistry; Progesterone/metabolism; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism |
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