TY - JOUR
T1 - The interaction of NADPH-P450 reductase with P450
T2 - An electrochemical study of the role of the flavin mononucleotide-binding domain
AU - Estabrook, Ronald W.
AU - Shet, Manjunath S.
AU - Fisher, Charles W.
AU - Jenkins, Christopher M.
AU - Waterman, Michael R.
N1 - Funding Information:
1This work was supported in part by grants from the National Institutes of Health (GM16488, GM37942, and ES07628). 2To whom correspondence should be addressed.
PY - 1996/9/1
Y1 - 1996/9/1
N2 - 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 17α-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 17α-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/mRa-tOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase.
AB - 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 17α-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 17α-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/mRa-tOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase.
UR - http://www.scopus.com/inward/record.url?scp=0030249221&partnerID=8YFLogxK
U2 - 10.1006/abbi.1996.0395
DO - 10.1006/abbi.1996.0395
M3 - Article
C2 - 8806785
AN - SCOPUS:0030249221
SN - 0003-9861
VL - 333
SP - 308
EP - 315
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -