31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.
31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.
Narayanasami
R
R
Department of Biochemistry, University of Texas Health Science Center-San Antonio 78284-7760.
Otvos
J D
JD
Kasper
C B
CB
Shen
A
A
Rajagopalan
J
J
McCabe
T J
TJ
Okita
J R
JR
Hanahan
D J
DJ
Masters
B S
BS
D000818:Animals; D003001:Cloning, Molecular; D003902:Detergents; D004926:Escherichia coli / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese; D016297:Mutagenesis, Site-Directed; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry; D051381:Rats; D012995:Solubility; D013552:Swine
D003902:Detergents; D010743:Phospholipids; D008345:Manganese; D009251:NADPH-Ferrihemoprotein Reductase