31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase

Ramani Narayanasami, James D. Otvos, Charles B. Kasper, Anna Shen, Jayanthi Rajagopalan, Timothy J. McCabe, Janice R. Okita, Donald J. Hanahan, Bettie Sue Siler Masters

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Abstract

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 ∼0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phosphohpids. 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.

Original languageEnglish (US)
Pages (from-to)4210-4218
Number of pages9
JournalBiochemistry
Volume31
Issue number17
StatePublished - 1992

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Flavin Mononucleotide
NADPH-Ferrihemoprotein Reductase
Flavin-Adenine Dinucleotide
Nuclear magnetic resonance
Detergents
Prosthetics
Liver
Phospholipids
Oxidoreductases
Flavins
Tissue
Mutagenesis
Biochemistry
Enzymes
Site-Directed Mutagenesis
Escherichia coli
Nuclear magnetic resonance spectroscopy
Rats
Catalyst activity
Peptide Hydrolases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Narayanasami, R., Otvos, J. D., Kasper, C. B., Shen, A., Rajagopalan, J., McCabe, T. J., ... Masters, B. S. S. (1992). 31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. Biochemistry, 31(17), 4210-4218.

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. / Narayanasami, Ramani; Otvos, James D.; Kasper, Charles B.; Shen, Anna; Rajagopalan, Jayanthi; McCabe, Timothy J.; Okita, Janice R.; Hanahan, Donald J.; Masters, Bettie Sue Siler.

In: Biochemistry, Vol. 31, No. 17, 1992, p. 4210-4218.

Research output: Contribution to journalArticle

Narayanasami, R, Otvos, JD, Kasper, CB, Shen, A, Rajagopalan, J, McCabe, TJ, Okita, JR, Hanahan, DJ & Masters, BSS 1992, '31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase', Biochemistry, vol. 31, no. 17, pp. 4210-4218.
Narayanasami R, Otvos JD, Kasper CB, Shen A, Rajagopalan J, McCabe TJ et al. 31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. Biochemistry. 1992;31(17):4210-4218.
Narayanasami, Ramani ; Otvos, James D. ; Kasper, Charles B. ; Shen, Anna ; Rajagopalan, Jayanthi ; McCabe, Timothy J. ; Okita, Janice R. ; Hanahan, Donald J. ; Masters, Bettie Sue Siler. / 31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. In: Biochemistry. 1992 ; Vol. 31, No. 17. pp. 4210-4218.
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AU - Narayanasami, Ramani

AU - Otvos, James D.

AU - Kasper, Charles B.

AU - Shen, Anna

AU - Rajagopalan, Jayanthi

AU - McCabe, Timothy J.

AU - Okita, Janice R.

AU - Hanahan, Donald J.

AU - Masters, Bettie Sue Siler

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N2 - 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 ∼0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phosphohpids. 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.

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