/sup 31/P NMR evidence for localization of the semiquinone radical on the FMN moiety of NADPH-cytochrome P-450 reductase

Journal Article · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:7025132

Krum, D P; Otvos, J D; Masters, B S.S.

The formation of air-stable semiquinone of NADPH-cytochrome P-450 reductase, the only mammalian flavoprotein known to contain both FAD and FMN, was first reported by Masters et al. /sup 31/P NMR spectra were recorded (101.2 MHz) at 10+/-2/sup 0/C with a 0.82s pulse repetition rate for the acquisition of 90-170,000 transients per spectrum. Oxidized reductase showed five resolved resonances, four of which correspond to tightly-bound FAD and FMN. Upfield resonances at -7.33 (T/sub 1/ = 3.6s) and at -11.25 (T/sub 1/ = 6.5s)ppm are characteristic of FAD and downfield resonances at 3.98 and 4.40 (T/sub 1/ = 3.0s)ppm are attributed to FMN. The fifth resonance, at 1.70 (T/sub 1/ = 2.9s)ppm, is attributed to 2'-AMP which binds tightly to the enzyme and results from the preparative procedure. The reductase semiquinone was generated by NADPH, NADH or photoreduction under anaerobic conditions to permit stoichiometric reduction and minimize reoxidation of the flavoprotein over the 20-39-hour data acquisition period. The FMN signals of the semiquinone generated by three different reductants were line broadened while the FAD resonances were unaffected. These results represent the first direct evidence for localization of the free radical of the air-stable semiquinone in the FMN moiety and support the earlier studies of Vermilion and Coon. In addition, the accessibility of the /sup 31/P-containing moieties was probed with Mn.

• Research Organization: Medical College of Wisconsin, Milwaukee
• OSTI ID: 7025132
• Report Number(s): CONF-8606151-
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
• Country of Publication: United States
• Language: English

Related Subjects

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550600 -- Medicine
59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
AMP
AROMATICS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL LOCALIZATION
COENZYMES
CYTOCHROMES
ELEMENTS
ENZYMES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAGNETIC RESONANCE
MANGANESE
METALS
NADH2
NADP
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
OXIDOREDUCTASES
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
PIGMENTS
PROTEINS
QUINONES
RADICALS
REACTION KINETICS
RESONANCE
SPECTRA
STABLE ISOTOPES
TRANSITION ELEMENTS