Reconstitution of FMN-free NADPH-cytochrome P-450 reductase with a phosphorothioate analog of FMN: T P NMR studies of the reconstituted protein

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6285870

Krum, D P; Otvos, J D; Calhoun, J P; Miziorko, H M; Masters, B S.S.

A phosphorothioate analog of FMN (FMNS) has been synthesized and shown to be completely competent in reconstituting the FMN-free form of NADPH-cytochrome P-450 reductase as evidenced by flavin determinations and cytochrome c reductase activity assays. The FMNS-reconstituted FMN-free reductase gives rise to an air-stable semiquinone, and the fluorescence of FMNS is quenched upon addition of FMN-free reductase. T P NMR spectra of the FMN-free reductase reveal only two resonances (-7.3 and -11.3 ppm), which are attributable to FAD. This result confirms the assignments of Otvos et al, and demonstrates unequivocally that there are no phosphate residues other than those of FMN and FAD attached to the steapsin-solubilized reductase. The addition of FMN to the FMN-free reductase resulted in the appearance of one additional resonance at 3.9 ppm. Addition of FMNS to the FMN-free reductase caused no change, surprisingly, in the T P NMR spectrum until Mn(II) was added, after which a peak centered at approx. 45 ppm was observed. This unexpected result may be explained if the T1 for the phosphate of FMNS is significantly longer than that of FMN, and suggests that the sulfur atom of FMNS may perturb the interaction of the phosphate with its protein environment. These results demonstrate the utility of phosphorothioate analogs as mechanistic probes for proteins containing nucleotide cofactors.

• Research Organization: Medical Collge of Wisconsin, Milwaukee
• OSTI ID: 6285870
• Report Number(s): CONF-870644-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ENZYME ACTIVITY
ENZYMES
ESTERS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MOLECULAR STRUCTURE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXIDOREDUCTASES
PHOSPHORIC ACID ESTERS
PHOSPHORUS 31
PHOSPHORUS ISOTOPES
RESONANCE
SPECTRA
STABLE ISOTOPES