31P- and 13C-NMR studies on the flavin-protein and flavin-ligand interactions in brewer's yeast old yellow enzyme.
The 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corresponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE(0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C]FMN or [4,10a-13C2]FMN, while apo OYE-I was reconstituted with [4a-13C]FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols.(ABSTRACT TRUNCATED AT 250 WORDS)
Miura
R
R
Yamano
T
T
Miyake
Y
Y
D003605:D-Amino-Acid Oxidase; D005486:Flavin Mononucleotide / Q000032:analysis / Q000378:metabolism*; D005415:Flavins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D009252:NADPH Dehydrogenase / Q000032:analysis*; D010100:Oxygen; D010636:Phenols / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005415:Flavins; D008024:Ligands; D010636:Phenols; D005486:Flavin Mononucleotide; D003605:D-Amino-Acid Oxidase; D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase; C032037:4-bromophenol; D010100:Oxygen