Nuclear magnetic resonance studies of the old yellow enzyme. 1. 15N NMR of the enzyme recombined with 15N-labeled flavin mononucleotides.
The apoenzyme of NADPH oxidoreductase, 'old yellow enzyme', was reconstituted with specifically 15N-labeled flavin mononucleotide and investigated by 15N NMR spectroscopy in the oxidized and reduced state. The results indicate that in the oxidized state a hydrogen bond is formed between the N(5) atom and the apoprotein. In addition, hydrogen bonds exist between the N(1) and N(3) atoms of FMN and the apoprotein. The resonance position of N(10) indicates that this atom is somewhat sp3-hybridized, i.e. lifted out of the molecular plane of the isoalloxazine ring system. In the reduced state the N(1) atom is negatively charged and the N(3) atom forms a hydrogen bond with the apoprotein. The N(10) atom in protein-bound FMN exhibits about the same hybridization state as in free anionic reduced FMN, i.e. it is located in the plane of the isoalloxazine ring. The chemical shift of the N(5) resonance indicates that this atom is almost completely sp3-hybridized. This interpretation can also be derived from the 15N(5)-1H coupling constant. Among the flavoproteins thus far studied by NMR techniques, old yellow enzyme is the only protein that shows a conformation of the reduced prosthetic group with the N(5) atom lifted out of the molecular plane. The isoelectric focussing properties of old yellow enzyme and a new easy method for the preparation of the apoprotein are also reported.
D001051:Apoenzymes / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004563:Electrochemistry; D005486:Flavin Mononucleotide / Q000032:analysis*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D009252:NADPH Dehydrogenase / Q000032:analysis*; D009587:Nitrogen Isotopes; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation
D001051:Apoenzymes; D009587:Nitrogen Isotopes; D005486:Flavin Mononucleotide; D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase