Open AccessNuclear magnetic resonance studies of the old yellow enzyme
Author(s) -
BEINERT WolfDieter,
RÜTERJANS Heinz,
MÜLLER Franz
Publication year - 1985
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1985.tb09234.x
Subject(s) - chemistry , ring (chemistry) , crystallography , flavin group , hydrogen bond , flavin mononucleotide , resonance (particle physics) , hydrogen atom , covalent bond , stereochemistry , enzyme , molecule , organic chemistry , atomic physics , group (periodic table) , physics
The apoenzyme of NADPH oxidoreductase, ‘old yellow enzyme’, was reconstituted with specifically 15 N‐labeled flavin mononucleotide and investigated by 15 N 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 sp 3 ‐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 sp 3 ‐hybridized. This interpretation can also be derived from the 15 N(5)‐ 1 H 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.