title: 15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger.
abstract: The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.
authors.affiliation: Institut für Biophysikalische Chemie, Universität Frankfurt, Federal Republic of Germany.
authors.forename: C
authors.initials: C
authors.lastname: Sanner
authors.forename: P
authors.initials: P
authors.lastname: Macheroux
authors.forename: H
authors.initials: H
authors.lastname: Rüterjans
authors.forename: F
authors.initials: F
authors.lastname: Müller
authors.forename: A
authors.initials: A
authors.lastname: Bacher
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology*; D005949:Glucose Oxidase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction
chemical_list: D005949:Glucose Oxidase