¹⁵N‐ and ¹³C‐NMR investigations of glucose oxidase from Aspergillus niger

Abstract

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically lSN-and 13C-enriched FAD derivatives and investigated by "N-and I3C-NMR spectroscopy. On the basis of the "N-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 spz 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 I3C-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(l) position is anionic at pH 5.6. The pK, 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

Catalysis of electron transfer during activation of O 2 by the flavoprotein glucose oxidase

“…A recent mechanistic study by Xu and Gunner (61) of photosynthetic reaction centers proposes that electron transfer between quinones is gated by a pH-dependent conformational change. With regard to GO, x-ray studies at pH 5.6 and 7.4 show nearly identical active-site conformations consistent with the majority of enzyme existing in the E(FADH Ϫ , HisH ϩ ) form (50). At elevated pH where the catalytic His-516 is deprotonated, loss of this key charge is likely to cause an increase in conformational flexibility at the active site and, consequently, an increase in the reorganization energy for electron transfer.…”

“…A one-electron reduction potential for [E(FADH • , HisH ϩ )] of E°Ј ϭ Ϫ0.065 V vs. NHE has been determined at pH 5.3. This value was originally attributed to a proton-coupled process because at the time of measurement it was not known that FADH Ϫ existed as an anion at this pH (50). The reported E°Ј ϭ Ϫ0.242 V vs. NHE at pH 9.3 corresponds to a proton-coupled reduction.…”

Influence of Flavin Analogue Structure on the Catalytic Activities and Flavinylation Reactions of Recombinant Human Liver Monoamine Oxidases A and B

“…Apoglucose oxidase was prepared from 80 mg (total weight) of the Aspergillus niger holoenzyme (Sigma type V) as described (26) with the following changes. Apoenzyme eluting from the desalting column was collected in 3-ml fractions into 9 ml of 0.5 M potassium phosphate, pH 7.0.…”

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

“…In Aspergillus niger glucose oxidase, an increase in sp 3 hybridization of N(5) was observed for the reduced enzyme. 25 Besides APSR, the only other crystal structure available of a flavoprotein flavin N(5)-sulfite adduct is that of alditol oxidase (AldO; PDB ID: 2VFV 26 ). The enzyme has the FAD 8-methyl group covalently linked to His46 N δ1 , and the deposited model shows a heavily distorted isoalloxazine ring with an unusual conformation at the N(5) locus, as well as N(5) almost fully sp 2 hybridized.…”