G-Tensors of the Flavin Adenine Dinucleotide Radicals in Glucose Oxidase:  A Comparative Multifrequency Electron Paramagnetic Resonance and Electron−Nuclear Double Resonance Study

Abstract

The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD*-) and neutral (FADH*) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD*- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX=2.00429(3), gY=2.00389(3), gZ=2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso=2.00345(3). The gY-component of FAD*- from GO is moderately shifted upon deprotonation of FADH*, rendering the g-tensor of FAD*- slightly more axially symmetric as compared to that of FADH*. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH* radical into the anionic FAD*- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.

The Electronic State of Flavoproteins: Investigations with Proton Electron–Nuclear Double Resonance

“…2, pulsed proton X-band ENDOR spectra from different flavoproteins are presented. More specifically, data from Xenopus laevis (6–4) photolyase ( Xl 64PL) [26], Synechocystis species cryptochrome-DASH ( Ss Cry), Thermus thermophilus cyclobutane pyrimidine dimer (CPD) photolyase ( Tt CPDPL), Arabidopsis thaliana CPD photolyase ( At CPDPL), Arabidopsis thaliana cryptochrome 1 ( At Cry1) [20], Drosophila melanogaster cryptochrome ( Dm Cry) [13], Desulfovibrio vulgaris flavodoxin ( Dv FD) and Aspergillus niger glucose oxidase ( An GO) [10], the latter measured at two proton concentrations, at pH 5 and 10, have been compiled. On the left side of Fig.…”

“…The investigated protein samples were recombinantly expressed and purified according to the published procedures [10, 13, 20–24]. To prepare the radical state of the flavin cofactor, purified protein samples were supplemented with 5 mM ethylenediaminetetraacetic acid or 5 mM dithiothreitol, illuminated with blue light (Halolux 30HL, Streppel, Wermelskirchen-Tente, Germany) filtered by a 420–470-nm band filter (Schott, Mainz) at 4°C for times adequate for radical generation.…”

New roles of flavoproteins in molecular cell biology: Blue‐light active flavoproteins studied by electron paramagnetic resonance

“… High magnetic field/high microwave frequency continuous‐wave EPR spectra (first derivatives) of various flavin radicals. Left, 360.04 GHz EPR spectrum of the stable anionic FAD radical of Aspergillus niger glucose oxidase (pH 10) recorded at 140 K [35]. Middle, 360.03 GHz EPR spectrum of the neutral FAD radical of E. coli CPD photolyase [30].…”

“… Principal values of the g ‐tensor of flavin anion and neutral radicals. The values listed here were compiled from recent EPR experiments on flavoproteins using microwave frequencies of at least 90 GHz [30,31,33–36]. For the neutral FAD radical, the principal axes X , Y and Z of the g ‐tensor were determined with respect to the molecular axes of the 7,8‐dimethyl isoalloxazine moiety [30,32].…”

“… ENDOR spectroscopy on neutral versus anionic flavin radicals. Pulsed ENDOR spectra of the flavin radical in Aspergillus niger glucose oxidase obtained at pH 10 (upper, anionic radical) and pH 5 (lower, neutral radical), recorded at 80 K [35]. …”