G-tensors of the flavin adenine dinucleotide radicals in glucose oxidase: a comparative multifrequency electron paramagnetic resonance and electron-nuclear double resonance study.

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: g x = 2.00429(3), gy = 2.00389(3), g z = 2.00216(3) (X, Y, and Z are the principal axes of g) yielding g iso = 2.00345(3). The g y -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.