The flavin adenine dinucleotide (FAD) cofactor of Aspergillus nigerglucose 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 ghave been established with high precision: gX2.00429(3),gY2.00389(3), gZ2.00216(3) (X, Y, and Zare the principal axes of g) yielding giso2.00345(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 gagainst 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. [ABSTRACT FROM AUTHOR]