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.