Identification of neutral and anionic 8 alpha-substituted flavin semiquinones in flavoproteins by electron spin resonance spectroscopy

Electron paramagnetic resonance spectral studies of protein-bound 8α-substituted FMN neutral semiquinones (in a complex with Azotobacter apoflavodoxin) show peak-to-peak linewidths (~ 20–23 G) similar to those of normal flavoenzymes which form neutral semiquinones. The anionic form of the 8α-substituted flavin semiquinone of thiamine dehydrogenase shows a narrower peak-to-peak linewidth (12 G) than normal anionic flavin semiquinones (14–15 G). The electron paramagnetic resonance spectral properties of the 8α-N(3)-histidyl FAD semiquinone of succinate dehydrogenase showed a peak-to-peak linewidth of 12 G through the pH range of 6.1 to 9.1 and remained unchanged whether the buffer medium was H2O or D2O. From these observations, it is concluded that the covalent flavin semiquinone in succinate dehydrogenase exists in the anionic (red) form.