Identification of neutral and anionic 8α-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 H 2O or D 2O. From these observations, it is concluded that the covalent flavin semiquinone in succinate dehydrogenase exists in the anionic (red) form.