Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin.

The oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin are substantially different from those of the flavin mononucleotide (FMN) containing native protein, with the midpoint potential for the semiquinone-hydroquinone couple for the riboflavin complex being 180 mV less negative. This increase has been attributed to the absence in the riboflavin complex of unfavorable electrostatic effects of the dianionic 5'-phosphate of the FMN on the stability of the flavin hydroquinone anion. In this study, 15N and 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopic studies demonstrate that when bound to the flavodoxin, (1) the N1 of the riboflavin hydroquinone remains anionic at pH 7.0 so the protonation of the hydroquinone is not responsible for this increase, (2) the N5 position is much more exposed and may be hydrogen bonded to solvent, and (3) that while the hydrogen bonding interaction at the N3H appears stronger, that at the N5H in the reduced riboflavin is substantially weaker than for the native FMN complex. Thus, the higher reduction potential of the riboflavin complex is primarily the consequence of altered interactions with the flavin ring that affect hydrogen bonding with the N5H that disproportionately destabilize the semiquinone state of the riboflavin rather than through the absence of the electrostatic effects of the 5'-phosphate on the hydroquinone state.