The functional role of glutamate-59 in the modulation of the oxidation-reduction potentials of the Clostridium beijerinckii flavodoxin

The structural simplicity of flavodoxin makes it a good model system in which to investigate how the oxidation-reduction properties of the flavin cofactor are modulated in more complex flavoproteins. The role of the hydrogen bonding interaction with the N(3)H of the cofactor in the regulation of the redox potentials is not well understood. In the Clostridium beijerinckii flavodoxin, the ycarboxylate group of Glu59 serves as a dual "bridging" hydrogen bond acceptor between an adjacent polypeptide backbone amide group and the N(3)H of the flavin mononucleotide cofactor (FMN) in all three oxidation states. The disruption of this interaction through the placement of Glu59 with glutamine, asparagines, aspartate, or alanine resulted in the weakening of binding of FMN for each replacement, particularly for the semiquinone state, affecting the midpoint potentials of both couples in opposite directions. In addition, the glutamine and alanine substitutions had very little apparent affect on the pH dependency for the midpoint potential of the semiquinone/hydroquinone couple, suggesting the Glu59 is not the single redox-linked group responsible for the observed pH dependency as had been previously proposed. 15 N NMR and 1H15N HSQC NMR studies indicated that not only the N(3)H interaction, but also the hydrogen bonding interaction between the backbone carbonyl of Gly57 and N(5)H are weakened by each of the Glu59 substitutions. Using the temperature coefficients for the N(5)H values to correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each FMN redox state was isolated. It was observed that this interaction was most important in the stability of the oxidized state, followed by the semiquinone state, and finally the fully reduced state, providing roughly a 20--25 mV contribution to the lowering of the midpoint potentials for each redox couple. It is concluded that Glu59 primarily affects the redox properties of the C. beijerinckii flavodoxin through the anchoring of the FMN to the 50's loop, promoting the formation of proper flavin-protein contacts, particularly at N(5)H, that serve to stabilize the semiquinone rather than through the effects transmitted directly via the interaction with N(3)H itself.