The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.