The cumulative electrostatic effect of aromatic stacking interactions and the negative electrostatic environment of the flavin mononucleotide binding site is a major determinant of the reduction potential for the flavodoxin from Desulfovibrio vulgaris [Hildenborough].

Flavodoxins are typified by the very low one-electron reduction potential for the semiquinone/hydroquinone couple (Esq/hq) of the flavin mononucleotide (FMN) cofactor. In the Desulfovibrio vulgaris flavodoxin, the elimination of the side chain of Tyr98, which flanks the outer or si face of the flavin, through the Y98A mutation results in a substantial increase in Esq/hq of 139 mV, representing about one-half of the total shift in Esq/hq in this flavodoxin [Swenson, R. P., & Krey, G. D. (1994) Biochemistry 33, 8505-8514]. The extent to which this large effect was the result of the elimination of unfavorable coplanar aromatic stacking interactions or to the greater solvent exposure of the flavin ring was not known. The significance of the latter effect was heightened by the characterization of the Fld+6 mutant which demonstrated that the unfavorable interaction between the negative electrostatic environment provided by the asymmetric clustering of acidic residues surrounding the cofactor and the FMN hydroquinone anion is responsible for about one-third of the total decrease in Esq/hq in this flavodoxin [Zhou, Z., & Swenson, R. P. (1995) Biochemistry 34, 3183-3192]. In this study, a flavodoxin mutant was generated in which an alanine was substituted for Tyr98 while at the same time the negative electrostatic surface was partially neutralized by the substitution of the six acidic amino acid residues with their amide equivalents. The Esq/hq value of this mutant was found to have increased by 221 mV relative to wild type, which accounts for 70-80% of the total shift in Esq/hq in this flavodoxin. This increase is very similar to the sum of the individual changes in Esq/hq introduced independently in the Y98A and Fld+6 mutants. The similarity in the magnitude of the effect of the neutralization of the six acidic residues in the context of an alanine residue at position 98 (Y98A) relative to an aromatic tyrosine residue (wild type) suggests that the increase in Esq/hq observed for the Y98A mutant is more likely due to the elimination of unfavorable pi-pi interactions between Tyr98 and the FMN hydroquinone rather than to the increased solvent exposure of the flavin. This study provides further support for the concept that the cumulative effect of the unfavorable electrostatic interactions introduced by coplanar aromatic or pi-pi stacking interactions and the negative electrostatic environment of the FMN binding site is a major determinant of the low one-electron reduction potential of the flavodoxin.