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]

Abstract

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.

Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones

“…4A), indicating that the Sq functions as a redox cofactor in the scaffold of MtrC. This conclusion is in harmony with the Sq stabilization in flavodoxins as a redox cofactor, which is reported for a model FMN-binding protein (24,30). In flavodoxins the E p of the Ox/Sq couple is generally shifted to more positive potential from that of FMN in aqueous solution as high as −50 mV (30), and it has been demonstrated by sitedirected mutagenesis that the E p shift in flavodoxins in Desulfovibrio vulgaris is promoted by the nonpolar environments of the protein pocket and π-π stacking interactions with aromatic amino acids (24).…”

“…2B, trace 1), which is consistent with an n of 2 (21). When fully oxidized flavin (Ox) is reduced in one-and two-electron reactions, the resulting flavin derivatives are called semiquinone (Sq) and hydroquinone (Hq) species, respectively (22)(23)(24), as shown in [1] and [2]:…”

The Roles of Two Amino Acid Residues in the Active Site of l-Lactate Monooxygenase

“…The present data are thus entirely consistent with the close proximity of arginine 187 to the flavin. Indeed, the effects of the present mutation on the redox properties of the flavin are comparable to the effects of a series of mutations made of flavodoxin from Desulfovibrio vulgaris (33,34). Tryosine 98 in flavodoxin from D. vulgaris is known to make extensive van der Waals contacts with the isoalloxazine ring of the FMN cofactor.…”

Influence of the Protein Environment on the Redox Potentials of Flavodoxins from Clostridium beijerinckii

“…Likewise, the present study also demonstrates that the low E1 value in the C. beijerinckii flavodoxin is a result of the absence of solvation in the protein dielectric environment rather than any specific negatively charged groups ( Table 2). This may also hold true for the significant increase in the E1 value (140 mV) upon mutation of Tyr-98 with Ala in the D. vulgaris flavodoxin (37). The balky residue Tyr-98 has a van der Waals contact with the FMN ring (38).…”