THE CUMULATIVE ELECTROSTATIC EFFECT OF AROMATIC STACKING INTERACTIONSAND 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 potent ial for the semiquinone/hydroquinone couple (E(sq/hq)) of the flavin m ononucleotide (FMN) cofactor. In the Desulfovibrio vulgar is flavodoxi n, the elimination of the side chain of Tyr98, which flunks the outer or si face of the flavin, through the Y98A mutation results in a subst antial increase in E(sq/hq) of 139 mV, representing about one-half of the total shift in E(sq/hq) in this flavodoxin [Swenson, R, P., & Krey , G. D. (1994) Biochemistry 33, 8505-8514]. The extent to which this l arge effect was the result of the elimination of unfavorable coplanar aromatic stacking interactions or to the greater solvent exposure of t he flavin ring was not known. The significance of the latter effect wa s heightened by the characterization of the Fld(+6) mutant which demon strated that the unfavorable interaction between the negative electros tatic environment provided by the asymmetric clustering of acidic resi dues surrounding the cofactor and the FMN hydroquinone anion is respon sible for about one-third of the total decrease in E(sq/hq) in this fl avodoxin [Zhou. Z., & Swenson, R. P. (1995) Biochemistry 34, 3183-3192 ]. In this study, a flavodoxin mutant was generated in which an alanin e was substituted for Tyr98 while at the same time the negative electr ostatic surface was partially neutralized by the substitution of the s ix acidic amino acid residues with their amide equivalents. The E(sq/h q) 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 E(sq/hq ) in this flavodoxin. This increase is very similar to the sum of the individual changes in E(sq/hq) introduced independently in the Y98A an d Fld(+6) mutants. The similarity in the magnitude of the effect of th e neutralization of the six acidic residues in the context of an alani ne residue at position 98 (Y98A) relative to an aromatic tyrosine resi due (wild type) suggests that the increase in E(sq/hq) observed for th e Y98A mutant is mon likely due to the elimination of unfavorable pi-p i interactions between Tyr98 and the FR IN hydroquinone rather than to the increased solvent exposure of the flavin. This study provides fur ther support for the concept that the cumulative effect of the unfavor able electrostatic interactions introduced by coplanar aromatic pi-pi stacking interactions and the negative electrostatic environment of th e FMN binding site is a major determinant of the low one-electron redu ction potential of the flavodoxin.