Site-Directed Mutagenesis of Tyrosine-98 in the Flavodoxin from Desulfovibrio vulgaris (Hildenborough): Regulation of Oxidation-Reduction Properties of the Bound FMN Cofactor by Aromatic, Solvent, and Electrostatic Interactions

Abstract

The contributions made by tyrosine-98 in establishing the redox properties of the flavodoxin from Desulfovibrio vulgaris were investigated by substituting a number of amino acids at this position using site-directed mutagenesis. Tyr98, which makes extensive van der Waals contacts with the isoalloxazine ring of the flavin mononucleotide cofactor, is often found in the cofactor binding site of flavodoxins and related flavoproteins. Solution studies suggest that tyrosine may assist in the stabilization of the neutral flavin semiquinone through preferential complex formation relative to the other oxidation states. In this study, the midpoint potentials of the oxidized/semiquinone couple of the Y98W and Y98F mutants were found to be very similar to the wild-type flavodoxin. However, significantly more negative midpoint potentials (by 25-60 mV) were observed in the Y98A, Y98H, and Y98R mutants. These results imply that it is the general apolar environment provided by the aromatic amino acids rather than preferential affinities suggested by solution studies that is at least partially responsible for the thermodynamic stabilization of the neutral flavin semiquinone in this flavodoxin. The midpoint potential of the semiquinone/hydroquinone couple is profoundly dependent on the properties of the amino acid at this position. Compared to phenylalanine, the more electron-rich aromatic side chains of tryptophan and tyrosine decrease the midpoint potential of this couple by 30-40 mV. Greater solvent exposure of the isoalloxazine ring in the Y98A mutant increases the midpoint potential by 140 mV relative to wild type. The positively charged amino acids increase the midpoint potential of this couple by > 180 mV, most probably through favorable electrostatic interactions with the flavin hydroquinone anion. These observations strongly support the proposition that the functional role of the electron-rich, apolar aromatic amino acid residues adjacent to the flavin isoalloxazine ring is to substantially destabilize the flavin hydroquinone anion, resulting in the very low oxidation-reduction potentials for the semiquinone/hydroquinone couple that typify the flavodoxin family.

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).…”

Redox Properties of Human Medium-Chain Acyl-CoA Dehydrogenase, Modulation by Charged Active-Site Amino Acid Residues

“…The human MCADH appears to be more flexible, and more breathable, which in turn affects the active-site pK a s and redox potential. Second, the difference in potential might result from a variation in the balance of charges in the extended surroundings of the isoalloxazine between the two enzymes, as found by Swenson for flavodoxins (29,30).…”

Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP+ oxidoreductase activity toward NADPH

“…2b, dashed line). In the equivalent Fld mutants, a longwavelength band extending from 500 nm to 700 nm was assigned as a charge transfer band (Swenson and Krey 1994;Lostao et al 1997). In contrast to WT and the other mutants, the absorption coefficients of the FAD transition band II of the Y50W mutant were significantly decreased (Fig.…”

“…In the case of flavodoxins from Desulfovibrio vulgaris and Anabaena PCC7119, the si-face Tyr plays a role in adjusting the stability of the prosthetic group in different redox states (Swenson and Krey 1994;Lostao et al 1997). …”