X-ray analyses of wild-type and mutant flavodoxins from Clostridium beijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxidation state of the FMN prosthetic group. The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen of Gly57 (O57) points toward the flavin ring; trans O-down, in which O57 points away from the flavin; and cis O-down. Interconversions among these conformers that are linked to oxidation-reduction of the flavin can modulate the redox potentials of bound FMN. In the semiquinone and reduced forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up conformation and accepts a hydrogen bond from the flavin N5H [Smith, W. W., Burnett, R. M., Darling, G. D., & Ludwig, M. L. (1977) J. Mol. Biol. 117, 195-225; Ludwig, M. L., & Luschinsky, C. L. (1992) in Chemistry and Biochemistry of Flavoenzymes III (Müller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL]. Analyses reported in this paper confirm that, in crystals of wild-type oxidized C. beijerinckii flavodoxin, the Gly57-Asp58 peptide adopts the O-down orientation and isomerizes to the cis conformation. This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137. Structures for the mutant Asn137Ala indicate that a mixture of all three conformers, mostly O-down, exists in oxidized C. beijerinckii flavodoxin in the absence of intermolecular hydrogen bonds. Redox potentials have been manipulated by substitutions that alter the conformational energies of the bend at 56M-G-D-E. The mutation Asp58Pro was constructed to study a case where energies for cis-trans conversion would be different from that of wild type. Intermolecular interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when the flavin is oxidized. Reduction of the flavin induces rearrangement to the trans O-up conformation. Redox potential shifts reflect the altered energies associated with the peptide rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol). Further, the results of mutation of Gly57 agree with predictions that a side chain at residue 57 should make addition of the first electron more difficult, by raising the energy of the O-up conformer that forms when the flavin is reduced to its semiquinone state. The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by approximately 60 mV (1.3 kcal/mol). Introduction of the beta-branched threonine side chain at position 57 has much larger effects on the conformations and potentials. The Thr57-Asp58 peptide adopts a trans O-down conformation when the flavin is oxidized; upon reduction to the semiquinone, the 57-58 peptide rotates to a trans O-up conformation resembling that found in the wild-type protein. Changes in FMN-protein interactions and in conformational equilibria in G57T combine to decrease the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol) and to inc...

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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.

The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor. These proteins function as low-potential one-electron transferases in bacteria. A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function. The structural basis of this phenomenon is not yet thoroughly understood. In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated. These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor. Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate. Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations. There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted. However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type. These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution. Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion.

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.