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.
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.
Department of Biochemistry, Ohio State University, Columbus 43210-1292.
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Swenson
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Krey
D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D016969:Desulfovibrio vulgaris / Q000737:chemistry*; D004355:Drug Stability; D004563:Electrochemistry; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011809:Quinones / Q000737:chemistry / Q000378:metabolism; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D005418:Flavodoxin; D011809:Quinones; D012997:Solvents; D014443:Tyrosine; D005486:Flavin Mononucleotide