Structural changes caused by site-directed mutagenesis of tyrosine-98 in Desulfovibrio vulgaris flavodoxin delineated by 1H and 15N NMR spectroscopy: implications for redox potential modulation.

Flavodoxins mediate electron transfer at low redox potential between the prosthetic groups of other proteins. Interactions between the protein and the flavin mononucleotide cofactor shift both the oxidized/semiquinone and semiquinone/hydroquinone redox potentials significantly from their free-in-solution values. In order to investigate the possible role that the tyrosine at position 98 plays in this process, we have used heteronuclear three-dimensional NMR spectroscopy to determine the solution conformation of wild-type and four position-98 mutants, Y98W, Y98H, Y98A, and Y98R, of Desulfovibrio vulgaris flavodoxin. Assigned 1H and 15N resonances indicate that the secondary structure and topology of the proteins are identical. However, residues that undergo substantial mutation-induced changes in chemical shift are spread throughout the flavin cofactor binding site. Distance and dihedral angle constraints were used to generate solution structures for the wild-type and mutant proteins. Collectively, the mutant proteins have no gross conformational changes in the flavin binding site. The changes that do occur are minor and result from the different packing interactions required to accommodate the new side chain at position-98. The solvent accessibility and electrostatic nature of the flavin binding site in the mutant proteins are compared to those of the wild-type structure. The structural data support the hypothesis that the very low midpoint of the semiquinone/hydroquinone couple in the wild-type protein is modulated to a large extent by the energetically unfavorable formation of the flavin hydroquinone anion in the apolar environment of the flavin binding site.