STRUCTURAL-CHANGES CAUSED BY SITE-DIRECTED MUTAGENESIS OF TYROSINE-98IN DESULFOVIBRIO-VULGARIS FLAVODOXIN DELINEATED BY H-1 AND N-15 NMR-SPECTROSCOPY - IMPLICATIONS FOR REDOX POTENTIAL MODULATION

Flavodoxins mediate electron transfer at low redox potential between t he prosthetic groups of other proteins. Interactions between the prote in and the flavin mononucleotide cofactor shift both the oxidized/semi quinone and semiquinone/hydroquinone redox potentials significantly fr om their free-insolution values. In order to investigate the possible role that the tyrosine at position 98 plays in this process, we have u sed 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 H-1 and N-15 resonances indicate that the secondary structure and topo logy of the proteins are identical. However, residues that undergo sub stantial mutation-induced changes in chemical shift are spread through out the flavin cofactor binding site. Distance and dihedral angle cons traints were used to generate solution structures for the wild-type an d mutant proteins. Collectively, the mutant proteins have no gross con formational changes in the flavin binding site. The changes that do oc cur are minor and result from the different packing interactions requi red to accommodate the new side chain at position-98. The solvent acce ssibility and electrostatic nature of the flavin binding site in the m utant proteins are compared to those of the wild-type structure. The s tructural data support the hypothesis that the very low midpoint of th e semiquinone/hydroquinone couple in the wild-type protein is modulate d to a large extent by the energetically unfavorable formation of the flavin hydroquinone anion in the apolar environment of the flavin bind ing site.