BACKBONE DYNAMICS OF OXIDIZED AND REDUCED D-VULGARIS FLAVODOXIN IN SOLUTION

Recombinant Desulfovibrio vulgaris flavodoxin was produced in Escheric hia coli. A complete backbone NMR assignment for the two-electron redu ced protein revealed significant changes of chemical shift values comp ared to the oxidized protein, in particular for the flavine mononucleo tide (FMN)-binding site. A comparison of home-and heteronuclear NOESY spectra for the two redox slates led to the assumption that reduction is not accompanied by significant changes of the global fold of the pr otein. The backbone dynamics of both the oxidized and reduced forms of D. vulgaris flavodoxin were investigated using two-dimensional N-15-H -1 correlation NMR spectroscopy. T-1, T-2 and NOE data are obtained fo r 95% of the backbone amide groups in both redox states. These values were analysed in terms of the 'model-free' approach introduced by Lipa ri and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4559, 4559-4570]. A comparison of the two redox states indicates that in the reduced speci es significantly more flexibility occurs in the two loop regions enclo sing FMN. Also, a higher amplitude of local motion could be found for the N(3)H group of FMN bound to the reduced protein compared to the ox idized state.