Two-dimensional NMR studies of the flavin binding site of Desulfovibrio vulgaris flavodoxin in its three redox states.

The riboflavin 5'-monophosphate (FMN) binding site of Desulfovibrio vulgaris flavodoxin in the diamagnetic oxidized and two-electron reduced form was investigated using two-dimensional proton NMR. The NMR results are compared to existing X-ray crystallographic data. In the paramagnetic one-electron reduced redox state resonances of protons which are close to the FMN ring are strongly broadened due to the paramagnetic properties of the flavin ring. From comparison of the NMR spectra of the three redox states it could be concluded that outside the FMN binding site no structural changes occur upon reduction. Strong hydrogen bonds are observed between the N(1) and C(2) carbonyl of the isoalloxazine ring and the amide protons of D95 and C102, respectively. The amide resonances of D95 and C102 are strongly downfield shifted upon two-electron reduction, caused by the negative charge in the N(1)-C(2) carbonyl region in the two-electron reduced FMN. It is suggested that the ring current of the central pyrazine ring of the FMN molecule in the two-electron reduced flavodoxin is decreased compared to the oxidized flavodoxin. The decrease in ring current is apparently caused by the loss of aromaticity of this pyrazine moiety due to protonation of N(5). Strong hydrogen bonds between the flavin phosphate group and amide and hydroxyl protons of the apoprotein are observed. Resonances of protons involved in this hydrogen bonding network are downfield shifted up to 3.5 ppm. It is suggested that the negative charges of the dianionic FMN phosphate group are stabilized by local peptide dipoles. On reduction of the protein from the oxidized to the one-electron reduced form, a conformational change occurs in the FMN binding region. No conformational change can be observed between the one-electron and the two-electron reduced state.