Chemical modification of the N-10 ribityl side chain of flavins. Effects on properties of flavoprotein disulfide oxidoreductases.

Three flavin derivatives modified at the 2'-position of the flavin N-10 ribityl side chain were synthesized: arabinoflavin, 2'-F-2'-deoxyarabinoflavin, and 2'-deoxyriboflavin. These were converted to the FAD level with FAD synthetase. Apoproteins of lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, a family of flavoprotein oxidoreductases, were reconstituted with these flavins. Significant reduction of the catalytic activities was observed with the modified enzymes. During anaerobic reduction of the modified enzymes with substrate or dithiothreitol, decreased thermodynamic stability of the two-electron reduced enzyme forms (EH2) and the accumulation of the four-electron reduced forms (EH4) noted. This effect was more pronounced in case of arabino-FAD-reconstituted enzymes than with the other two. It was found that NAD+ binding influences the interaction between the flavin and the reduced disulfide in the 2'-F-arabino-FAD-lipoamide dehydrogenase, presumably by altering the relative oxidation-reduction potentials. 19F NMR data were obtained for different forms of the 2'-F-arabino-FAD-lipoamide dehydrogenase, which suggest marked conformational changes from one form to the other. The 19F NMR data for the oxidized forms of all three 2'-F-arabino-FAD proteins suggest that the fluorine experiences very similar chemical environments at the active sites.