FLUORESCENCE OF THE COENZYME ANALOG NICOTINAMIDE FORMYCIN DINUCLEOTIDE

Abstract— Emission spectra of unbound reduced nicotinamide formycin dinucleotide (NFDH) revealed the presence of two major conformations of the coenzyme in solution: when examined at excitation wavelengths at or below 307 nm, emission spectra contained peaks at 343 and 447 nm; when excited above 307 nm, an additional maximum appeared at 355 nm and the peak of the dihydronicotinamide emission band shifted from 447 to 440 nm. Both conformers are probably detected at the longer wavelengths since the emission peak at 343 nm was retained. Identical changes occurred in the emission spectra of NFD⁺, however, the dihydronicotinamide emission between 440 and 447 nm was absent. Several mechanisms which may account for the presence of these conformers have been considered. The choice has been narrowed to conformations with ring-ring interactions of the formycin and nicotinamide moieties resulting from (a) formycin tautomerization or (b) heterogeneity of glycosidic bond angles in the structures. The efficiency of intramolecular energy transfer from the formycin to the dihydronicotinamide moiety for free NFDH in aqueous solution was 84% and declined slightly (to 77%) when measured in 1,2-propanediol. NFD⁺ has coenzyme activity for NAD-specific isocitrate dehydrogenase (Plaut et al. , 1979). The emission spectrum of enzyme bound NFDH was altered markedly in the presence of manganese isocitrate; emission intensity at 343 and 355 nm decreased while the emission from the dihydronicotinamide ring at 433 nm increased, when NFDH was excited at 310 nm. This shift in emission intensity was indicative of an increase in energy transfer within the NFDH molecule, caused by a change in coenzyme conformation upon binding to the enzyme-substrate complex.