Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin

The reduction potential of flavin bearing a methylsulfonyl moiety (MeS02) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (1 03-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8- (methylsulfony1)riboflavin exhibits two intense, well-resolved absorption bands (A,,, = 3 10, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of *-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeS02, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeS02-substituted flavins with thiols results in nucleophilic displacement of MeS02- in a reaction that is about 1 03-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeS02-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeS02FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeS02FMN flavodoxin exhibits spectral properties (A,,, = 323,382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5- dihydro-FMN flavodoxin (A,,, = 31 1, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeS02FMN. This suggests that the protein environment may favor enhanced resonance delocalization of *-electron density into the benzene ring of bound 1,5-dihydro- FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeS02FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite. Covalent attachment of 8-MeS02FMN to the protein, via reaction with a cysteine residue, was not detected during prolonged storage of the reconstituted enzyme. The results are consistent with crystallographic data which show that the 8-position of FMN in the native enzyme is accessible to solvent, that solvent access to N(5) is hindered, and that none of the protein's three cysteine residues is in direct contact with the flavin.