PROPERTIES OF A HIGH-POTENTIAL FLAVIN ANALOG AND ITS USE AS AN ACTIVE-SITE PROBE WITH CLOSTRIDIAL FLAVODOXIN

The reduction potential of flavin bearing a methylsulfonyl moiety (MeS O2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompani ed by a substantial increase in reactivity with various reductants, in cluding NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8(methylsulfonyl)r iboflavin exhibits two intense, well-resolved absorption bands (lambda (max) = 310, 362 nm) in a region where most other reduced flavins exhi bit weak, characterless absorption. This unusual spectrum is attributa ble to a shift of pi-electron density from the N(5) atom into the benz ene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, lik e sulfite, which interferes with sp2 hybridization at N(5). Reaction o f 8-MeSO2-substituted flavins with thiols results in nucleophilic disp lacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-s ubstituted flavins. The flavin ring acts as a redox switch in controll ing electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. I nitial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP . 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorba nce and fluorescence changes similar to those observed for FMN binding . 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lamb da(max) = 323, 382 nm) similar to those of the corresponding free flav in, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, t he spectral properties of 1,5-dihydro-FMN flavodoxin (lambda(max) = 31 1, 362 nm) appear to lie about midway between those observed for the f ree 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi -electron density into the benzene ring of bound 1,5-dihydro-FMN, as c ompared with the free flavin. This hypothesis is consistent with previ ous NMR studies and with a proposal that electron transfer from reduce d flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous th iols but does not react with sulfite. Covalent attachment of 8-MeSO2FM N to the protein, via reaction with a cysteine residue, was not detect ed during prolonged storage of the reconstituted enzyme. The results a re consistent with crystallographic data which show that the 8-positio n of FMN in the native enzyme is accessible to solvent, that solvent a ccess to N(5) is hindered, and that none of the protein's three cystei ne residues is in direct contact with the flavin.