C-13-NMR OF THE CYANYLATED APOFLAVODOXIN AND FLAVODOXIN FROM CLOSTRIDIUM-PASTEURIANUM

The thiol group of the flavodoxin from Clostridium pasteurianum has be en cyanylated in a single step using [cyanato-C-13]2-nitro-5-thiocyana tobenzoic acid. This chemical modification increases the dissociation constant of the apoflavodoxin-FMN complex 10-fold from 0.33 +/- 0.15 n M to 2.9 +/- 1.3 nM. The thiocyanate carbons of the cyanylated cystein e residue of apoflavodoxin and flavodoxin had chemical shift values of 114.7 and 112.3 p.p.m. respectively. From these chemical shifts we co nclude that the binding of FMN by the cyanylated apoflavodoxin causes a decrease in the polarity and/or hydrogen bonding capacity of the env ironment of the thiocyanate group. We compare these results with those obtained from similar studies on the cyanylated apoflavodoxin and fla vodoxin from Megasphaera elsdenii and we discuss how FMN binding and c yanylation perturb the structures of apoflavodoxins from Megasphaera e lsdenii and Clostridium pasteurianum.