Bba 66291 Studies on Flavin Binding in Flavodoxins

I. Stable apoproteins have been prepared from Peptostreptococcus elsdenii, C. pasteurianum and Clostridium MP flavodoxins by dialysis of the native proteins against 2 M KBr at pH 3.9 and 3" lO-4 M EDTA. The apoproteins each bind I molecule of FMN to give complexes identical with the native flavodoxins. 2. Binding causes almost complete quenching of both protein and FMN fluorescence. This property has been used to determine the dissociation constant for the dissociation of P. elsdenii flavodoxin into apoprotein and FMN, and to follow the kinetics of the interaction. The dissociation constant determined at pH 6.55 and 20 ° was 4.26" lO -l° M. The maximum rate of binding occurs at pH 4.4 and is described by a second order rate constant of 2.6.1o 5 M -1 .cm -1 at 0.5 °. The rate is markedly influenced by the salt composition of the solution. 3. Addition of excess apoprotein to FAD, riboflavin or lumiflavin causes only a small decrease in the flavin fluorescence, suggesting that binding of these compounds is weak. 4-Two derivatives of FMN, iso-FMN and 3,4-dihydro FMN form strong, complexes with apoflavodoxin. In the case of iso-FMN and P. elsdenii apoflavodoxin the complex is catalytically active, and it is reduced by systems which reduce native flavodoxin. The complex of 3,4-dihydro FMN is catalytically inactive. 5. C. pasteurianum, P. elsdenii and Clostridium MP flavodoxins contain I, 2 and 3 sulflaydryl groups, respectively. Experiments with p-chloromercuribenzoate and N-ethylmaleimide indicate that at least one sulflaydryl group is important for flavin binding.