Effects of substituting asparagine for glycine-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough).

Gly-61 in flavodoxin from Desulfovibrio vulgaris (Hildenborough) has been changed to Asn by site-directed mutagenesis of the cloned gene. Values determined for the dissociation constant for the dissociation of the mutant protein into apoprotein and FMN, and for the redox potentials of the two 1-electron steps in the reduction of the bound flavin showed that FMN in all three redox states is bound more weakly in the mutant protein than in the wild-type flavodoxin. However, the greatest effect was on the semiquinone, for which Kd is 920 times larger in the mutant. The side-chain of Asn-61 in the mutant may hinder a redox-linked conformational change that occurs in this region of the protein, and which is thought to lead to formation of a hydrogen bond between N(5)H of FMNH and the backbone carbonyl group of amino acid-61.