A Residue Critical for Flavin Binding in Flavocytochrome b2 from Baker's Yeast

The reagent 2-keto-3-butynoic acid is the product formed in the reaction between the suicide reagent 2-hydroxy-3-butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin-free flavocytochrome b2 by 2-keto-3-butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue. We also describe in this paper a simple enzymatic method for preparing 2-keto-3-butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2-keto-3-butynoate with other proteins.