2-Thioflavins as active site probes of flavoproteins.

Several representative flavoproteins have been re- constituted with 2-thioflavin (sulfur replacing oxygen substituent at position 2) at the appropriate FMN, FAD, or riboflavin level. Reconstitution with this modified coenzyme provides a useful probe of the flavin-binding site of the flavoprotein. The visible absorption spectra of free and protein-bound 2-thioflavins undergo char-acteristic changes with pH, reflecting ionization of the flavin N(3) proton. The corresponding pK, values for free as well as various protein-bound 2-thioflavins have been determined. In addition, the 2-thioflavin anion reacts readily with the thiol reagent methyl methanethiolsulfonate (MMTS) to yield the corresponding 2-SSCH3 flavin disulfide; this reaction is accompanied by a substantial change in the visible absorbance spectrum which af-fords a convenient means of monitoring MMTS reactivity with free and protein-bound 2-thioflavins under nondenaturing conditions. the flavin pyrimidine subnuclei in 2-thio p-hydroxybenzoate hydroxylase and 2-thio ri- boflavin-binding protein are accessible to solvent. In 2-thio flavodoxin and the 2-thio p-hydroxybenzoate hy-droxylase-p-hydroxybenzoate complex, however, there is considerable burial of the flavin pyrimidine subnucleus.