Several representative flavoproteins have been reconstituted 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 characteristic changes with pH, reflecting ionization of the flavin N(3) proton. The corresponding pK, values for free as

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... l 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 affords a convenient means of monitoring MMTS reactivity with free and protein-bound 2-thioflavins under nondenaturing conditions. A Brqnsted basicity-reactivity correlation of these data indicates that the flavin pyrimidine subnuclei in 2-thio p-hydroxybenzoate hydroxylase and 2-thio riboflavin-binding protein are accessible to solvent. In 2thio flavodoxin and the 2-thio p-hydroxybenzoate hydroxylase-p-hydroxybenzoate complex, however, there is considerable burial of the flavin pyrimidine subnucleus. 2-Thio Old Yellow Enzyme, 2-thio lactate oxidase, and 2-thio D-amino acid oxidase react only poorly with MMTS; the flavin pyrimidine subnuclei in these cases must be well buried within their respective protein matrices. The relevance of these findings to recent observations on the three-dimensional structures of some of these proteins is discussed (Burnett, R.