CYSTEINE-254 CAN COOPERATE WITH ACTIVE-SITE CYSTEINE-247 IN REACTIVATION OF 5,5'-DITHIOBIS(2-NITROBENZOIC ACID)-INACTIVATED RHODANESE AS DETERMINED BY SITE-DIRECTED MUTAGENESIS

Sulfhydryl substitution mutants of rhodanese (thiosulfate:cyanide sulf urtransferase; EC 2.8.1.1) were used to determine whether the 4 cystei ne residues in the native structure could cooperate in reactions. The sulfhydryl reactivity of persulfide-containing (ES) rhodanese was not significantly changed when cysteine residues at positions 63, 254, and 263 were replaced by serine, either individually or in combination. H owever, the sulfhydryl reactivity of persulfide-free (E) rhodanese was enhanced when Cys-254 was mutated. One sulfhydryl group, presumably t he active site Cys-247, reacted rapidly in the E forms of these protei ns with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4'-dipyridyl d isulfide (4-PDS). After reaction with DTNB or 4-PDS, proteins with Cys -254 retained >95% of their original activities as compared with Ser-2 54-containing proteins, which retained <6% of their activities. Cyanid e treatment could release thionitrobenzoate from rhodanese-thionitrobe nzoate complexes with an approximate 1:1 stoichiometry. After this tre atment, only the wild-type and C263S enzymes were fully active. Cyanid e-treated rhodanese-thionitrobenzoate complexes of the C254S and C254S /C263S mutants could be fully reactivated using an exogenously added t hiol, beta-mercaptoethanol. These results are consistent with the form ation of a Cys-247-thiocyano derivative that is inactive but capable o f being reactivated by intramolecular transfer of cyanide to Cys-254. In the absence of Cys 254, beta-mercaptoethanol can serve as the trans ferring sulfhydryl group.