Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activi ty upon refolding due to the formation of aggregates and disulfide-linked m isfolded states unless a large excess of reductant such as 200 mM beta -ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Che m. 265, 5967]. Even then, refolding is incomplete. We have studied the unfo lding and refolding of three rhodanese forms whose crystal structures are k nown: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the ac tive site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in soluti on differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimu m three-state folding model of CMR (N<->I<->U) followed by fluorescence at 363 nm, (N<->I) by fluorescence at 318 nm, and CD (I<->U) is consistent wit h the presence of a thermodynamically stable molten globule intermediate in 5-6 M urea. We conclude that the active-site sulfhydryl group in the persu lfide form is very reactive; therefore, its modification leads to the succe ssful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete rev ersibility of CMR suggests that oxidation among the three non-active-site S H groups can represent a minor trap for refolding through species that can be easily reduced.