RHODANESE CONFORMATIONAL-CHANGES PERMIT OXIDATION TO GIVE DISULFIDES THAT FORM IN A KINETICALLY DETERMINED SEQUENCE

When the structure of the monomeric enzyme rhodanese is perturbed by u rea or SDS and heat, sulfhydryl assays combined with SDS gel analyses reveal that intrachain disulfides are formed rapidly. Two intrachain d isulfide bonded species can be distinguished. One contains a single di sulfide and comigrates on SDS gels with fully reduced rhodanese (Band I), while a second species contains two disulfides and migrates faster than the reduced enzyme (Band II). The kinetic path and identity of t he participating sulfhydryl groups are suggested by the results with s ulfhydryl mutants. On mild oxidation or perturbation, a single disulfi de forms that involves two of the three sulfhydryl groups in C-termina l domain of the protein, i.e., two of the sulfhydryl groups from among the three residues: the active-site Cys-247, Cys-254 and Cys-263. The se disulfides are the same as those that are formed upon oxidation of the native enzyme. The remaining sulfhydryl group of these three, in a kinetically slower process, can form a disulfide with Cys-63 which is in the N-terminal domain in native rhodanese. The resulting looped st ructure is so conformationally constrained that its shape and/or alter ed SDS binding gives rise to the 'fast' Band II on the SDS gels. The c onformationally constrained species with two disulfides may be related to oxidized rhodanese species that are difficult to reduce.