The molecular mechanism by which the homodimeric enzyme Cu͞Zn superoxide dismutase (SOD) causes neural damage in amytrophic lateral sclerosis is yet poorly understood. A striking, as well as an unusual, feature of SOD is that it maintains intrasubunit disulfide bonds in the reducing environment of the cytosol. Here, we investigate the role of these disulfide bonds in folding and assembly of the SOD apo protein (apoSOD) homodimer through extensive protein engineering. The results show that apoSOD folds in a simple three-state process by means of two kinetic barriers: 2Dº2MºM 2. The early predominant barrier represents folding of the monomers (M), and the late barrier the assembly of the dimer (M 2). Unique for this mechanism is a dependence of protein concentration on the unfolding rate constant under physiological conditions, which disappears above 6 M Urea where the transition state for unfolding shifts to first-order dissociation of the dimer in accordance with Hammond-postulate behavior. Although reduction of the intrasubunit disulfide bond C57-C146 is not critical for folding of the apoSOD monomer, it has a pronounced effect on its stability and abolishes subsequent dimerization. Thus, impaired ability to form, or retain, the C57-C146 bond in vivo is predicted to increase the cellular load of marginally stable apoSOD monomers, which may have implications for the amytrophic lateral sclerosis neuropathology.protein folding ͉ protein stability ͉ disulfide bond ͉ transition-state shifts ͉ protein engineering T he mechanism by which mutant superoxide dismutase (SOD) leads to neural damage in the familial form of amyotrophic lateral sclerosis (ALS) is yet unknown (1-3). In analogy with other neurodegenerative disorders (4), however, an increasing body of observations (5-16) suggest that the ALS disease mechanism is coupled to destabilization or misfolding of the SOD structure (17), manifested ultimately by cellular inclusions of SOD aggregates (3,18). From a strictly energetic perspective, the native SOD structure is distinctive by containing one oxidized disulfide bond per monomer (C57-C146) in the reducing environment of the cytosol (19) (Fig. 1). Normally, disulfide bonds are maintained only under oxidizing conditions in the extracellular space (20, 21) where they increase protein stability by confining the configurational entropy of the denatured ensemble (22). Indications that the integrity of the C57-C146 bond may have bearing on the molecular events in ALS were recently provided by Tiwari and Hayward (15), who demonstrated that ALS-associated SOD mutants are more susceptible to chemical disulfide reduction than the wild-type protein. More detailed elucidation of the structural and energetic effects accompanying mutational perturbations and disulfide reduction has so far been prevented by scarce knowledge about how the SOD homodimer folds (23).In this study, we shed further light on this issue by mapping out the folding and assembly reaction of metal-depleted SOD through kinetic and thermodynamic analysis of...