Folding of human superoxide dismutase: disulfide reduction prevents dimerization and produces marginally stable monomers

Abstract

The molecular mechanism by which the homodimeric enzyme Cu/Zn 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: 2D<==>2M<==>M(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.

Fig. 1.

Structural comparison of the SOD homodimer and monomer. (Left) Structure of the human SOD homodimer (1SPD), showing the positions of the cysteines, the loop containing residues 49–84, and the Cu and Zn ions. (Right) Loop displacements upon dissociation of the SOD homodimer (illustrated on the right-hand-side monomer), obtained by structural superposition of holoSOD^wt (blue) (51) and holoSOD^50/51/E133Q (orange) (41).

Fig. 2.

Size-exclusion chromatograms showing the dimer/monomer composition of wild-type and mutant SOD with and without the C57–C146 disulfide linkage. Dimers elute at 10–11 ml and folded monomers at 12 ml. (a) Oxidized apoSOD^wt (black), reduced apoSOD^wt (red), and oxidized apoSOD^50/51 (blue). (b) Oxidized apoSOD^wt (black), fully cysteine-depleted apoSOD (red), and fully cysteine-depleted holoSOD (blue). (c) Oxidized apoSOD^6/111 (black) and reduced apoSOD^6/111 (red). AU, absorbance unit.

Scheme 1.

Fig. 4.

Stability histogram for 27 single-domain proteins adapted from ref. . The apparent stabilities of the reduced apoSOD^wt monomer and the native holoSOD^wt dimer are found on the extreme sides of the distribution.

Fig. 3.

Chevron plots of wild-type and mutant SOD monomers display simple two-state folding behavior. Units are in s^–1. (a) Reduced apoSOD^wt (•) and reduced apoSOD^50/51 (○). (b) Reduced apoSOD^wt (•), reduced apoSOD^C6A (▵), reduced apoSOD^C111A (▴), and reduced apoSOD^6/111 (▾). (c) Reduced apoSOD^50/51/6/111 (□) and oxidized apoSOD^50/51/6/111 (▪).

Fig. 5.

Chevron plots for the apoSOD^6/111 dimer at protein concentrations of 4 μM (blue) and 70 μM (red). For comparison, shown are the data for the monomer apoSOD^50/51/6/111 (black). Units are in s^–1. The fits are from a three-state model according to Scheme 2 (52). Below are the corresponding folding free-energy profiles at 0, 2.7, and 6.5 M urea, as estimated from transition-state theory with prefactor of 10⁶ s^–1 (53), and the apparent stabilities f_D/(1 – f_D) of M and M₂ by using chevron and equilibrium data, respectively. At low [urea], the dimeric and monomeric proteins seem to fold over the same transition state (‡′), where the offset in logk_f is due to the F50E/G51E mutation. Above the transition midpoint, logk_u decreases with increasing protein concentrations as the free-energy difference between ‡′ and M₂ goes up. This concentration dependence vanishes at high [urea] where the rate-limiting step shifts to the first-order dissociation of M₂→‡′′ according to Hammond-postulate behavior (33).

Scheme 2.