Solution Structure of Reduced Monomeric Q133M2 Copper, Zinc Superoxide Dismutase (SOD). Why Is SOD a Dimeric Enzyme?^,

Abstract

Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa) has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonance NMR experiments. The solution structure is represented by a family of 36 structures, with a backbone rmsd of 0.81 +/- 0.13 A over residues 3-150 and of 0.56 +/- 0.08 A over residues 3-49 and 70-150. This structure has been compared with the available X-ray structures of reduced SODs as well as with the oxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greek key beta-barrel. In general, the backbone and the metal sites are not affected much by the monomerization, except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorder is present. Significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymatic reaction under nonsaturating conditions. In the present monomer, its conformation is less favorable for the diffusion of the substrate to the reaction site. The structure of the copper center is well-defined; copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines. The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.

Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

“…In addition, the backbone oxygen atoms of residues Cys-57 and Gly-61 in this loop form three hydrogen bonds that correctly orient the side chain of Arg-143, an important determinant of copper ion accessibility in SOD1 (27,28,(45)(46)(47)(48). These structural features increase the likelihood that disulfide reduction at Cys-57, and the disorder of this loop could facilitate partial unfolding, monomerization, metal ion loss, or altered reactivity of bound copper.…”

“…Structural properties of SOD1 that contribute to its extreme thermochemical stability include an eight-stranded ␤-barrel motif, binding sites for copper and zinc ions, hydrophobic interactions associated with dimerization, and an unusual intrasubunit disulfide bond bridging a loop residue, Cys-57, and Cys-146 of the ␤-barrel (24, 26). The loop that includes Cys-57 also strongly influences the conformation of Arg-143, which regu-lates steering of superoxide anion and reactivity of the copper ion via a local hydrogen bond network and the disulfide linkage to Cys-146 (27)(28)(29). Furthermore, portions of this loop contribute to the dimer interface and form the zinc ion binding site (26).…”

The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status

“…The 15 N-labeled protein in which the non-conserved cysteine residues, Cys 6 and Cys 111 , were mutated to Ser was obtained by growing the cells in the M9 minimal medium with N 15 -NH 4 Cl following a reported procedure (16), whereas LB medium was used for the nonlabeled protein. The cells were grown at 37°C until A 600 ϭ 0.6 and induced with 1.0 mM isopropyl 1-thio-␤-D-galactopyranoside for 6 h. The protein was isolated and purified according to previously published protocols (16). Fully reduced and demetallated hSOD1 (E,E-hSOD1 SH ) was prepared by treating the isolated protein with dithiothreitol at 37°C for 1 h in an anaerobic chamber to reduce the disulfide bond (13).…”

Diffusion NMR spectroscopy

“…2 Consequently, quantitative measurements of the effective diffusivity provide information on the shape and the interactions of the diffusing molecule. This property is widely exploited in in vitro MRS studies of biomolecules in solution to assess various biophysical and biochemical parameters, including the hydrodynamics and the folding/unfolding of proteins, 10 proton exchange with biomacromolecules, 11 the oligomerization state of biomacromolecules 12 and protein-ligand interactions. 13 Equation (8) shows that diffusion strongly depends on temperature which should therefore ideally be kept constant throughout the diffusion measurement, unless temperature per se is the parameter of interest.…”

“…It should be noted that eqn (12) ignores the effects of displacement during the gradient pulse (the so-called short gradient pulse limit). Experimentally, this condition is approximated by keeping ( D (Fig.…”