A subset of individuals with familial amyotrophic lateral sclerosis (FALS) possesses dominantly inherited mutations in the gene that encodes copper-zinc superoxide dismutase (CuZnSOD). A4V and G93A, two of the mutant enzymes associated with FALS, were shown to catalyze the oxidation of a model substrate (spin trap 5,5'-dimethyl-1-pyrroline N-oxide) by hydrogen peroxide at a higher rate than that seen with the wild-type enzyme. Catalysis of this reaction by A4V and G93A was more sensitive to inhibition by the copper chelators diethyldithiocarbamate and penicillamine than was catalysis by wild-type CuZnSOD. The same two chelators reversed the apoptosis-inducing effect of mutant enzymes expressed in a neural cell line. These results suggest that oxidative reactions catalyzed by mutant CuZnSOD enzymes initiate the neuropathologic changes in FALS.

Over 90 different mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) cause ϳ2% of amyotrophic lateral sclerosis (ALS) cases by an unknown mechanism. We engineered 14 different human ALSrelated SOD1 mutants and obtained high yields of biologically metallated proteins from an Sf21 insect cell expression system. Both the wild type and mutant "as isolated" SOD1 variants were deficient in copper and were heterogeneous by native gel electrophoresis. By contrast, although three mutant SOD1s with substitutions near the metal binding sites (H46R, G85R, and D124V) were severely deficient in both copper and zinc ions, zinc deficiency was not a consistent feature shared by the as isolated mutants. Eight mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) exhibited normal SOD activity over pH 5.5-10.5, per equivalent of copper, consistent with the presumption that bound copper was in the proper metal-binding site and was fully active. The H48Q variant contained a high copper content yet was 100-fold less active than the wild type enzyme and exhibited a blue shift in the visible absorbance peak of bound Cu(II), indicating rearrangement of the Cu(II) coordination geometry. Further characterization of these as-isolated SOD1 proteins may provide new insights regarding mutant SOD1 enzyme toxicity in ALS.Amyotrophic lateral sclerosis (ALS, 1 Lou Gehrig's disease) is an age-dependent, degenerative disorder of motor neurons in the spinal cord and brain. Progressive dysfunction of both upper and lower motor neurons causes death from respiratory paralysis, usually within 5 years. Investigation of the causes of familial ALS, which comprises ϳ10% of cases, may contribute insights relevant to the pathophysiology of sporadic ALS and of other motor neuron diseases (1, 2).A subset of autosomal dominant ALS is caused by over 90 mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) (3, 4) (see an updated list of all mutations on the World Wide Web at www.alsod.org). SOD1 is a 32-kDa homodimeric enzyme that functions as an antioxidant, converting two molecules of superoxide anion (O 2 . ) to O 2 and H 2 O 2 .This redox cycle involves alternate reduction (reaction 1) and reoxidation (reaction 2) of the catalytic copper ion by O 2 . .REACTIONS 1 and 2 SOD1 contains an eight-stranded ␤-barrel motif, an intrasubunit disulfide bond, and a zinc binding site that contribute to its extreme thermochemical stability (Fig. 1). The mutant residues are scattered throughout the protein, including some residues important for copper or zinc coordination, others located near the dimer interface or at either pole of the ␤-barrel, and several in the charged loop near the C terminus that may guide O 2 . to the active site. Although most are missense substitutions, some are predicted to truncate the C terminus of the protein, including the charged loop. No null mutations have been described. Mutant SOD1 most likely causes motor neuron death by gain of an unknown toxic property rather than by deficiency of dismutase a...

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The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala 4 3 Val, Gly 93 3 Ala, and Leu 38 3 Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZn-SOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS.Copper-zinc superoxide dismutase (CuZnSOD) 1,2 is an abundant cytosolic eukaryotic enzyme that catalyzes the disproportionation of superoxide anion to dioxygen and hydrogen peroxide (Reaction 1) (1-4).More than 70 different point mutations of CuZnSOD have been implicated in the fatal motor neuron disease, familial amyotrophic lateral sclerosis (FALS) (5). It has been demonstrated that the disease is initiated not by a loss of function, i.e. decrease in catalytic SOD activity, but by the gain of a new and toxic property. Suggestions for the nature of this property include the increased ability of mutant CuZnSODs to catalyze oxidation reactions by hydrogen peroxide (6 -8), to promote the formation of nitrotyrosine (9 -12), and to induce protein aggregation (13-15), each of which might be acting alone or in combination. Many theories concerning the gain of function of FALS mutant CuZnSOD are based on the assumption that the new toxic property is related to the enzyme-bound metal ions, particularly the copper ions. The detailed mechanism for the catalytic disproportionation of O 2 Ϫ is well understood for human, bovine, and yeast CuZnSODs (16 -22). The mechanism involves sequential reduction (Reaction 2) and reoxidation (Reaction 3) of the copper(II) center, where both reactions are relatively pH-independent over the pH range of 5.0 -9.5 and proceed with diffusion-controlled rate constants of 2 ϫ 10

A series of mutant human and yeast copperzinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.