Kinetic data for the superoxide radical (HO2⇄O−2 +H+, pK=4.8) in aqueous solution have been critically assessed. Rate constants for reactions of O−2 and HO2 with more than 300 organic and inorganic ions, molecules and other transient species have been tabulated.

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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...

The enzyme Cu, Zn superoxide dismutase (SOD) protects against oxidative damage by dismuting the superoxide radical O2-. to molecular oxygen and hydrogen peroxide at the active-site Cu ion in a reaction that is rate-limited by diffusion and enhanced by electrostatic guidance. SOD has evolved to be one of the fastest enzymes known (V(max) approximately 2 x 10(9) M-1 s-1). The new crystal structures of human SOD show that amino-acid site chains that are implicated in electrostatic guidance (Glu 132, Glu 133 and Lys 136) form a hydrogen-bonding network. Here we show that site-specific mutants that increase local positive charge while maintaining this orienting network (Glu----Gln) have faster reaction rates and increased ionic-strength dependence, matching brownian dynamics simulations incorporating electrostatic terms. Increased positive charge alone is insufficient: one charge reversal (Glu----Lys) mutant is slower than the equivalent charge neutralization (Glu----Gln) mutant, showing that the newly introduced positive charge disrupts the orienting network. Thus, electrostatically facilitated diffusion rates can be increased by design, provided the detailed structural integrity of the active-site electrostatic network is maintained.