Kinetic analysis of product inhibition in human manganese superoxide dismutase.

Manganese superoxide dismutase (MnSOD) cycles between the Mn(II) and Mn(III) states during the catalyzed disproportionation of O(2)(*-), a catalysis that is limited at micromolar levels of superoxide by a peroxide-inhibited complex with the metal. We have investigated the role in catalysis and inhibition of the conserved residue Trp161 which forms a hydrophobic side of the active site cavity of MnSOD. Crystal structures of mutants of human MnSOD in which Trp161 was replaced with Ala or Phe showed significant conformational changes on adjacent residues near the active site, particularly Gln143 and Tyr34 which in wild-type MnSOD participate in a hydrogen bond network believed to support proton transfer during catalysis. Using pulse radiolysis and observing the UV absorbance of superoxide, we have determined rate constants for the catalytic dismutation of superoxide. In addition, the rates of formation and dissociation of the product-inhibited complex of these mutants were determined by direct observation of the characteristic visible absorption of the oxidized and inhibited states. Catalysis by W161A and W161F MnSOD was associated with a decrease of at least 100-fold in the catalytic rate of reduction of superoxide, which then promotes a competing pathway leading to product inhibition. The structural changes caused by the mutations at position 161 led to small changes, at most a 6-fold decrease, in the rate constant for formation of the inhibited complex. Solvent hydrogen isotope effects support a mechanism in which formation of this complex, presumably the peroxide dianion bound to the manganese, involves no rate-contributing proton transfer; however, the dissociation of the complex requires proton transfer to generate HO(2)(-) or H2O2.

The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin.

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state. The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.

2-methoxyestradiol does not inhibit superoxide dismutase.

It has been reported in the literature that the endogenous estrogen metabolite 2-methoxyestradiol (2-ME) inhibits both manganese and copper,zinc superoxide dismutases (Mn and Cu,Zn SODs) and that this mechanism is responsible for 2-ME's ability to kill cancer cells. In fact, as demonstrated using several SOD assays including pulse radiolysis, 2-ME does not inhibit SOD but rather interferes with the SOD assay originally used. Nevertheless, as confirmed by aconitase inactivation measurements and lactate dehydrogenase release in human leukemia HL-60 cells, 2-ME does increase superoxide production in these cells and is more toxic than its non-O-methylated precursor 2-hydroxyestradiol. Other mechanisms previously suggested in the literature may explain 2-ME's ability to increase intracellular superoxide levels in tumor cells.

Multiple replacements of glutamine 143 in human manganese superoxide dismutase: effects on structure, stability, and catalysis.

Glutamine 143 in human manganese superoxide dismutase (MnSOD) forms a hydrogen bond with the manganese-bound solvent molecule and is investigated by replacement using site-specific mutagenesis. Crystal structures showed that the replacement of Gln 143 with Ala made no significant change in the overall structure of the mutant enzyme. Two new water molecules in Q143A MnSOD were situated in positions nearly identical with the Oepsilon1 and Nepsilon2 of the replaced Gln 143 side chain and maintained a hydrogen-bonded network connecting the manganese-bound solvent molecule to other residues in the active site. However, their presence could not sustain the stability and activity of the enzyme; the main unfolding transition of Q143A was decreased 16 degrees C and its catalysis decreased 250-fold to k(cat)/K(m) = 3 x 10(6) M(-)(1) s(-)(1), as determined by stopped-flow spectrophotometry and pulse radiolysis. The mutant Q143A MnSOD and other mutants at position 143 showed very low levels of product inhibition and favored Mn(II)SOD in the resting state, whereas the wild type showed strong product inhibition and favored Mn(III)SOD. However, these differences did not affect the rate constant for dissociation of the product-inhibited complex in Q143A MnSOD which was determined from a characteristic absorbance at 420 nm and was comparable in magnitude ( approximately 100 s(-)(1)) to that of the wild-type enzyme. Hence, Gln 143, which is necessary for maximal activity in superoxide dismutation, appears to have no role in stabilization and dissociation of the product-inhibited complex.

Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis.

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) --> Val, Gly(93) --> Ala, and Leu(38) --> 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 CuZnSOD 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.

Role of tryptophan 161 in catalysis by human manganese superoxide dismutase.

Tryptophan 161 is a highly conserved residue that forms a hydrophobic side of the active site cavity of manganese superoxide dismutase (MnSOD), with its indole ring adjacent to and about 5 A from the manganese. We have made a mutant containing the conservative replacement Trp 161 --> Phe in human MnSOD (W161F MnSOD), determined its crystal structure, and measured the catalysis of the resulting mutant using pulse radiolysis to produce O(2)(*)(-). In the structure of W161F MnSOD the phenyl side chain of Phe 161 superimposes on the indole ring of Trp 161 in the wild type. However, in the mutant, the hydroxyl side chain of Tyr 34 is 3.9 A from the manganese, closer by 1.2 A than in the wild type. The tryptophan in MnSOD is not essential for the half-cycle of catalytic activity involving reduction of the manganese; the mutant W161F MnSOD had k(cat)/K(m) at 2.5 x 10(8) M(-)(1) s(-)(1), reduced only 3-fold compared with wild type. However, this mutant exhibited a strong product inhibition with a zero-order region of superoxide decay slower by 10-fold compared with wild type. The visible absorption spectrum of W161F MnSOD in the inhibited state was very similar to that observed for the inhibited wild-type enzyme. The appearance of the inhibited form required reaction of 2 molar equiv of O(2)(*)(-) with W161F Mn(III)SOD, one to form the reduced state of the metal and the second to form the inhibited complex, confirming that the inhibited complex requires reaction of O(2)(*)(-) with the reduced form of the enzyme. This work suggests that a significant role of Trp 161 in the active site is to promote the dissociation of product peroxide, perhaps in part through its effect on the orientation of Tyr 34.

Examination of the nickel site structure and reaction mechanism in Streptomyces seoulensis superoxide dismutase.

Superoxide dismutases are metalloenzymes involved in protecting cells from oxidative damage arising from superoxide radical or reactive oxygen species produced from superoxide. Examples of enzymes containing Cu, Mn, and Fe as the redox-active metal have been characterized. Recently, a SOD containing one Ni atom per subunit was reported. The amino acid sequence of the NiSOD deduced from the nucleotide sequence of the structural gene sodN from Streptomyces seoulensis is reported and has no homology with other SODs. X-ray absorption spectroscopic studies coupled with EPR of the Ni center show that the Ni in the oxidized (as isolated) enzyme is in a five-coordinate site composed of three S-donor ligands, one N-donor, and one other O- or N-donor. This unique coordination environment is modified by the loss of one N- (or O-) donor ligand in the dithionite-reduced enzyme. The NiSOD activity was determined by pulse radiolysis, and a value of kcat = 1.3 x 10(9) M-1 s-1 per Ni was obtained. The rate is pH sensitive and drops off rapidly above pH 8. The results characterize a novel class of metal center active in catalyzing the redox chemistry of superoxide and, when placed in context with other nickel enzymes, suggest that thiolate ligation is a prerequisite for redox-active nickel sites in metalloenzymes.

Reactions of hydrogen peroxide with familial amyotrophic lateral sclerosis mutant human copper-zinc superoxide dismutases studied by pulse radiolysis.

Mutations in copper-zinc superoxide dismutase (CuZn-SOD) have been implicated in the familial form of the motor neuron disease amyotrophic lateral sclerosis (Lou Gehrig's disease). We have expressed and purified recombinant human wild type (hWT) and G93A (hG93A) CuZn-SOD, and we have used pulse radiolysis to measure their superoxide dismutase activities and their rates of deactivation upon exposure to hydrogen peroxide or heat. Both hG93A and hWT CuZn-SOD were found to have high SOD activities in their copper and zinc containing as-isolated forms as well as when remetallated entirely with copper (CuCu). Rates of deactivation by hydrogen peroxide of the as-isolated hWT and hG93A enzymes were determined and were found to be similar, suggesting that the FALS mutant enzyme is not inactivated at a higher rate than wild type by generation of and subsequent reaction with hydroxyl radical, .OH, when it is in the CuZn form. However, rates of deactivation by hydrogen peroxide of the CuCu derivatives of both hWT and hG93A were significantly greater than those of the copper and zinc containing as-isolated enzymes. Rates of thermal deactivation were also similar for the mutant and hWT as-isolated CuZn forms but were greater for the CuCu derivatives of both enzymes. Reactions of hydrogen peroxide with the Cu(II)Cu(II) derivative of the WT enzyme demonstrate that the copper ion in the copper site is reduced much more rapidly than the copper in the zinc site, leading to the conclusion that reaction of hydrogen peroxide with Cu(I) in the copper site is the source of deactivation in the CuCu as well as the CuZn enzymes.

Computational, pulse-radiolytic, and structural investigations of lysine-136 and its role in the electrostatic triad of human Cu,Zn superoxide dismutase.

Key charged residues in Cu,Zn superoxide dismutase (Cu,Zn SOD) promote electrostatic steering of the superoxide substrate to the active site Cu ion, resulting in dismutation of superoxide to oxygen and hydrogen peroxide, Lys-136, along with the adjacent residues Glu-132 and Glu-133, forms a proposed electrostatic triad contributing to substrate recognition. Human Cu,Zn SODs with single-site replacements of Lys-136 by Arg,Ala, Gln, or Glu or with a triple-site substitution (Glu-132 and Glu-133 to Gln and Lys-136 to Ala) were made to test hypotheses regarding contributions of these residues to Cu,Zn SOD activity. The structural effects of these mutations were modeled computationally and validated by the X-ray crystallographic structure determination of Cu,Zn SOD having the Lys-136-to-Glu replacement. Brownian dynamics simulations and multiple-site titration calculations predicted mutant reaction rates as well as ionic strength and pH effects measured by pulse-radiolytic experiments. Lys-136-to-Glu charge reversal decreased dismutation activity 50% from 2.2 x 10(9) to 1.2 x 10(9) M-1 s-1 due to repulsion of negatively charged superoxide, whereas charge-neutralizing substitutions (Lys-136 to Gln or Ala) had a less dramatic influence. In contrast, the triple-mutant Cu,Zn SOD (all three charges in the electrostatic triad neutralized) surprisingly doubled the reaction rate compared with wild-type enzyme but introduced phosphate inhibition. Computational and experimental reaction rates decreased with increasing ionic strength in all of the Lys-136 mutants, with charge reversal having a more pronounced effect than charge neutralization, implying that local electrostatic effects still govern the dismutation rates. Multiple-site titration analysis showed that deprotonation events throughout the enzyme are likely responsible for the gradual decrease in SOD activity above pH 9.5 and predicted a pKa value of 11.7 for Lys-136. Overall, Lys-136 and Glu-132 make comparable contributions to substrate recognition but are less critical to enzyme function than Arg-143, which is both mechanistically and electrostatically essential. Thus, the sequence-conserved residues of this electrostatic triad are evidently important solely for their electrostatic properties, which maintain the high catalytic rate and turnover of Cu,Zn SOD while simultaneously providing specificity by selecting against binding by other anions.

Function of the Greek key connection analysed using circular permutants of superoxide dismutase.

Human Cu,Zn superoxide dismutase (SOD) is a single domain all beta-sheet protein with its eight beta-strands arranged as a Greek key beta-barrel or immunoglobulin fold. Three circularly permuted variants of SOD were made by joining the native amino- and carboxy-termini, and introducing new termini at sites originally within connections between beta-strands. The locations of the new termini were chosen to interrupt beta-turns between the two N-terminal beta-hairpins and the short cross-barrel Greek key connection. Expression levels in the Escherichia coli periplasm were indistinguishable from that of native SOD. Reaction rates for the purified proteins were similar to those of the native enzyme, indicating that the permutants are correctly folded. Interrupting the covalent cross-bracing provided by the Greek key connection reduced the stability of the protein by approximately 1.0 kcal/mol, indicating only a slight contribution to conformational stability. The experiments test and eliminate two hypotheses for folding pathways for Greek key beta-barrels that require N-terminal beta-hairpins or covalent attachment across the short Greek key connection.

Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface.

Human manganese superoxide dismutase (MnSOD) is a homotetrameric enzyme which protects mitochondria against oxygen-mediated free radical damage. Within each subunit, both the N-terminal helical hairpin and C-terminal alpha/beta domains contribute ligands to the catalytic manganese site. Two identical four-helix bundles, symmetrically assembled from the N-terminal helical hairpins, form a novel tetrameric interface that stabilizes the active sites. The 2.5 A crystallographic structure of the naturally occurring polymorphic variant Ile58Thr MnSOD reveals that the helical hairpin mutation Thr58 causes two packing defects in each of the two four-helix bundles of the tetrameric interface. Similar mutations, expected to cause packing defects in the Cu,ZnSOD dimer interface, are associated with the degenerative disease amyotrophic lateral sclerosis. Ile58Thr MnSOD is primarily dimeric in solution and is significantly less thermostable than the normal enzyme, with decreases of 15 degrees C in the main melting temperature and 20 degrees C in the heat-inactivation temperature. Consequently, this mutant MnSOD is compromised at normal body temperatures: thermal inactivation, predicted from the decrease in thermal stability, occurs with a theoretical half-life of only 3.2 h at 37 degrees C (1.4 h at 41 degrees C), compared with 3.1 years for native MnSOD. This prediction is supported by direct measurements: incubation at 41.7 degrees C for 3 h has no effect on the activity of native MnSOD but completely inactivates mutant MnSOD. Rapid inactivation of Ile58Thr MnSOD at the elevated temperatures associated with fever and inflammation could provide an early advantage by killing infected cells, but also would increase superoxide-mediated oxidative damage and perhaps contribute to late-onset diseases.

The role of arginine 143 in the electrostatics and mechanism of Cu,Zn superoxide dismutase: computational and experimental evaluation by mutational analysis.

Cu,Zn superoxide dismutase protects cells from oxidative damage by removing superoxide radicals in one of the fastest enzyme reactions known. The redox reaction at the active-site Cu ion is rate-limited by diffusion and enhanced by electrostatic guidance. To quantitatively define the electrostatic and mechanistic contributions of sequence-invariant Arg-143 in human Cu,Zn superoxide dismutase, single-site mutants at this position were investigated experimentally and computationally. Rate constants for several Arg-143 mutants were determined at different pH and ionic strength conditions using pulse radiolytic methods and compared to results from Brownian dynamics simulations. At physiological pH, substitution of Arg-143 by Lys caused a 2-fold drop in rate, neutral substitutions (Ile, Ala) reduced the rate about 10-fold, while charge-reversing substitutions (Asp, Glu) caused a 100-fold decrease. Position 143 mutants showed pH dependencies not seen in other mutants. At low pH, the acidic residue mutations exhibited protonation/deprotonation effects. At high pH, all enzymes showed typical decreases in rate except the Lys mutant in which the rate dropped off at an unusually low pH. Increasing ionic strength at acidic pH decreased the rates of the wild-type enzyme and Lys mutant, while the rate of the Glu mutant was unaffected. Increasing ionic strength at higher pH (> 10) increased the rates of the Lys and Glu mutants while the rate of the wild-type enzyme was unaffected. Reaction simulations with Brownian dynamics incorporating electrostatic effects tested computational predictability of ionic strength dependencies of the wild-type enzyme and the Lys, Ile, and Glu mutants. The calculated and experimental ionic strength profiles gave similar slopes in all but the Glu mutant, indicating that the electrostatic attraction of the substrate is accurately modeled. Differences between the calculated and experimental rates for the Glu and Lys mutants reflect the mechanistic contribution of Arg-143. Results from this joint analysis establish that, aside from the Cu ligands, Arg-143 is the single most important residue in Cu,Zn superoxide dismutase both electrostatically and mechanistically, and provide an explanation for the evolutionary selection of arginine at position 143.

An essential role for the conserved Glu-133 in the anion interaction with superoxide dismutase.

Negatively charged glutamic acid residues at positions 132 and 133 in human Cu2Zn2SOD are located at the entrance to the active site cavity and affect electrostatic interactions with the negatively charged substrate. The mutants in which these residues have been neutralized separately and together by conversion to glutamine residues or changed to a positive group on position 133 have been characterized through a variety of biophysical techniques. The structure around the metal ions, as monitored by spectroscopic measurements, is the same in the mutants and native enzyme. The mutants have been characterized with respect to the affinity for the anion N3-. The mutants have larger affinity for azide than the WT, as a result of the removal of one or two negative charges or of the introduction of a positive group. The pattern of the azide affinity constants parallels that of the rate of O2- dismutation. The substitution of the negative Glu-133 with a positive group does not induce a larger increase in the affinity as well as in the catalytic rates with respect to its neutralization. These patterns cannot, therefore, be rationalized only in terms of electrostatic interactions. The behavior of the mutants towards the substrate (O2-) and substrate analogue (N3-) is discussed on the basis of theoretical predictions available in the literature.

Faster superoxide dismutase mutants designed by enhancing electrostatic guidance.

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.

Thermostabilization of recombinant human and bovine CuZn superoxide dismutases by replacement of free cysteines.

Human CuZn superoxide dismutase (HSOD) has two free cysteines: a buried cysteine (Cys6) located in a beta-strand, and a solvent accessible cysteine (Cys111) located in a loop region. The highly homologous bovine enzyme (BSOD) has a single buried Cys6 residue. Cys6 residues in HSOD and BSOD were replaced by alanine and Cys111 residues in HSOD by serine. The mutant enzymes were expressed and purified from yeast and had normal specific activities. The relative resistance of the purified proteins to irreversible inactivation of enzymatic activity by heating at 70 degrees C was HSOD Ala6 Ser111 greater than BSOD Ala6 Ser109 greater than BSOD Cys6 Ser109 (wild type) greater than HSOD Ala6 Cys111 greater than HSOD Cys6 Ser111 greater than HSOD Cys111 (wild type). In all cases, removal of a free cysteine residue increased thermostability.

Highlights of current research involving superoxide and perhydroxyl radicals in aqueous solutions.

Selected topics of current research on HO2/O2- radical reactions in aqueous solutions are described. The subject is introduced by a brief review of the fundamental background chemistry of HO2/O2-, including a description of the rates and mechanisms of formation and decay as well as their spectral properties, current research on self-exchange rates, etc. This is followed by examples of their reactivity with simple metal complexes of iron, manganese and copper and extended to some biological significant metal-containing systems such as porphyrins, haem enzymes and superoxide dismutases. Some recent results on the interaction of these oxy-radicals with antioxidants, specifically vitamin C and E, are discussed. A brief review of some topical chemistry describing HO2/O2- involvement in atmospheric and aquatic chemistry is given. Finally, some new trends in the areas of heterogeneous catalysis and reactor chemistry that involve these species are described.

A characterization of copper/zinc superoxide dismutase mutants at position 124. Zinc-deficient proteins.

Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site-directed mutagenesis on the human enzyme. Asp124 is H-bonded to the NH of two histidines, one of which is bound to copper and the other to zinc. The mutant proteins, as expressed in Escherichia coli, result in an essential zinc-free enzyme which is similar to that obtained from the wild-type derivative through chemical manipulation. Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximately 50% of the molecules in the Cu2Zn2 or Cu2Co2 form. The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques. The Cu2Cox derivatives (x approximately 1) were used to monitor, through electronic and 1H-NMR spectroscopies, the metal sites which are found to be similar to those of the wild type. In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125. The behavior is similar to that of the other mutants in most respects. The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8-10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives. The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme. A full profile up to pH 10.5 was obtained for the mutants.

Advances in the understanding of the structure-function relationship in Cu,Zn superoxide dismutase.

The structure-function relationship in Cu,Zn superoxide dismutase has been partially elucidated by the combined use of many spectroscopic techniques (electronic spectroscopy, circular dichroism, EPR and NMR) and site-directed mutagenesis techniques. The comparison of the spectroscopic and catalytic properties of various mutants, in which some active site residues have been substituted through site-directed mutagenesis, allowed us to establish that the activity is in general more sensitive to electrostatic effects rather than to steric effects or changes in the copper hydration or coordination geometry.

The interaction between Cu(I) superoxide dismutase and hydrogen peroxide.

The interaction between superoxide dismutase (SOD) and peroxide, under anaerobic conditions in the presence of an OH radical scavenger, formate, and an indicator, nitro blue tetrazolium, involves five reactions and an equilibrium: (table; see text) Reaction 3 occurs at a rate that is proportional to both peroxide and enzyme with no kinetic evidence for any intermediate peroxide-enzyme complex. Rate studies as a function of pH corroborate previously published work (Fuchs, H. J. R., and Borders, C. L., Jr. (1983) Biochem Biophys. Res. Commun. 116, 1107-1113; Blech, D. M., and Borders, C. L., Jr. (1983) Arch. Biochem. Biophys. 224, 579-586) suggesting that HO2-, and not H2O2, is the active species in this system: k(HO2- + superoxide dismutase-Cu+) = 2.6 x 10(3) M-1 s-1. Evidence is presented which suggests that HO2-, like O2-, reacts at rates that are affected by the electrostatic forces of the enzyme.