Known for 30 years as hemocuprein, the copper-zinc superoxide dismutase from red cells was originally thought to be a storage protein for the metal ions.  However, in 1969 McCord and Fridovich discovered that the protein removes the harmful radical, superoxide anion (McCord & Fridovich, 1969) by converting it to hydrogen peroxide and dioxygen (2 O₂^-  +  2 H⁺ --->  H₂O₂   +  O₂).  The discovery of this activity solved the mystery of the oxygen paradox because superoxide anion is the first product in a series of chemical reactions causing oxygen toxicity.  Superoxide anion is now known to have a profound influence on biological and pathological processes (Valentine, et al., 1998).  There are several types of superoxide dismutases but all protect aerobic cells from superoxide anion (Bannister, et al., 1987).  Toxic reactions of oxygen are correlated with aging (Knight, 1998) and damage from poisonous substances such as the agrochemical , paraquat, and the anticancer drug, daunorubicin that is extremely toxic to the heart (Winterbourn, 1991).  Because of their protective activity, superoxides dismutases have been used as therapeutic agents to reduce toxic reactions of oxygen that are stimulated in strokes and heart attacks and in organs during removal and transplantation (Fan, 1999).

    Research has focused on characterizing changes that occur in the enzyme during its oxidative modification of hydrogen peroxide, one of the products of the reaction catalyzed by the dismutase.  One of our important findings is that copper, present in the dismutase and required for biological activity, is lost during the peroxide reaction (Jewett, et al., 1989).  More recently we have discovered that it is lost as copper (I) (Jewett, et al., 1999).  Any loss in a cell would compromise the protective function of the enzyme.  Recent reports have linked a genetic defect in the dismutase with familial amyotrophic lateral (FALS), or Lou Gehrig's disease (Hosle & Brown, 1996).  Some investigators have suggested that the mutant dismutases may be more susceptible to oxidative damage that results in the release of copper, a known neurotoxin (Bredesen, 1996).  Others report that aberrant copper chemistry mediates oxygen toxicity in ALS (Gabbianelli, 1999).  Our in vitro work with the bovine native enzyme has clearly indicated that hydrogen peroxide causes the loss of copper even at the lowest amounts of peroxide so that this oxidative damage in the cell could be contributing to the aberrant copper chemistry.

    Recent evidence indicates that muscular exercise results in the production of radicals and other reactive oxygen species within skeletal muscle (Alessio, 1993; Davies et al., 1982; Neill et al., 1996).  Specifically, muscular contraction has been shown to generate several reactive radicals, such as superoxide (O₂^-), hydrogen peroxide (H₂O₂), nitric oxide (NO^.) and hydroxyl radicals (HO^.) (Davies et al., 1982; ONeill et al., 1996; Reid et al., 1992a,b).  Because of these increases, antioxidant enzymes would be expected to increase in exercise-adapted muscle, and indeed, there is growing evidence for such increases as review by Powers et al. (Powers, et al., 1999).  Further evidence suggests that the majority of the oxidants produced within contracting myocytes are due to an elevated rate of mitochondrial respiration (Turrens, 1997).  Unscavanged oxidants modify  macromolecules in the cell including nucleic acids, proteins and lipids (Halliwell & Gutteridge, 1979; Yu, 1994).  These oxidations most likely occur when an imbalance exists between oxidants and antioxidants.  Oxidative stress occurs when local antioxidant defenses are depleted because the rates of radical reactions are greater than the rates of antioxidant defense mechanisms.  This stress could occur in skeletal muscle during acute exercise conditions when oxidant/antioxidant balance shifts toward the pro-oxidant state.  Evidence exists implicating oxidants as contributing to both exercise-induced enzyme down regulation and to muscle fatigue (Reid et al., 1992a).  Increased production of reactive oxygen species has also associated with various pathogenic conditions of old age (Knight, 1998; Meyandi & Evans, 1993).  It is well established that the antioxidant defense systems of many mammalian tissues are capable of adaptation in response to chronic exposure to oxidants.
 
 

    The 2:1 and 3:1 complexes of the catecholamines, epinephrine and norephinephrine, as well as the less easily oxidized catechol, and iron(III), form at physiological pHs in air-saturated buffers at 25^o C starting with iron(II).  No spectrophotometrically observable complexes are formed in deoxygenated solutions.  Kinetic analyses of the formation of the 2:1 complex from pH 6.0 to 6.4 indicate that the reaction is first order in both iron(II) and catechol(amine) and has a pH dependence of 1/[H⁺]².  At pH 6.0 the rate constant for the consumption of dioxygen is essentially identical to the rate constant for formation of the 2:1 complexes suggesting the participation of dioxygen in the rate determining step.  Kinetic data are interpreted in terms of a mechanism involving the formation of a 1:1 iron(II)-catechol(amine) dianion complex followed by the rate determining step of oxidation by dioxygen to the iron(III) complex.  This step is assumed to be followed by the rapid addition of a second (and presumably third) catechol(amine) to give the 2:1 complex (and 3:1 complex).

    The equilibrium constant for the 2:1 to 3:1 conversion, which occurs for the catecholamines from pH 6.5 to 8.5 and for catechol from 7 to 9, can be determined from absorbance data at the end of reactions carried out at constant iron and catechol(amine) using new buffer solutions and new aliquots of reagents at each desired pH.  The equilibrium constant for epinephrine is 10 ± 5 x 10⁹, for norepinephrine, 10 ± 2 x 10⁹ (using the same pKa values as for epinephrine), and for catechol, 2 ± 0.2 x 10⁹.   The constant for catechol is exactly in line with the one calculated from the data of K. A. Raymond (Bioinorganic Chemistry-II, Amer. Chem. Soc., Washington, D. C., pp. 33-54 (1977)).

    Similar spectra for the 2:1 and the 3:1 complexes were obtained using iron(III), however absorbances were less stable especially in the more alkaline solutions and the rate of formation of the complexes was much slower. With either form of iron, evidence for the formation of the four-electron oxidation products, adrenochrome or noradrenochrome could not be assessed because of overlapping spectra.  The catecholamines are finally oxidized to insoluble products. At pHs 7 to 9, the iron(II) is oxidized to iron (III) with an iron(II)/dioxygen stoichiometry of 2.2 ± 0.2 consistent with the reduction of dioxygen to peroxide.  Peroxide accumulates in the solutions and can be decomposed by catalase.  At the more acidic pH 6.0 in the presence of catechol, the iron(II) is oxidized with an iron(II)/dioxygen stoichiometry of 3.9 ± 0.2 consistent with the reduction of dioxygen towater whereas as this pH, the iron(II)/dioxygen ratio in the presence of epinephrine is 3.1 ± 0.2 suggesting a mixed stoichiometric system.   As peroxide accumulates, it is partly decomposed by the 2:1 complex, but not by the 3:1 complex, suggesting that an open coordination site is required for peroxide decomposition.  Spectra of the complexes are unaffected as peroxide accumulates or as it is decomposed by the 2:1 complex or by added catalase.

REFERENCES

McCord, J. M. and Fridovich, I. (1969) Superoxide Dismutase.  An Enzymatic Function for Erythrocuprein.  J. Biol. Chem. 244, 6049-6055.

Bannister, J. V., Bannister, W. H., and Rotilio, G. (1987)  "Aspects of the Structure, Function, and Applications of Superoxide Dismutase," in Critical Reviews in Biochemistry, Fasman, G. D. Ed., CRC Press, Boca Raton, FL, 111-180.

Valentine, J. S., Wertz, D. L., Lyons, T. H., Liou, L-L., Goto, J., and Gralla, E. B. (1998)  Curr. Opin in. Chem. Biol. 2: 253-262.

Knight, J. A . (1998)  Free Radicals: Their History and Current Status in Aging and Disease.  Ann. Clin. Lab. Sci. 28:  331-346.

Winterbourn, C. C., Vile, G. F., and Monteiro, H. P. (1991)  Ferritin, Lipid Peroxidation and Redox-Cycling Xenobiotics.  Free Radic. Res. Commun.  12-13:  107-114 .

Fan , C., Zwacka, R. M., and Engelhardt, J. F. (1999) Therapeutic Approaches for Ischemia/Reperfusion Injury in the Liver.  J. Mol. Med. 77:  577-592.

Jewett,  S. L., Cushing, S., Gillespie, F., Smith, D., and Sparks, S. (1989)  Reaction of Bovine-Liver Copper-Zinc Superoxide Dismutase with Hydrogen Peroxide: Evidence for reaction with H₂O₂ and HO₂^- Leading to Loss of Copper.  Eur. J. Biochem.  180:  569-575.

Jewett, S. L., Rocklin, A. M., Ghanevati, M., Abel, J. M., and Marach, J. A. (1999)  A New Look at at Time-Worn System:  Oxidation of CuZn-SOD by H₂O₂.  Free Rad. Biol. Med. 26:  905-918.

Hosler, B., A. and Brown, R. H., Jr.  (1996)  Superoxide Dismutase and Oxygen Radical Neurotoxicity. Curr. Opin. Neurol. 9:  486-491

Bredesen, D. E., Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Roe, J. A., Gralla, E. B., Ellerby, L. M., and Valentine, J. S. (1996) Cell Death Mechanisms in ALS.  Neurology 47:  S36-39.

Gabbianelli, R., Ferri, A., Rotilio, G., and Carri, M. T. (1999) Aberrant Copper Chemistry as a Mjor Mediator of Oxidative Stress in a Human Cellular Model of Amyotrophic Lateral Sclerosis.  J. Neurochem. 73:  1175-1180 .

Turrens,  J. F. (1997)  Superoxide Production by the Mitochondrial Respiratory Chain.  Biosci. Rep. 17:  3-8.

Powers, S. K., Ji, L. L., and Leeuwenburgh, C. (1999)  Exercise Training-Induced Alterantions in Skeletal Muscle Antioxidant Cpacity:  a Brief Review.  Med. Sci. Sports Exerc.  31:  987-997.

Jewett, S. L. and Rocklin, A. M. (1993).  Variation of One Unit of Activity with Oxidation Rate of Organic Substrate in Indirect Superoxide Dismutase Assays.  Anal. Biochem. 212:  555-559.