Copper-zinc superoxide dismutase mechanism

The transfer of a quadridentate N2S2-donor ligand from M2+ (M = Cr, Mn, Fe, Co or Ni) to Cu2+ (271), already mentioned in Section V.A.l, has a formal connection with an investigation of the mechanism of copper delivery to metalloproteins, such as copper zinc superoxide dismutase. Both are ligand exchange reactions of the type ML + CuL ML + CuL (300). 

Guanine is the most easily oxidizable natural nucleic acid base [8] and many oxidants can selectively oxidize guanine in DNA [95]. Here, we focus on the site-selective oxidation of guanine by the carbonate radical anion, COs , one of the important emerging free radicals in biological systems [96]. The mechanism of COs generation in vivo can involve one-electron oxidation of HCOs at the active site of copper-zinc superoxide dismutase [97, 98], and homolysis of the nitrosoperoxycarbonate anion (0N00C02 ) formed by the reaction of peroxynitrite with carbon dioxide [99-102]. 

The enzyme copper, zinc superoxide dismutase (Cu,Zn-SOD, EC 1.15.1.1) catalyzes the disproportionation of superoxide anion to dioxygen and hydrogen peroxide (equations 1 and 2). Crystallographic data can be found in References 41-46. This antioxidant enzyme is present in the cytosol and mitochondrial intermembrane space of eukaryotic cells and in the periplasmic space of bacterial cells as a homodimer of 32 kDa. Each monomer binds one copper and one zinc ion. The reaction mechanism involves the... 

Smirnov V. V. Roth J. P. Mechanisms of electron transfer in catalysis by copper zinc superoxide dismutase. J. Am. Chem. Soc. 2006, 128, 16424—16425. 

CD spectroscopy has also provided valuable insight into the chemical stability and chemical denaturation of proteins. A recent study by Rumfeldt etal. examines the guanidinium-chloride induced denaturation of mutant copper-zinc superoxide dismutases (SODs). These mutant forms of the Cu, Zn-SOD enzyme are associated with toxic protein aggregation responsible for the pathology of amyotrophic lateral sclerosis. In this study, CD spectroscopy was used in conjunction with tryptophan fluorescence, enzyme activity, and sedimentation experiments to study the mechanism by which the mutated enzyme undergoes chemical denaturation. The authors found that the mutations in the enzyme structure increased the susceptibihty of the enzyme to form partially unfolded destabilized monomers, rather than the stable metaUated monomer intermediate or native metallated dimer. 

Stine-Elam J, Malek K, Rodriguez JA, Doucette PA, Taylor AB, Hayward LW, Cabelli DE, Valentine JS, Hart PJ. (2003) Mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutases. J Biol Chem 278 21032-21039. 

The generation of O2 from potassium superoxide was also applied to stop-flow procedures. In this method O2 was dissolved in dimethyl sulfoxide and stabilized in 18-crown-6-polyether. This method is useful for mechanistic studies indeed, McClune and Fee (1976) were able to obtain catalytic rate constants for bovine copper/zinc superoxide dismutase as a function of pH in various buffers. More recently the mechanism of catalysis and of anion inhibition of iron superoxide dismutase from E. coli have been examined by this method using a specially constructed stop-flow spectrophotometer (Bull and Fee, 1985). A limitation of the method is that the pre-equilibrium state cannot be properly investigated because of the time resolution of the stop-flow equipment (== 5 msec). 

Copper is an essential element to most life forms. In humans it is the third most abundant trace element only iron and zinc are present in higher quantity. Utilization of copper usually involves a protein active site which catalyzes a critical oxidation reaction, e.g., cytochrome oxidase, amine oxidases, superoxide dismutase, ferroxidases, dopamine-/ -hydrox-ylase, and tyrosinase. Accordingly, animals exhibit unique homeostatic mechanisms for the absorption, distribution, utilization, and excretion of copper (J). Moreover, at least two potentially lethal inherited diseases of copper metabolism are known Wilson s Disease and Menkes s Kinky Hair Syndrome (I). 

Forman and Fridovich (1973) using an indirect assay whereby O2 was generated either by the action of xanthine oxidase on xanthine or by the mechanical infusion of potassium superoxide in tetrahydrofuran. The generated OJ was allowed to react with ferricytochrome c or with tetra-nitromethane and the product formation was monitored spectroscopically. Details of the two assays are given in Section 11.3. Addition of superoxide dismutase inhibits the formation of products. A rate constant of 2 X 10 M sec was determined for all three enzymes. This value agreed with the rate constant determined by pulse radiolysis for the copper/zinc enzyme (Klug-Roth et al., 1973 Fielden et al., 1974). The mechanism of action of the superoxide dismutases has been investigated by the technique of pulse radiolysis which is described in Section II.2. The bovine erythrocyte copper/zinc enzyme is the most studied form as far as the molecular and catalytic properties are concerned (Rotilio and Fielden,... 

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