Copper-zinc superoxide dismutase bovine

R.N. Iyer and W.E. Schmidt, Observations on the direct electrochemistry of bovine copper-zinc superoxide dismutase. Bioelectrochem. Bioenerg. 27, 393 104 (1992). 

The simplest direct assay was described by Marklund (1976). Superoxide was generated from its potassium salt at alkaline pH where its stability increases. The decay at 250 nm in the presence and absence of superoxide dismutase is measured in a standard spectrophotometer. The assay is carried out in such a way that the rate of a decay in the presence of the enzyme is within the time response of the apparatus (Fig. 1). One unit of activity is arbitrarily taken as the enzyme activity that results in the dismutation of O2 at a rate of 0. lsec . This rate of dismutation corresponds to 5.5 ng bovine copper/zinc superoxide dismutase and to 64.5 ng bovine manganese superoxide dismutase under the conditions described by Marklund (1976). The rate constants calculated for both enzymes were... 

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). 

Viglino et al., (1979) have studied the interaction of with the copper of bovine copper/zinc superoxide dismutase. They have shown that in this case the Luz-Meiboom equations can be written in a simplified form ... 

Jl. Jewett, S. L., Cushing, S., Gillespie, F., Smith, D., and Sparks, S., Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HOj leading to loss of copper. Eur. J. Biochem. 180, 569-575 (1989). 

Another direct assay method is based on decay kinetics of pulse generated O2 (Takahashi and Asada, 1981). Superoxide was produced within 10 msec by a flash of light through the excitation of flavin mononucleotide in the presence of A,A,A, A -tetramethylethylenedi-amine and oxygen. The kinetics of O2 decay in the presence and absence of enzyme were followed at 240 nm. The catalytic rate constant for bovine erythrocyte copper/zinc superoxide dismutase was found to be 1.75 x... 

Kamnakaran, C., Zhang, H., Crow, J.P., Anthohne, W.E., Kalyanaraman, B., 2004. Direct probing of copper active site and free radical formed during bicarbonate-dependent peroxidase activity of bovine and human copper, zinc-superoxide dismutases. Low-temperature electron paramagnetic resonance and electron nuclear double resonance studies. J. Biol. Chem. 279, 32534—32540. 

Water soluble protein with a relative molecular mass of ca. 32600, which particularly contains copper and zinc bound like chelate (ca. 4 gram atoms) and has superoxide-dismutase-activity. It is isolated from bovine liver or from hemolyzed, plasma free erythrocytes obtained from bovine blood. Purification by manyfold fractionated precipitation and solvolyse methods and definitive separation of the residual foreign proteins by denaturizing heating of the orgotein concentrate in buffer solution to ca. 65-70 C and gel filtration and/or dialysis. 

J.S. Valentine, M.W. Pantoliano, PJ. Mcdonnell, A.R. Burger, and S.J. Lippard, pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 76, 4245-4249 (1979). 

J.A. Fee and R.G. Briggs, Reconstitution of bovine erythrocyte superoxide dismutase. V. Preparation and properties of derivatives in which both zinc and copper sites contain copper. Biochim. Biophy. Acta. 400, 439 150 (1975). 

Sette, M., Paci, M., Desideri, A. and Rotillo, G. (1992) Formate as an NMR probe of anion binding to copper-zinc and copper-cobalt bovine erythrocyte superoxide dismutase. Biochemistry, 31, 2410-2415. 

The structure and enzyme kinetics of bovine erythrocyte superoxide dismutase are reviewed. The protein has a novel imidazolate-bridged copper(II)-zinc(II) catalytic center in each of two identical subunits. Since a C /Cu1 redox couple is responsible for the dismutase activity of the enzyme, the role of zinc is of interest. Both 220-MHz NMR measurements of the exchangeable histidine protons and chemical modifications using diethylpyrocarbonate demonstrate that zinc alone can fold the protein chain in the region of the active site into a conformation resembling that of the native enzyme. Other possible roles for zinc are discussed. Synthetic, magnetic, and structural studies of soluble, imidazolate-bridged copper complexes of relevance to the 4 Cu(II) form of the enzyme have been made. 

Superoxide dismutase catalyzes the disproportionation of superoxide as 2O2 -i- 2H — O2 -I- H2O2. The Cu,Zn-superoxide dismutases are widely distributed in both plant and animal kingdoms and are found in eukaryotic cytosols. The vast majority of studies on superoxide dismutases have been made on the enzyme from bovine erythrocytes this enzyme contains 151 amino acid residues and two copper and two zinc atoms per molecule of —32,000 Da. The crystal structure of this protein has been reported (97), and the active site was shown to consist of a bimetallic (Cu,Zn) assembly (see Fig. 6). A type-2 copper center is... 

It has been concluded from a study of the optical and e.p.r. spectra of Co —Cu bovine superoxide dismutase, in which zinc has been replaced by cobalt, that the cobalt site reactivity should be described in terms of reaction of the Co-imidazolate-Cu system as a whole the crystal structure reported last year indicated that the metals were linked by a common histidine residue. There is an exchange interaction between the cobalt and copper however, this is abolished when the linking imidazole is protonated. Further evidence for the close proximity and interactive dependence of the zinc and copper binding sites was obtained from a study of the 4 Cu protein a two-fold enhancement of the activity of 2 Cu dismutase was observed upon occupation of the zinc sites by the Cu ". On the basis of C1 n.m.r. studies, Fee and Ward have suggested that one co-ordination position of Cu in superoxide dismutase is normally occupied by water they further suggest that superoxide can displace the solvent to form a cupric peroxide complex. 

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,... 

The dismutation of O2 by iron superoxide dismutase was found to be similar to that for the copper/zinc bovine superoxide dismutase. The results obtained by Lavelle et al. (1977) showed that catalysis of dismutation of O2 by the iron superoxide dismutase from Photobacterium leiognathi is first order with respect to substrate concentration for all ratios of substrate to enzyme concentrations reported. Although the enzyme is stable between pH 6.0 and 10.0, the value of the rate constant decreases as the pH increases. The second-order rate constant for the reaction be-... 

Cadmium-113 nuclear magnetic resonance studies of the cadmium substituted bovine superoxide dismutase were carried out Only a very small chemical-shift difference between the 2 Cd(Il) protein (Cd(II) is bound to the zinc site and the copper site is unoccupied) and the 2 Cd(ll)—2 Cu(I) enzyme (analogous to the reduced form of the native protein) was found. This was interpreted in that the imidazolate bridge is protonated at the Cu site after reduction. 

Scheme 1. pH-dependent migration of copper to the vacant zinc-binding site of zinc free bovine erythrocyte superoxide dismutase... 

In the superoxide dismutase of bovine erythrocytes, and possibly elsewhere, zinc and copper are both linked to a histidine moiety. Analogous enzymes containing manganese instead of copper occur in both mammalian and bacterial organisms (Oberley and Buettner, 1979). For further reading on this highly unusual metalloenzyme, see Oberley (1982) and Rodgers and Powers (1981). 

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