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The binding of copper ions to copper-free bovine superoxide dismutase. Properties of the protein recombined with increasing amounts of copper ions.

ABSTRACT: 1. E.p.r. (electron-paramagnetic-resonance), proton-relaxation and u.v.-absorption parameters, and enzyme activity of samples of Cu2+-free bovine superoxide dismutase recombined with different amounts of Cu2+ up to the stoicheiometric [Cu2+]/protein] ratio were investigated after attainment of equilibrium in the recovery process. 2. The e.p.r. spectra were identical with the spectrum of the native protein at all [Cu2+]/[protein] ratios. The relaxation rate of the water protons (T1) and the u.v. absorption increase as linear functions of the added Cu2+. 3. On the other hand, in recombination experiments in the range pH 7.6-10.5 the enzyme activity shows a non-linear increase as the [Cu2+]/[protein] ratio rises. The experimental curves can be interpreted in terms of the model of co-operative binding of Cu2+ to the two sites proposed on the basis of the electrophoretic analyses of the samples, and show that the specific activity of the molecules containing only one Cu2+ ion is twice as high as that of the molecules with two Cu2+ ions. 4. These results support the hypothesis of an anti-co-operative interaction between the two sites during the activity, which allows only one Cu2+ ion to function in catalysis.

SUBMITTER: Rigo A

PROVIDER: S-EPMC1164471 | biostudies-other | 1977 Jan

REPOSITORIES: biostudies-other

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Project description:Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms.Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

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The binding of copper ions to copper-free bovine superoxide dismutase. Properties of the protein recombined with increasing amounts of copper ions.

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