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Protein Science (2004), 13:1347-1355. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Mitchel D. de Beus, Jinhyuk Chung and Wilfredo Colón

Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, USA

(RECEIVED January 9, 2004; FINAL REVISION February 12, 2004; ACCEPTED February 14, 2004)



Abstract

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.

Keywords: Cu/Zn superoxide dismutase; SOD; cysteine modification; persulfide; copper; oxidative damage; ALS

Abbreviations: ALS, amyotrophic lateral sclerosis • ANS, 1-anilinonaphthalene-8-sulfonate • BMe, 2-mercaptoethanol • EDTA, ethylenediamine tetraacetic acid • ES-MS, electrospray mass spectrometry • FALS, familial amyotrophic lateral sclerosis • GuHCl, guanidine hydrochloride • H2O2, hydrogen peroxide • KCN, potassium cyanate • MALDI, matrix-assisted laser desorption ionization mass spectrometry • MW, molecular weight • NTA, nitrilotriacetic acid • PAR, 4-pyridylazaresorcinol • PB, potassium phosphate buffer • SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis • SOD, Cu/Zn superoxide dismutase • WT, wild type

Reprint requests to: Wilfredo Colón, Department of Chemistry, Rens-selaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA; e-mail: colonw{at}rpi.edu; fax: 518-276-4887.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03576904.


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