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Role of copper, zinc-superoxide dismutase in S-nitrosation and denitrosation of cysteine residues


Role of copper, zinc-superoxide dismutase in S-nitrosation and denitrosation of cysteine residues

Ye, Mengwei (2007) Role of copper, zinc-superoxide dismutase in S-nitrosation and denitrosation of cysteine residues. PhD thesis, Concordia University.

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Selective inhibition of the S -nitroso- L -glutathione (GSNO) reductase activity but not the superoxide dismutase (SOD) activity of Cu,Zn-superoxide dismutase (CuZnSOD) by the commonly used polyaminocarboxylate metal-ion chelators, EDTA and DTPA, is systematically investigated here. The effects of EDTA and DTPA on the Cu II d-d absorption of Cu II ZnSOD at 680-nm, on the reduction of Cu II ZnSOD by GSH, on the metal-loading of the enzyme, on the chemical modification of catalytic Arg141, and on the SOD and GSNO-reductase activities were studied. Binding to the enzyme was probed by measuring heat changes with ITC on titration of CuZnSOD and the metal-free EESOD with the chelators. The results reveal that EDTA and DTPA bind to the solvent-exposed active-site copper of one subunit without removing the metal. This induces a conformational change at the second active site that inhibits the GSNO-reductase but not the SOD activity of the enzyme. The effects of the copper colorimetric reagents, neocuproine (neo), diethyldithiocarbamate (DDC), and cuprizone (cup), on the GSNO-reductase and the SOD activities of CuZnSOD were also examined. These chelators alter the Cu II d-d absorption of Cu II ZnSOD at 680 nm and give the absorption characteristic of their CuL 2 complexes. The GSNO-reductase activity of CuZnSOD was inhibited by the three colorimetric reagents in the order: DDC > neo > cup which mirrors the extent of CuL 2 formation. Incubation of CuZnSOD with 5 mM DDC also resulted in 90% loss of its SOD activity due to removal of {598}73% of the active-site copper. No loss of SOD activity resulted from CuZnSOD exposure to neo or cup. These results indicate that removal of the active-site copper is necessary for loss of SOD activity whereas association of the chelators with the active-site copper is sufficient for loss of GSNO-reductase activity. Chemical modification at pH 8.0 of Arg- and Cys-containing peptides and CuZnSOD by phenylglyoxal (PGO), an arginine specific reagent, was re-evaluated. ESI-MS analysis shows that doubly PGO-labelled Arg and Cys residues were formed in the peptides but only singly PGO-labelled Arg residues formed in CuZnSOD. Loss of SOD activity of PGO-modified CuZnSOD was observed due to derivatization of its Arg residues by PGO. Labelling of multiple Arg residues in the enzyme is in disagreement with previous reports that Arg141, which is essential for SOD activity, was selectively PGO-modified. Two to four free Cys residues per tetramer of commercial rat hemoglobin (Hb) from Sigma and of Hb isolated from rat red blood cells (RBCs) were determined by 5,5'-dithiolbis(2-nitrobenzoic acid) (DTNB) titration and by ESI mass spectrometry using N -ethylmaleimide (NEM). Both CysÝ125 and CysÝ93 in rat RBC oxyHb were S -nitrosated and S -glutathiolated in GSNO/oxyHb (10:1) incubations with and without 20 oM bovine CuZnSOD indicating that this enzyme is not essential for S -nitrosation of rat Hb under the conditions used here. Based on the Saville assay, NO release from rat Hb-SNO in the presence of GSH was catalyzed by free copper ions and by CuZnSOD. As observed for the GSNO-reductase activity of CuZnSOD, Hb-SNO denitrosation was inhibited by the metal chelators confirming a catalytic role for copper. Combining the results presented in thesis with those obtained from the literature helps us understand the role of CuZnSOD in the formation of Hb-SNO and the release of NO from Hb-SNO.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Ye, Mengwei
Pagination:xxix, 272 leaves : ill. ; 29 cm.
Institution:Concordia University
Degree Name:Ph. D.
Thesis Supervisor(s):English, Ann
Identification Number:LE 3 C66C54P 2007 Y4
ID Code:975690
Deposited By: Concordia University Library
Deposited On:22 Jan 2013 16:12
Last Modified:13 Jul 2020 20:08
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