Mechanism of S-Nitrosothiol Formation and Degradation Mediated by Copper Ions

Abstract

Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu 2؉ , but not Fe 3؉ , induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu ؉ is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu 2؉ , the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu 2؉ in S-nitrosothiol formation with the known destabilizing effect of Cu ؉ , providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation. S-Nitrosothiols (RS-NOs)1 have a variety of biological activities, which are mostly attributed to their ability to release NO (1-3). RS-NOs are not only synthesized and administered clinically (2) but are also produced endogenously. Stamler et al. (4) reported that human plasma contains ϳ7 M RS-NOs, mostly as S-nitroso-albumin, a level unexpectedly high as the basal cellular NO level is in the low nanomolar range (5, 6). Thus, RS-NOs are considered as NO pools buffering the level of NO, which may be targeted at different sites (7). RS-NOs are also reported to be involved in the trans-S-nitrosation of proteins by transferring the NO ϩ moiety (8, 9), a process suggested to be a reversible post-translational modification regulating the activity of enzymes and receptors (3, 10, 11). (21), respectively. It is worth noticing that the reaction with water efficiently competes with direct thiol nitrosation by N 2 O 3 , due to the large molar excess of water over thiols. NAD ϩ substituting oxygen for the electron acceptor can also accelerate the reaction of NO with thiols (22). Several authors also suggested that S-nitrosation of thiols occurs by reaction with nitrosonium ions (NO ϩ ) formed either via metalcatalyzed oxidation of NO or via dinitrosyl-iron-cysteine complexes (8,21,23,24); efficiency and physiological relevance of these reactions remain unclear.In this study we have examined by spectroscopic and amperometric techniques the interaction of NO and thiols in the presence of cupric and ferric ions. Experiments have been carried out using the small tripeptide GSH (low millimolar amounts in the cell), bovine serum albumin (BSA, which is the most abundant plasma protein), and human hemoglobin (Hb). BSA and GSH both bear only one reduced cysteine per molecule (Cys-34 in BSA; Refs. 25 and 26), but, as shown below, they display in the presence of Cu 2ϩ a very different reactivity with NO...

Nitric Oxide-dependent Processing of Heparan Sulfate in Recycling S-Nitrosylated Glypican-1 Takes Place in Caveolin-1-containing Endosomes

“…1C, detection of total Gpc-1 protein was unaffected by ascorbate (GPC ϩ Ascorbate), whereas the S1 epitope was destroyed (GPC-S1 ϩ Ascorbate). Re-S-nitrosylation with NO is dependent on a Cu 2ϩ /Cu ϩ redox cycle (34). Accordingly, when NO donor and Cu 2ϩ were provided after ascorbate treatment, the S1 epitope was partially restored (GPC-S1 ϩ Ascorbate ϩ SNP ϩ CuCl 2 ).…”

“…Then, by using NO released from the intrinsic SNO groups, anMan-containing HS-oligosaccharides are generated by deaminative cleavage at the GlcNH 3 ϩ residues. The triggering mechanism for NO release remains unknown, but both S-nitrosylation and NO release are dependent on a Cu 2ϩ /Cu ϩ redox cycle (34). The redox state of the cell may therefore regulate NO release.…”

S-Nitrosylation — another biological switch like phosphorylation?

“…Studies have shown that in the presence of NO, Cu 2+ ion induces a fast S-nitrosation of bovine serum albumin and human hemoglobin, and this reaction is prevented by thiol blocking reagents. During this reaction, Cu 2+ is accumulated and causes destabilization of the S-nitrosothiols formed (Stubauer et al, 1999). In another study it was shown that lower concentration Fe 2+ favored formation and stabilization of S-nitrosothiols however higher concentrations (higher than 10µM) abolished the effect.…”

Copper dependence of the biotin switch assay: Modified assay for measuring cellular and blood nitrosated proteins

“…It appears that the success of the biotin switch assay is quite dependent on the contamination of this and other metals in the solutions. It is known that reduced copper can reduce RSNO [25,29,30,42,43]. The mechanism for the decomposition of S-nitrosothiols by Cu(I) ions likely involves the one-electron reduction of the RSNO group to give the free thiol, NO and Cu(II).…”