Mechanistic investigation of the denitrosylation activity of a water-soluble copper(ii) compound probed by experimental and computational approaches

Abstract

Overproduction of nitric oxide (˙NO) with subsequent formation of peroxynitrite has a major impact on cell death, inflammation, and disease development. ˙NO exerts a significant part of its biological activity through S-nitrosylation, leading to the formation of protein and low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO). Excessive oxidative/nitrosative stress contributes to disease by S-nitrosylating multiple targets, culminating in the accumulation of misfolded proteins and cytotoxicity. We here report on the discovery that a water-soluble copper(ii) compound [Cu(HL)Cl₂] (1) (HL = N-2[(pyridine-2-ylmethyl)aminoethanol]) promotes homolytic cleavage of the S–NO bond to form ˙NO and glutathione disulfide (GSSG), following initial coordination of the sulfur atom of GSNO to the Cu(ii) center. The X-ray crystal structure of complex 1 is presented. Potentiometric titration studies indicate that the species [Cu(HL)(H₂O)₂]²⁺ is the major species at physiological pH (K_f = 9.33 × 10⁶). Monitoring the reaction between 1 and GSNO by EPR spectroscopy revealed that the Cu(ii) signal remains stable throughout the reaction. The release of ˙NO was confirmed using the spin trap [Fe(DETC)₂], while formation of the GS˙ intermediate was detected using DMPO. EPR analysis also confirmed the formation of a Cu–S bond, as evidenced by superhyperfine coupling, further corroborated by Raman spectroscopy and UV–Vis studies. The latter indicates that two equivalents of GSNO coordinate to the copper centers, in agreement with the kinetic studies. The formation of several intermediate species and production of GSSG was confirmed by ESI-(+)-MS. Gas-phase chemiluminescence experiments performed under both aerobic and anaerobic conditions showed that the amounts of ˙NO produced are independent of the presence of oxygen. Taken together, our experimental data suggest that, during GS–NO bond cleavage, the Cu(ii) center does not undergo reduction, allowing the proposal of a novel reaction mechanism, the feasibility of which is supported by theoretical calculations. Thus, copper(ii) complexes with tridentate ligands promote efficient denitrosylation without undergoing bulk electron transfer of the transition metal, potentially opening new avenues for modulating S-nitrosothiol stability in disease conditions known to be associated with excessive nitrosation.

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