Thiolate-based dinitrosyl iron complexes: Decomposition and detection and differentiation from S-nitrosothiols

Abstract

Dinitrosyl iron complexes (DNIC) spontaneously form in aqueous solutions of Fe(II), nitric oxide (NO), and various anions. They exist as an equilibrium between diamagnetic, dimeric (bi-DNIC) and paramagnetic, monomeric (mono-DNIC) forms. Thiolate groups (e.g., on glutathione or protein cysteine residues) are the most biologically relevant anions to coordinate to Fe(II). Low molecular weight DNIC have previously been suggested to be important mediators of NO biology in cells, and emerging literature supports their role in the control of iron-dependent cellular processes. Recently, it was shown that DNIC may be one of the most abundant NO-derived products in cells and may serve as intermediates in the cellular formation of S-nitrosothiols. In this work, we examined the stability of low molecular weight DNIC and investigated issues with their detection in the presence of other NO-dependent metabolites such as S-nitrosothiols. By using spectrophotometric, Electron Paramagnetic Resonance, ozone-based chemiluminesence, and HPLC techniques we established that at neutral pH, bi-DNIC remain stable for hours, whereas excess thiol results in decomposition to form nitrite. NO was also detected during the decomposition, but no S-nitrosothiol formation was observed. Importantly, mercury chloride accelerated the degradation of DNIC; thus, the implications of this finding for the diagnostic use of mercury chloride in the detection of S-nitrosothiols were determined in simple and complex biological systems. We conclude S-nitrosothiol levels may have been substantially overestimated in all methods where mercury chloride has been used.

Hemodynamic Effects of Glutathione-Liganded Binuclear Dinitrosyl Iron Complex: Evidence for Nitroxyl Generation and Modulation by Plasma Albumin

“…Others have reported the production of SNOs from cellular DNICs under hypoxic or anoxic conditions, thus minimizing the possibility of NO • oxidation reactions. However, these previous studies used HgCl 2 to selectively detect SNO concentrations, a method recently found to artifactually detect DNICs as SNOs (Keszler et al, 2017). Although recent closer examinations (Hickok et al, 2011(Hickok et al, , 2012 of this issue agree with our results that production of SNOs from DNICs is not a predominant mode of action of DNICs, if it occurs at all, we would submit that a validated method that selectively detects SNOs in the presence of DNICs is needed to address this question rigorously.…”

Detection and quantification of nitric oxide–derived oxidants in biological systems

“…The sensitivity is in the nanomolar range. The method can be used with any type of gas or liquid sample, including cell lysates and tissue homogenates (68,69).…”

“…3). If buffer at neutral pH is used, only NO ⅐ as such will give a signal (69). One of the most popular reactants for chemiluminescent detection of NO ⅐ and its derivatives is an acidic triiodide solution (65).…”

“…Cellular DNICs can also be quantified through ozone-based chemiluminescence, using the triiodide method. Care should be taken in quantification because the signal is time-sensitive and decays within hours, and also because DNICs are sensitive to HgCl 2 , analogously to S-nitrosothiols (69). To distinguish between signals from S-nitrosothiols and DNICs, it was proposed to stabilize S-nitrosothiols in the cell lysate using a buffer containing diethylenetriaminepentaacetic acid and N-ethylmaleimide and to analyze the sample immediately after extraction and 20 h later to ensure the full decay of DNICs (69).…”

“…Regarding the mechanisms of biological thiol nitrosation, there is evidence supporting the intermediacy of nitrosyl-iron complexes (DNICs) (69,121), as well as the intermediacy of cytochrome c (126).…”