Nitric Oxide Physiological Responses and Delivery Mechanisms Probed by Water-Soluble Roussin’s Red Ester and {Fe(NO)₂}¹⁰ DNIC

Abstract

Dinitrosyl-iron complexes (DNICs) are stable carriers for nitric oxide (NO), an important biological signaling molecule and regulator. However, the insolubility of synthetic DNICs, such as Roussin's red ester (RRE), in water has impaired efforts to unravel their biological functions. Here, we report a water-soluble and structurally well-characterized RRE [Fe(mu-SC2H4COOH)(NO)2]2 (DNIC-1) and a {Fe(NO)2}(10) DNIC [(PPh2(Ph-3-SO3Na))2Fe(NO)2] (DNIC-2), their NO-induced protein regulation, and their cellular uptake mechanism using immortalized vascular endothelial cells as a model. Compared with the most common NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), the in vitro NO release assay showed that both DNICs acted as much slower yet higher stoichiometric NO-release agents with low cytotoxicity (IC50 > 1 mM). Furthermore, L-cysteine facilitated NO release from SNAP and DNIC-1, but not DNIC-2, in a dose- and time-dependent manner. EPR spectroscopic analysis showed, for the first time, that intact DNIC-1 can either diffuse or be transported into cells independently and can transform to either paramagnetic protein bound DNIC in the presence of serum or [DNIC-(Cys)2] with excess L-cysteine under serum-free conditions. Both DNICs subsequently induced NO-dependent upregulation of cellular heat shock protein 70 and in vivo protein S-nitrosylation. We conclude that both novel water-soluble DNICs have potential to release physiologically relevant quantities of NO and can be a good model for deciphering how iron-sulfur-nitrosyl compounds permeate into the cell membrane and for elucidating their physiological significance.

X‐Ray Emission Spectroscopy: A Spectroscopic Measure for the Determination of NO Oxidation States in Fe–NO Complexes

“…Endogenous formation of DNICs derived from nitrosylation of CIP triggers the subsequent transnitrosation to afford cellular S‐nitrosothiol 3a. Induction of NO‐dependent upregulation of celluar heat shock protein 70 and in vivo protein S‐nitrosylation by treatment of exogenous DNICs was also reported 4…”

Nitrogen Monoxide (NO) Storage and Transport by Dinitrosyl-Dithiol-Iron Complexes: Long-lived NO That Is Trafficked by Interacting Proteins

“…Collectively, it can be suggested that, at lower concentrations, DNICs may function as NO carriers and release it (59,60), which can occur by spontaneous decomposition of DNICs in vitro (61), to acceptors with higher affinity for NO (e.g. the heme center of sGC).…”

“…the heme center of sGC). In fact, for DNICs to be a storage form of NO would imply that NO is liberated and/or transferred at some point (49,60,62). Although yet to be directly demonstrated, this could assist NO-dependent signaling in vivo.…”

Electronic and Spatial Structures of Water-Soluble Dinitrosyl Iron Complexes with Thiol-Containing Ligands Underlying Their Ability to Act as Nitric Oxide and Nitrosonium Ion Donors

“…This hypothesis was first stated in experiments illustrating the ability of M-DNIC with cysteine for in vivo S-nitrosation of intracellular proteins in EA.hy926 cells obtained by fusion of primary human umbilical vein endothelial cells with human lung carcinoma cells [71]. The concentration of S-nitrosated proteins determined by the biotin method increased after incubation of cells with DNIC, reaching the maximum level after 24 h. The {Fe(NO) 2 } 9 structure of M-DNIC (note: the structure of the core complex appears as [Fe + (NO) 2 ]) led the authors to conclude that S-nitrosating activity of DNIC with cysteine is determined by NO released from DNIC and its subsequent oxidation to nitrogen dioxide.…”