Nitric oxide (NO^•) is a gaseous free radical produced naturally by our body. It is involved in many physiological and pathological processes, playing important roles in vasodilation, inflammation and immune response. Nitric oxide metabolism generates nitrite, peroxynitrite, dinitrosyl iron complexes (DNIC) and nitrosothiols. Among these derivatives, DNICs are the most abundant in the intracellular environment and presumably have physiological functions that are being intensively studied, including their role in the S-nitrosation of proteins.

In an article published in the journal Chemical Communications of the Royal Society of Chemistry, Daniela Ramos Truzzi, a professor at the Instituto de Química of the Universidade de São Paulo and a member of the RIDC Redoxoma, described the mechanism of dinitrosyl iron complexes (DNICs) formation, identifying thiyl radicals, detected by electronic paramagnetic resonance (EPR), as co-products of this process. The results of the research provide a new route for the formation of nitrosothiols.

“The literature has already shown that increases in DNIC cellular levels are concomitant to increases in nitrosothioles levels and it was believed that DNICs, once formed, promoted S-nitrosation of biothiols, but there was no explanation for this. Our work shows that the DNIC synthesis itself generates thiyl radicals in a nitric oxide rich environment, and in this condition nitrosation reactions are inevitable. For the first time it has been shown that the mechanism of DNIC formation can lead to the formation of nitrosothiols,” said Truzzi, who carried out part of the work at the University of California, Santa Barbara, USA, as a postdoc under the supervision of chemist Peter C. Ford.

S-nitrosation is a post-translational modification and consists of the addition of a nitroso group to a thiol, affecting the activity of proteins of different functional classes and influencing various physiological processes. Thiols are organosulfur compounds of the form R–SH. Thiol-proteins (Cys–SH) have been increasingly studied as redox adapters that regulate specific molecular targets, with multiple cellular functions. Thus, proteins S-nitrosation can also be considered a redox signaling mechanism dependent on nitric oxide.

DNICs and S-nitrosation

A large number of recent studies have shown the occurrence of S-nitrosation in cells and tissues, associated with various physiological processes such as vascular homeostasis, autophagy, and immune response. Dysregulation of protein S-nitrosation has been associated with several diseases, including neurodegenerative disorders, various cancers, and diabetes.

Since nitric oxide does not react directly with thiols, several mechanisms for the formation of nitrosothiols have been suggested, and DNICs, which are considered to be nitric oxide reservoirs and carriers, have been proposed as intermediates of S-nitrosation reactions.

DNICs are known to inorganic chemists for a long time, and they have been detected in cells and tissues since 1965, even before the discovery that nitric oxide is produced endogenously. Their physiological roles are related to nitric oxide. It has been shown that these complexes induce vasodilation, inhibit platelet aggregation, accelerate wound healing and have therapeutic potential. However, according to Truzzi, their chemical nature remains unknown and their biochemical properties have been little explored. The researcher explains that the study of DNICs is an interface between biochemistry and inorganic chemistry.

To investigate the mechanism of the complexes formation, the researchers worked with cysteine and glutathione, which are low-molecular-weight thiols abundant in cells, in addition to iron and nitric oxide. The experiments were performed in the absence of oxygen, to avoid secondary reactions, and in aqueous media, pH 7.4, which approximates physiological conditions. “Because the reactions are very fast – in 140 milliseconds we already see what is happening – we used a flow cavity in the EPR, where the solutions are mixed and quickly directed to the detector,” explained the researcher. “We started from iron II and the final complex is formed by iron I, because an oxidoreduction reaction occurs, in which the iron is reduced and the thiol is oxidized. To identify the thiyl radical, we used spin traps, which are molecules that bind to specific radicals, making them more stable and allowing their detection.” Kinetic studies, not yet published, were also performed.

According to the researcher, the next step is to study proteins, whose structural complexity provides a varied chemical environment and may confer a greater specificity for the formation of nitrosothiols.

The article Thiyl Radicals Are Co-products of Dinitrosyl Iron Complexes (DNICs) Formation, by Daniela R. Truzzi, Ohara Augusto and Peter C. Ford, can be accessed at this link.