Dinitrosyliron complexes are the most abundant nitric oxide-derived cellular adduct: biological parameters of assembly and disappearance

Abstract

It is well established that nitric oxide (•NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all •NO-derived adducts (900 pmol/mg protein (45–90 μM)). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics of, DNIC assembly and disappearance from endogenous and exogenous sources of •NO in relation to iron and O2 concentrations. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of •NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic •NO concentrations (50–500 nM). Decreasing the O2 concentration reduced the rate of enzymatic •NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t½ = 80 min) and remained detectable long after the •NO source was removed (>24 h). These results demonstrate that DNIC will be formed under all cellular settings of •NO production and that the contribution of DNIC to the multitude of observed effects of •NO must always be considered.

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase

“…Similar to our result shown in Fig. 2, higher DNIC production from bolus ⅐ NO addition in hypoxic cells has been reported previously, and this increase was attributed to the higher ⅐ NO level (2). To test this possibility, we used an ⅐ NO electrode to follow the real-time ⅐ NO level after addition to the cell suspension pretreated with 1 h of anoxia or normoxia.…”

“…3A, the rapid ⅐ NO consumption indicates that significant anoxia has not been achieved under this condition. As explained under "Discussion," this could explain the overestimation of DNIC formation from the bolus ⅐ NO addition under "normoxia" (2).…”

“…It has been reported that the most abundant cellular adduct of ⅐ NO is dinitrosyliron complexes (DNIC) 5 (2). These complexes contain one iron, two nitrosyl groups, and two other ligands, all binding to iron ((L) 2 Fe(NO) 2 ) (3)(4)(5).…”

Mechanisms of Nitric Oxide Reactions Mediated by Biologically Relevant Metal Centers

“…These salts and certain Roussin's red esters (RREs) have been studied as potential sources of therapeutic NO either thermally [22] or photochemically activated [23][24][25][26][27][28][29]. In addition, both mononitrosyl iron complexes (MNICs) and dinitrosyl iron complexes (DNICs) are drawing increasing attention with regard to their potential roles in mammalian physiology [30][31][32][33][34]. The structures drawn in Fig.…”

“…Despite reports that dinitrosyl-iron complexes are the most abundant nitric oxide-derived cellular adducts [34], quantitative investigations of DNIC formation are quite limited. Vanin and coworkers [107] studied the reaction between Fe 2+ , nitrosothiol (RSNO), and thiol (RSH ¼ cysteine or glutathione) and demonstrated that first a mononitrosyl iron complex Fe(NO)(RS) n is formed followed by formation of the DNIC [Fe(NO) 2 …”