Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

Abstract

Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (•NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of •NO with O 2, reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of •NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 M) O2 concentrations and exhibits zero-order dependence on •NO concentration. Indeed, RSNO formation is not inhibited even at O 2 levels <1 M. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O 2-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of •NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by Ϸ50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free •NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells.iron ͉ nitrosation ͉ reactive nitrogen species ͉ reactive oxygen species ͉ chelatable iron N itric oxide (nitrogen monoxide, •NO) is a ubiquitous signaling molecule that originally was thought to exert its effects solely through interaction with transition metal ligands of proteins, most notably the heme group of soluble guanylate cyclase. However, it is now recognized that •NO is capable of affecting cell physiology by inducing oxidative and covalent modification of protein amino acid residues. One such modification, S-nitrosation, the addition of a nitroso group to a thiol to form a nitrosothiol (RSNO), has received considerable attention as a potentially important posttranslational protein modification. S-nitrosated products are found ubiquitously in vivo (1), and thiol nitrosation has been found to alter the activity of a diverse set of proteins and may therefore represent an important concept in cellular and organismal biology (2).A central issue in this paradigm is understanding the routes by which RSNO are formed inside cells. Importation of low molecular weight (LMW) RSNO through LAT transporters, followed by transnitrosation reactions, have been demonstrated to be a potent route for intracellular RSNO formation (3). However, mechanisms of de novo synthesis from free •NO are less clear. Proposed mechanisms include the reaction of •NO with O 2 (autoxidation), either in the aqueous phase (4) or in ...

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“…3C). S-nitrosation of C151 presumably proceeds by transnitrosation, possibly via dinitrosyliron complexes (26).…”

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation

“…Studies exploring the S-nitrosylation of proteins in cells indicated that more than 50% of the cellular formation of protein S-nitrosocysteine is derived by dinitrosyliron complexes (7). Within the liver S-nitrosoproteome, 13 Snitrosylated cysteine residues, which are directly involved in the chelation of metal ions, were identified (Table S3).…”

“…The identification of in vivo S-nitrosylated proteins has indicated that not all reduced cysteine residues and not all proteins with reduced cysteine residues are modified, implying a biased selection. Several biological chemistries have been proposed to account for the S-nitrosylation of proteins in vivo (1,7,8). Broadly, these include (i) oxidative S-nitrosation by higher oxides of NO, (ii) transnitrosylation by small molecular weight NO carriers such as S-nitrosoglutathione or dinitrosyliron complexes, (iii) catalysis by metalloproteins, and (iv) protein-assisted transnitrosation, as elegantly documented for the S-nitrosylation of caspase-3 by S-nitrosothioredoxin (9, 10).…”

Nitric Oxide Storage and Transport in Cells Are Mediated by Glutathione S-Transferase P1-1 and Multidrug Resistance Protein 1 via Dinitrosyl Iron Complexes

“…These effects are explained by the reactivity of NO with iron in iron-sulfur clusters, e.g. in the electron transport chain and other pools (1,3). The high affinity of NO for Fe(II) results in the interaction of NO with iron-sulfur clusters in proteins.…”