S-Nitrosation of Serum Albumin by Dinitrosyl-Iron Complex

Abstract

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO) 2 Fe(L-cysteine) 2 ], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO) 2 Fe(L-cysteine) 2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NO x -species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyliron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO) 2 Fe(Lcysteine) 2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyliron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Nitrogen monoxide (NO)-mediated iron release from cells is linked to NO-induced glutathione efflux via multidrug resistance-associated protein 1

“…5A). The nature of these EPR signals suggests the nitrosyl-Fe complex observed is formed by the interaction with protein thiols (14,29). We compared this spectrum with a synthetic low M r DNIC, namely a dinitrosyl-diglutathionyl-Fe complex (30,31), which gives much sharper, isotropic EPR signals at 293 K (Fig.…”

“…5-8 h (30, 31). DNICs can donate Fe to cells and transnitrosylate targets and increase the bioavailability and efficiency of NO transport (29,34,35).…”

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

“…After treatment with 10 M (1,19,20). These results show that the mechanism responsible for RSNO formation is only slowly reversible and that the increase in RSNO levels ( (21), which in acellular systems are capable of nitrosative chemistry (8,9). DNICs in cells exposed to •NO are formed from the CIP (22), representing a small (Ϸ5%) fraction of total iron thought to be in transit between the various cellular iron pools (23).…”

“…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 an accelerated manner in hydrophobic phases (5) (7), and the interaction between •NO and transition metals and/or metalloproteins (8)(9)(10)(11)(12)(13)(14).…”

“…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 an accelerated manner in hydrophobic phases (5) (7), and the interaction between •NO and transition metals and/or metalloproteins (8)(9)(10)(11)(12)(13)(14).In this study, we examine the contributions of potential nitrosative pathways in RAW 264.7 murine macrophages. We present evidence suggesting that dinitrosyliron complexes (DNICs) formed at least in part from the cellular chelatable iron pool (CIP), through an O 2 -independent transnitrosative process, are responsible for the majority of RSNO formation in cells, and that reactive oxygen species (ROS) enhance RSNO formation, possibly via increases in DNICs.…”