S-Nitrosation of Serum Albumin by Dinitrosyl-Iron Complex

Matthias Boese,Peter I Mordvintcev,Anatoly F Vanin,Rudi Busse,Alexander Mülsch

doi:10.1074/jbc.270.49.29244

Matthias Boese, Peter I Mordvintcev + Show 3 more

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https://doi.org/10.1074/jbc.270.49.29244

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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)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(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 NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)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 dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Highlights

S-Nitrosation of protein thiols by L-arginine- and nitrovasodilator-derived NO and/or subsequent further reaction of the S-nitrosothiol has been proposed to initiate widespread biological signal and effector pathways [1,2,3]
As a model reaction we assessed the S-nitrosation of bovine serum albumin (BSA) by low molecular weight dinitrosyl-iron-di-L-cysteine complex (DNIC), a representative of naturally occurring iron-nitrosyls generated by the L-arginine-NO pathway in mammalian cells [17,18,19,20,21,22,23,24]
Several lines of evidence indicated that S-nitrosation proceeded by an intermolecular reaction of the free DNIC with the protein thiol, rather than by an intramolecular rearrangement of protein-bound DNIC. 1) Removal of excessive L-cysteine from preformed BSA-DNIC prevented further S-nitrosation

Summary

Introduction

S-Nitrosation of protein thiols by L-arginine- and nitrovasodilator-derived NO and/or subsequent further reaction of the S-nitrosothiol has been proposed to initiate widespread biological signal and effector pathways [1,2,3]. Some back donation of electron density to NO might decrease its electrophilicity, which carries only a partial positive charge (␦ϩ), similar to the situation with mononitrosyl-metal complexes [33] This complex can be regarded as a natural source of NOϩ or NO␦ϩ, capable of S-nitrosation reactions according to Structure 1 (RS ϭ low mass thiol; RЈSH ϭ low mass or protein thiol). To test this hypothesis we assessed S-nitrosation of the free cysteine residue of bovine serum albumin (BSA) by the dinitrosyliron-di-L-cysteine complex. We observed that this complex Snitrosates albumin, in support of our hypothesis that dinitrosyl-iron complexes are intermediates in the biological NO:S-nitrosothiol pathway

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