NiII -ATCUN-Catalyzed Tyrosine Nitration in the Presence of Nitrite and Sulfite

Abstract

The nitration of tyrosine residues in proteins represents a specific footprint of the formation of reactive nitrogen species (RNS) in vivo. Here, the fusion product of orange protein (ATCUN-ORP) was used as an in vitro model system containing an amino terminal Cu(II)- and Ni(II)-binding motif (ATCUN) tag at the N-terminus and a native tyrosine residue in the metal-cofactor-binding region for the formation of 3-NO₂ -Tyr (3-NT). It is shown that Ni^II -ATCUN unusually performs nitration of tyrosine at physiological pH in the presence of the NO₂ ^- /SO₃ ^2- /O₂ system, which is revealed by a characteristic absorbance band at 430 nm in basic medium and 350 nm in acidic medium (fingerprint of 3-NT). Kinetics studies showed that the formation of 3-NT depends on sulfite concentration over nitrite concentration suggesting key intermediate products, identified as oxysulfur radicals, which are detected by spin-trap EPR study by using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). This study describes a new route in the formation of 3-NT, which is proposed to be linked with the sulfur metabolism pathway associated with the progression of disease occurrence in vivo.

Keywords: ATCUN motif; orange protein; radicals; sulfur metabolism; tyrosine nitration.