Hydrophilicity of heme derivatives influences site-specific tyrosine nitration in MnSOD.

Tyrosine nitration is a critical post-translational modification linked to various oxidative stress-related diseases. However, the factors governing site selectivity remain incompletely understood. Manganese superoxide dismutase (MnSOD) is particularly vulnerable to nitration at Tyr34 under pathological conditions, which significantly affects its enzymatic activity. Previous studies have shown that peroxynitrite efficiently nitrates Tyr34 in MnSOD, whereas lactoperoxidase (LPO)-mediated nitration in the H2O2-NO2- system shows limited reactivity at this site. Consequently, peroxynitrite-induced nitration is often associated with MnSOD inactivation in vivo. We hypothesized that the hydrophilicity of nitrating catalysts influences tyrosine site selectivity. To test this, we compared the nitration profiles induced by hydrophobic heme, a hydrophilic heme analogue FeTPPS, and a heme-hIAPP complex formed by heme and hydrophilic human islet amyloid polypeptide (hIAPP). All three nitrating agents induced nitration of four tyrosine residues (Tyr2, Tyr9, Tyr11, and Tyr34) in MnSOD. Among them, FeTPPS caused the highest Tyr34 nitration (40.1%), surpassing peroxynitrite (23.6%), while heme and heme-hIAPP yielded lower levels (16.8% and 19.4%, respectively), underscoring the influence of heme hydrophilicity on nitration site selectivity. MnSOD activity declined significantly after nitration, with FeTPPS causing greatest loss. Fluorescence spectroscopy and molecular docking indicated that both heme and FeTPPS bind near Tyr34, with heme engaging mainly through hydrophobic contacts, π-π stacking, and hydrogen bonding, whereas FeTPPS bound predominantly through electrostatic interactions. The heme-hIAPP complex also bound near Tyr34. Compared to FeTPPS, heme binding reduced hydrophilicity around Tyr34, suppressing its nitration. These results confirm that heme hydrophilicity plays a key role in protein tyrosine nitration selectivity.