Protein nitration induced by Hemin/NO: A complementary mechanism through the catalytic functions of hemin and NO-scavenging

Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide ((NO)-N-?) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the (NO)-N-?-binding affinity of hemin has been studied along with the influence of (NO)-N-? on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and (NO)-N-?, with an investigation of the effects of pH of the reaction medium, generation of H(2)O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its (NO)-N-?-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of (NO)-N-? and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and (NO)-N-? in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.

Automated summary

The study investigated the binding affinity of hemin with (NO)-N-?, the internalization of hemin into MDA-MB-231 cells, and the changes in intracellular nitrated proteins. It also examined the nitration of bovine serum albumin after treatment with hemin and (NO)-N-?, considering factors such as reaction medium pH, H2O2 generation, and tyrosine residue oxidation. The results showed that hemin nitrosylation enhanced cellular uptake, induced protein nitration, and depended on (NO)-N-? concentration and reaction medium pH, with protein scavenging effects playing a crucial role.