Oxidative HNO Formation Mechanisms from NH ₂ OH via Two Different Heme Proteins and Effects from Hemes, Nearby Residues, and Protein Environments

Nitroxyl (HNO) is a biologically active nitrogen oxide with therapeutic potential for several disorders, but its endogenous formation remains poorly understood. Previous experimental work showed that hydroxylamine (NH₂OH), a possible physiological precursor, was oxidized by hydrogen peroxide (H₂O₂) activated heme proteins to produce HNO. In this study, we employed both density functional theory (DFT) and hybrid quantum mechanics and molecular mechanics (QM/MM) calculations to investigate the reaction mechanisms of HNO generation from NH₂OH via two representative heme proteins, myoglobin (Mb) and catalase (CAT), with neutral and negative axial ligands. Various structural models to successively include more components of the proteins were studied to provide more detailed insights into the protein environment effect. Our results reveal a stepwise double hydrogen atom abstraction pathway initiated by the ferryl compound I (Cpd I) species after the H₂O₂ treatment, with the first H-abstraction as the rate-determining step. Mb, with a neutral histidine axial ligand, exhibited a reaction barrier lower than that of CAT (which contains a negatively charged tyrosine ligand), which was in good agreement with the observed experimental reactivity trend. Protein environment effects, including distal hydrogen bonding and steric hindrance, were found to modulate both proton affinity and electron transfer, influencing the reaction barriers. QM/MM calculations confirmed that while key residues near the heme center can enhance HNO formation through hydrogen bonding, spatial constraints in the full protein environment can raise the reaction barrier. These mechanistic insights explain experimentally observed differences in HNO yields and highlight how protein structure and heme coordination influence nitroxyl biosynthesis.