Abstract
HNO plays an important role in many biological processes related to vasodilation, regulation of enzyme activities, and neurological functions. The understanding of enzymatic oxidative HNO-releasing pathways in biology remains scarce. We investigated HNO formation from a well-established small-molecule drug hydroxyurea catalyzed by horseradish peroxidase (HRP). Density functional theory results reveal two sequential proton-coupled electron transfers from hydroxyurea to HRP Compound I as the most favorable mechanism for HNO generation, which was found to be kinetically feasible and thermodynamically favorable. This is consistent with its experimentally observed reactivity. Moreover, the large active site model study uncovered interesting conformation changes involving HRP's amino acid H-bonding network through the reaction pathway, which were employed to anchor the evolving substrate via its key CONH(2) moiety. Detailed computational analysis reveals some useful structural properties for future drug development. In addition, this study presents the first computational investigation of the full reaction mechanism of acyl nitroso hydrolysis for HNO generation, supporting the experimentally observed facile reactivity. Overall, this study reveals, for the first time, the detailed computational reaction mechanism of enzymatic oxidative and hydrolytic HNO generation from a clinical drug, providing structural features beneficial for future drug design.