Abstract
The active sites of heme enzymes have evolved to control the formation of highly reactive intermediates in oxidative catalysis. Proton delivery to the heme is essential, yet the mechanisms of proton delivery remain poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase (ascorbate peroxidase) using computational approaches that combine classical, quantum, and hybrid methods with enhanced sampling and local electric field (LEF) analyses. Our results show that networks of active-site water molecules facilitate proton exchange with Arg38, which may act as a transient proton carrier at the γ-heme edge where the substrate binds. The distal His42 residue aids proton transfer into the active site via solvent at the δ-edge. Molecular dynamics simulations of three heme peroxidases identify hydrated channels leading to both γ- and δ-edges, allowing solvent protons to reach the active site. Comparison with eight other heme peroxidases shows that these channels are conserved. LEF analyses reveal a continuous electrostatic funnel drawing protons toward the heme from the γ- and δ-edges, a feature that is broadly conserved across other peroxidases. These results suggest that nature pre-organizes electrostatic funnels and solvent channels to provide multiple well-defined routes for proton delivery in peroxidase catalysis.