Solvent channels and electric fields guide proton delivery to the active site of heme peroxidases

The active site architecture of heme enzymes has evolved to control the formation of highly reactive intermediates for oxidative catalysis. Finely tuned proton delivery to heme is essential, yet the mechanisms of proton delivery and the sources of protons are poorly understood. Here, we identify routes and drivers of proton delivery in a heme peroxidase enzyme (ascorbate peroxidase) employing a range of computational molecular modeling approaches, including molecular dynamics (MD) simulations, density functional theory (DFT) and combined quantum mechanics/molecular mechanics (QM/MM) calculations, enhanced sampling techniques, and local electric field (LEF) analyses. Our results show that networks of active site water molecules facilitate proton ex-change with Arg38, which may serve as a transient proton carrier at the γ-edge of the heme, where the substrate (ascorbate) binds. The distal His42 residue aids proton transfer into the active site via solvent from the δ-heme edge. MD simulations of three peroxidases identify hydrated channels in the structure – pointing towards both the γ- and δ-edges of the heme – through which protons from solvent can access the active site. Comparison with structures of 8 other heme peroxidases shows that these channels are conserved features. LEF calculations show a continuous electrostatic funnel, running from positive to negative, that pulls protons from the protein surface along the channels towards the heme iron from both the γ- and δ-heme edges. This electric field is also found to be a conserved feature in several peroxidases; its direction and shape is conserved but the gradient varies between differ-ent enzymes, suggesting that nature preorganises the electric field as a determinant of relative activity. Rather than a simplistic model for proton delivery (in which a single substrate provides a single proton (and an electron) to the heme) the data instead show conserved solvent channels and electrostatic funnels, which provide multiple well defined routes for proton delivery in peroxidase catalysis.