Solvent channels and proton transfer pathways in peroxidase catalysis

The active site architecture of all heme enzymes is designed for fully controlled formation of highly reactive heme intermediates for oxidative catalysis. Finely tuned proton delivery to and from the heme, substrates and active site water molecules is essential, yet the mechanisms of proton delivery and the sources of protons are poorly understood in all heme enzymes. This information is important if the power of heme enzymes is to be exploited for bespoke catalysis. Here, proton transfer pathways in a heme peroxi-dase enzyme (ascorbate peroxidase) are identified using molecular dynamics (MD) simulations, DFT calculations, QM(DFT)/MM calculations and QM(DFTB2)/MM MD simulations. The results show that an active site arginine residue, Arg38, is predominantly positively charged but can transiently deprotonate and adopt different tautomeric states, potentially accepting and donating pro-tons to nearby water molecules, depending on the instantaneous local environment. Extensive MD simulations identify two hy-drated tunnels through which protons from solvent can access the active site from the - and -edges of the heme. In the active site, dynamic proton exchange can occur, assisted by an active site distal histidine residue, and relayed to the substrate via Arg38. QM/MM free energy calculations demonstrate the feasibility of the transfer of a proton from solvent to His42 via distal water molecules. Rather than a simplistic model for proton delivery – where a single substrate provides a single proton (and an electron) – the data instead indicate multiple possible routes for proton delivery during reaction.