On the role of water in peroxidase catalysis: a theoretical investigation of HRP compound I formation.

We have investigated the dynamics of water molecules in the distal pocket of horseradish peroxidase to elucidate the role that they may play in the formation of the principal active species of the enzymatic cycle (compound I, Por(o+)-Fe(IV)=O) upon reaction of the resting Fe(III) state with hydrogen peroxide. The equilibrium molecular dynamics simulations show that, in accord with experimental evidence, the active site access channel is hydrated with an average of two to three water molecules within 5 A from the bound hydrogen peroxide. Although the channel is always hydrated, the specific conformations in which a water molecule bridges H(2)O(2) and the distal histidine, which were found (Derat; et al. J. Am. Chem. Soc. 2007, 129, 6346.) to display a low-energy barrier for the initial acid-base step of the reaction, occur with low probability but are relevant within the time scale of catalysis. Metadynamics simulations, which were used to reconstruct the free-energy landscape of water motion in the access channel, revealed that preferred interaction sites within the channel are separated by small energy barriers (<1.5 kcal/mol). Most importantly, water-bridged conformations lie on a shoulder just 1 kcal/mol above one local minimum and thus are easily accessible. Such an energy landscape appears as a requisite for the effectiveness of compound I formation, whereby the H-bonding pattern involving reactants and catalytic residues (including the intervening water molecule) has to rearrange to deliver the proton to the distal OH moiety of the hydrogen peroxide and thereby lead to heterolytic O-O cleavage. Our study provides an example of a system for which the "reactive configurations" (i.e., structures characterized by a low barrier for the chemical transformation) correspond to a minor population of the system and show how equilibrium molecular dynamics and free-energy calculations may conveniently be used to ascertain that such reactive conformations are indeed accessible to the system. Once again, the MD and QM/MM combination shows that a single water molecule acts as a biocatalyst in the cycle of HRP.