Quantum mechanics/molecular mechanics calculations of the vanadium dependent chloroperoxidase

Large quantum mechanics/molecular mechanics (QM/MM) calculations are used to probe the resting and initial protonated states of the vanadium dependent chloroperoxiclase from the pathogenic fungus Curvularia inaequalis. QSite was used to model 433 residues and 24 structural waters with molecular mechanics, while 8 active-site residues and the vanadate cofactor (161 atoms) were represented at the B3LYP/lacvp* level of theory. Our previous study of small model systems implied that the resting state of the enzyme contains a trigonal bipyramidal vanadate with one hydroxyl group in the equatorial plane and another in the axial position. This study uses a much larger model of the biological system at a higher level of theory to identify the location of the equatorial hydroxo group with respect to the enzyme active site. We also identify a second resting-state configuration with an axial water and three equatorial oxo moieties that is nearly isoenergetic with the previously identified state. We propose that the resting state is a hybrid of these two configurations, stabilized by the long-range electrostatic field of the protein environment. The first step in catalysis is believed to be protonation of the vanadate. Our previous small models indicated that there were two protonated configurations, but this study shows that the configuration containing an axial water and one hydroxo group in the equatorial plane is significantly lower in energy than any other configuration. Additionally, we can now assign an important role for lysine 353 in the catalytic cycle. Based on our calculations and other model studies, we provide an updated catalytic cycle for vanadium A dependent haloperoxidase activity. Further, we demonstrate the importance of system set up In particular, maintaining the proper electrostatic field at the active site is crucial for identifying the correct minima in a truncated protein model.