Quantum Mechanics/Molecular Mechanics Calculations of the Vanadium Dependent Chloroperoxidase

Abstract

Large quantum mechanics/molecular mechanics (QM/MM) calculations are used to probe the resting and initial protonated states of the vanadium dependent chloroperoxidase 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 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.

51V NMR Chemical Shifts Calculated from QM/MM Models of Peroxo Forms of Vanadium Haloperoxidases

“…28 The first step in the catalytic cycle is believed to be protonation of the vanadate, and the QM/MM studies on the native-form of VCPO showed that a configuration containing an axial water and one hydroxo group in the equatorial plane is significantly lower in energy than any other configuration. 26 We have applied a QM/MM scheme to study various …”

Preface: Recent Advances and Highlights of the 5th International Vanadium Symposium

“…In parallel, DFT calculations were carried out on large models of VC1PO active site (145). The above calculations suggest that in the resting state, at least one equatorial oxygen needs to be protonated to stabilize the metal cofactor (123,125,144,145). According to the DFT results, the equatorial hydroxo group is likely to be bound to Ser-402 (145).…”

“…Again we emphasize the observation that this active site is also observed for a series of acid phosphatases, indicating consequences of the chemical similarities of vanadate and phosphate (120). Enzymological (121), spectroscopic (122), and both synthetic (57,43) and computational models (48,(123)(124)(125) studies have elucidated key steps in the major catalytic steps in these systems (121). There is no evidence for redox cycling of the vanadate co-factor during catalysis {121, 122).…”

“…In fact, computational investigations of vanadium(V) complexes have been reported (48,(123)(124)(125)142).…”

“…Hybrid QM/MM calculations were conducted independently by two groups of investigators addressing extended active site models of VCPO treated quantum mechanically and significant portions of the protein treated with classical mechanics (125,144). In parallel, DFT calculations were carried out on large models of VC1PO active site (145).…”