Computational studies of the vanadium dependent haloperoxidase and vanadyl-imidazole complexes.

Abstract

This thesis describes computational studies of the protonation state of vanadate in the only known vanadium(V) dependent enzyme and the relationship between imidazole orientation and EPR parameters of vanadyl(IV) (V = O<super> 2+</super>) compounds. Vanadium dependent haloperoxidases, which catalyze the halogenation of organic substrates using peroxide to oxidize halides, have an absolute requirement for vanadium(V). This thesis presents quantum mechanical studies of small models of the vanadate cofactor and hybrid quantum mechanics/molecular mechanics calculations involving the majority of the protein, which clarify the protonation state of the vanadate cofactor in the resting form of the enzyme. In the resting state, there is an equilibrium between two anionic, trigonal bipyramidal vanadate structures, both of which contain histidine in one axial position. One structure contains a water moiety in the other axial position and three oxo groups in the equatorial plane. The second structure contains a hydroxide group trans to imidazole and two oxo groups and one hydroxo moiety in the equatorial positions. The location of the equatorial hydroxo group is identified. The latter part of this work is concerned with the hyperfine additivity relationship found in the EPR spectroscopy of vanadyl-imidazole compounds. The previously identified relationship between the orientation of an imidazole ligand and its contribution to the hyperfine coupling constant is examined. High field EPR spectroscopy is used to obtain more accurate values of A_|| and to resolve g_|| from g_&bottom; which is impossible at standard X-band frequency. The experimental data and the EPR parameters for a smaller bis-imidazole model system, incorporating imidazoles in all possible orientations relative to the vanadyl-oxygen bond, are calculated using DFT. These calculations accurately reproduce the experimental data and predict a relationship between g_|| and the imidazole orientation which has not been previously observed. The relative stability of complexes with imidazole in various orientations and their relationship to A_|| is explained based on the amount of electron delocalization observed in the molecular orbitals of the bis-imidazol complexes. The delocalization of electron density onto imidazoles which are antiparallel to the vanadyl-oxygen bond is greater than for parallel imidazoles which is greater than for perpendicular imidazoles.