title: ⁵¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.
abstract: Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.
authors: †Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
authors: Rupal
authors: R
authors: Gupta
authors: †Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
authors: Guangjin
authors: G
authors: Hou
authors: ‡Van't Hoff Institute for Molecular Science, University of Amsterdam, POSTBUS 94157, 1090 GD, Amsterdam, The Netherlands.
authors: Rokus
authors: R
authors: Renirie
authors: ‡Van't Hoff Institute for Molecular Science, University of Amsterdam, POSTBUS 94157, 1090 GD, Amsterdam, The Netherlands.
authors: Ron
authors: R
authors: Wever
authors: †Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, United States.
authors: Tatyana
authors: T
authors: Polenova
mesh_terms: D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D002711:Chloride Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003461:Crystallography; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017354:Point Mutation; D011487:Protein Conformation; D014638:Vanadates / Q000378:metabolism
chemical_list: D014638:Vanadates; C418625:vanadium chloroperoxidase; D002711:Chloride Peroxidase