<sup>51</sup>V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site Rupal Gupta Guangjin Hou Rokus Renirie Ron Wever Tatyana Polenova 10.1021/jacs.5b02635.s001 https://acs.figshare.com/articles/journal_contribution/_sup_51_sup_V_NMR_Crystallography_of_Vanadium_Chloroperoxidase_and_Its_Directed_Evolution_P395D_L241V_T343A_Mutant_Protonation_Environments_of_the_Active_Site/2172169 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 <sup>51</sup>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 <sup>51</sup>V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules. 2015-04-29 00:00:00 hydrogen peroxide pH 5.0 NMR crystallography approach Optimal sensitivity pH 7.3 Density Functional Theory VHPO cofactor environments VCPO MAS probe technologies pH values 51 V magic angle vanadate cofactor pH 8.3. Evolution P 395D Mutant gain insights vanadate cofactor changes P 395D quadruply protonated 51 V NMR Crystallography substrate specificity vanadium centers change oxidation state triply protonated vanadate protonation protonation environments coordination environments pH 6.3 NMR spectroscopy Protonation Environments coordination environment Vanadium Chloroperoxidase