@entity72148 NMR Crystallography of @entity5336 Chloroperoxidase and its Directed Evolution XXXX / @entity72150 / @entity72151 Mutant: Protonation Environments of the Active Site.

@entity5336 @entity48453 @entity72148 @entity273 @entity72149 @entity72150 @entity72151 @entity734 @entity8870
UNASSIGNED: @entity5336 -dependent haloperoxidases (VHPOs) perform two-electron oxidation of @entity48453 using @entity273 . Their mechanism, including the factors determining the substrate specificity and the pH- @entity734 of the catalytic rates, is poorly understood. The @entity8870 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 @entity72148 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 @entity5336 -dependent chloroperoxidases (VCPO). The cofactor environments in the wild type VCPO and its @entity72149 / @entity72150 / @entity72151 mutant exhibiting 5-100 fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained owing to the fast MAS probe technologies enabled the assignment of the location and number of protons on the @entity8870 as a function of pH. The @entity8870 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 @entity8870 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- @entity734 . This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of @entity5336 centers in large biological molecules.
