%0 Journal Article
%A Gupta, Rupal
%A Hou, Guangjin
%A Renirie, Rokus
%A Wever, Ron
%A Polenova, Tatyana
%D 2015
%T 51V NMR
Crystallography of Vanadium Chloroperoxidase
and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments
of the Active Site
%U 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
%R 10.1021/jacs.5b02635.s001
%2 https://acs.figshare.com/ndownloader/files/3806095
%K hydrogen peroxide
%K pH 5.0
%K NMR crystallography approach
%K Optimal sensitivity
%K pH 7.3
%K Density Functional Theory
%K VHPO
%K cofactor environments
%K VCPO
%K MAS probe technologies
%K pH values
%K 51 V magic angle
%K vanadate cofactor
%K pH 8.3.
%K Evolution P 395D Mutant
%K gain insights
%K vanadate cofactor changes
%K P 395D
%K quadruply protonated
%K 51 V NMR Crystallography
%K substrate specificity
%K vanadium centers
%K change oxidation state
%K triply protonated
%K vanadate protonation
%K protonation environments
%K coordination environments
%K pH 6.3
%K NMR spectroscopy
%K Protonation Environments
%K coordination environment
%K Vanadium Chloroperoxidase
%X 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 51V 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 51V-based NMR crystallography can be
used to derive the detailed coordination environments of vanadium
centers in large biological molecules.
%I ACS Publications