<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