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.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

“…52−56 NMR crystallography was initially introduced as a way to refine three-dimensional structures of crystalline molecular solids, 57 using agreement between first-principles predicted and experimental chemical shifts as a convergence criterion. This method has recently been applied to ordered and disordered extended solids 54,55 and to biological systems, 35,58−61 including work by our group and others on enzyme active sites. 35,58,61 For the TS 2AP quinonoid analogue, NMR crystallography provides direct, atomic-resolution support for the carbanionic form of the intermediate, ruling out a true quinonoid species and suggesting an equilibrium between the phenolic and protonated Schiff base tautomers that favors the phenolic form.…”

“…This method has recently been applied to ordered and disordered extended solids 54,55 and to biological systems, 35,58−61 including work by our group and others on enzyme active sites. 35,58,61 For the TS 2AP quinonoid analogue, NMR crystallography provides direct, atomic-resolution support for the carbanionic form of the intermediate, ruling out a true quinonoid species and suggesting an equilibrium between the phenolic and protonated Schiff base tautomers that favors the phenolic form. Natural bond orbital (NBO) calculations show the buildup of negative charge at the substrate C α for the protonated Schiff base form, implicating it as the catalytically significant tautomer.…”

A Doubly Biomimetic Synthetic Transformation: Catalytic Decarbonylation and Halogenation at Room Temperature by Vanadium Pentoxide

“…This suggests that the 5+ oxidation state of V was maintained throughout the reaction. A recent 51 V NMR study on vanadium haloperoxidases suggests the presence of spectroscopically silent V V during the catalytic cycle as revealed in our biomimetic models . The mechanism proposed has high relevance to biology particularly if vanadium haloperoxidase is found to halogenate thousands of natural products.…”

Functional Enzyme Mimics for Oxidative Halogenation Reactions that Combat Biofilm Formation

“…The similarity of the haloperoxidase reaction mechanism to that proposed for many homogeneous catalysts prompted the study of structural and functional model compounds. Figure shows the catalytic V‐BPO center of A. nodosum and the proposed reaction mechanism for HOX formation . The cofactor and catalytic center of V‐HPO, particularly the trigonal‐bipyramidally coordinated orthovanadate unit, is stabilized through a highly complex hydrogen bonded network of amino acids such as histidine, lysine, arginine, and serine (Figure , I ) .…”