Vanadium-51 solid-state magic angle spinning NMR spectroscopy of vanadium haloperixodases and oxovanadium (V) haloperoxidase mimics

One of the most stimulating challenges in biochemical sciences is understanding the fundamental roles trace metals play in biological systems. Vanadium has been recognized for its pharmacological and therapeutic importance. Yet its mechanism of action remains elusive, due to the lack of suitable spectroscopic techniques. This study demonstrates the use of 51V solid-state NMR as a novel tool to explore the diamagnetic vanadium sites in proteins.; Vanadium chloro- and bromoperoxidase (VCPO and VBPO) are the focus of this investigation. Despite abundant experimental data on vanadium haloperoxidases, many aspects of their function and mechanism remain a mystery. The factors governing whether the enzyme will or will not display chlorinating activity are unclear. A delicate balance of multiple interactions at the vanadium site is proposed to dictate the substrate specificity. We anticipate that by directly probing the vanadium site using solid-state NMR spectroscopy, valuable insight will be gained on the structural and electronic determinants of the substrate specificity.; The sensitivity of 51V solid-state NMR to subtle variations in the geometry and electronic environment was verified using a series of oxovanadium (V) haloperoxidase mimics.; The anisotropic tensors were obtained by numerical simulations of the spectra. For four crystal lographically characterized compounds, Density Functional Theory calculations and experimental NMR results were in excellent agreement.; 51V solid-state NMR spectroscopy revealed significant differences for both the anisotropic and isotropic parameters of the vanadium site in vanadium chloro- and bromoperoxidase, corroborating that the electronic environments are unique in each enzyme. The observed chemical shifts were found to be -520 ppm in VCPO and -687 ppm in VBPO. The quadrupolar anisotropy is larger in VBPO than in VCPO. Initial studies on the peroxo-intermediate of VCPO also revealed dramatically increased quadrupolar and chemical shielding anisotropies for the intermediate compared to the resting state.; This study is the first to demonstrate the direct detection of the vanadium site in proteins by 51V solid-state NMR. This work forms a basis for further investigations of the vanadium sites in haloperoxidases, which are expected to shed light on the substrate specificity and catalytic mechanism of these enzymes.