Spectroscopic and biochemical studies have also established that the oxidation state of vanadium remains +5 throughout the catalytic cycle ; therefore, it has been thought that the protonation state of the oxo groups coordinating vanadium may be responsible for redox tuning of the cofactor. Recently, Gupta et al. performed 51 V magic angle spinning (MAS) NMR spectroscopy and determined certain NMR parameters (i.e., the reduced anisotropy, the asymmetry parameter, and the electric field gradient (EFG) tensor parameters) which are influenced by the protonation state of vanadate in the resting state. Then, they constructed models of the active site with the vanadate in various protonation states, and calculated these NMR parameters by density functional theory using the B3LYP hybrid functional and 6-311G(d,p) basis set while constraining the position of all atoms except for the hydrogens and vanadate. They argued that the protonation state is dependent on the pH in the following way: diprotonated 3 (pH 8.3), triprotonated 10 or 13 (pH 7.3) and quadprotonated 16 (pH 6.3) (Figure ). At pH 7.3, the observed NMR parameters were consistent with either triprotonated 10 or 13.