However, for V(IV)=O, this isomerization is barrierless, and the minimum energy structure positions the oxo moiety between the axial position and equatorial plane, i.e., NHis-V=O angles: 147° (Figure ). The energetics of axial and equatorial oxo isomers observed along the isomerization RCs for Fe and V differ by ca. 4 kcal/mol, which is slightly higher than that observed during free optimizations (Figure and Figure ). The small increase in energetic differences along the RCs could be the result of additional constraints used during the construction of RCs and the differences in Glu orientation, i.e., the carboxylate group of Glu pointing towards succinate along the RCs vs pointing away from succinate during free optimizations (Figure and Figure ). We also obtain largely similar isomerization RCs for Fe and V where Glu is forced to remain monodentate while succinate is allowed to be monodentate or bidentate, and we find that succinate prefers to remain monodentate along these RCs even in the absence of constraints on succinate (Figure and Supporting Information Figure ). The second RC also connects the monodentate succinate isomers of axial oxo and equatorial oxo as a function of the NHis-M=O angle but, unlike the previous RC, allows Glu to bind the metal either in a monodentate or bidentate fashion (Supporting Information Figures ). For both Fe and V, Glu prefers to be bidentate when oxo is closer to the axial position or the equatorial plane but becomes monodentate when oxo is midway between the axial position and the equatorial plane (Supporting Information Figure ). The change in denticity of Glu is captured by the discontinuities in the isomerization energy landscape, i.e., at NHis-M=O angle of 143° for Fe and 149° for Fe V, where the sudden change to a more favorable energy corresponds to the change in Glu binding from monodentate to bidentate (Supporting Information Figure ). These modified isomerization RCs of Fe and V with flexible Glu binding are qualitatively similar to those obtained with both monodentate succinate and monodentate Glu except that the isomerization barrier for Fe(IV)=O is higher by 3.0 kcal/mol when Glu is not constrained to be monodentate (Figure and Supporting Information Figure ). This suggests that although the isomerization barrier could be higher in the enzyme active site if bidentate Glu coordination is enforced throughout the catalytic cycle by interactions with the protein environment, it can be lowered by forcing Glu to remain monodentate for the entirety of the reaction through alternative favorable interactions. For example, hydrogen bonding (HB) interactions between the carboxylate of Glu and nearby residues could force Glu to remain monodentate and enable isomerization with a lower barrier. For a representative hydroxylase, VioC, Glu bound to the metal center can potentially form HBs with the substrate, L-arginine, or a nearby Arg334 residue that would force Glu to remain monodentate. Similarly, in TauD 119 , Asp bound to the metal center can potentially HB with the nearby Arg270 residue, thus remaining monodentate.