To isolate the effect of bidentate coordination on M(IV)=O isomerization, we create artificial isomerization RCs where either succinate or Glu is constrained to its bidentate orientation along the RCs. We find that succinate/Glu cannot remain bidentate throughout the RC and must rearrange to structures with monodentate succinate/Glu, especially for NHis-M=O angles between 130° and 140°, to facilitate isomerization for both Fe and V (Figure ). We also observe that for the Fe(IV)=O intermediate, when bidentate structures are feasible, RCs with bidentate Glu show more favorable energies overall relative to RCs with bidentate succinate (Figure ). This suggests that oxo isomerization would most likely take place with bidentate Glu and monodentate succinate (Figure ). While monodentate Glu isomers are higher in energy than bidentate Glu isomers by only 3 kcal/mol for Fe, in the isomerization RCs, we observe a larger difference of 10 kcal/mol (Figures and). This is due to the additional constraints imposed on succinate and Glu during the isomerization RCs which result in unfavorable angles of binding between succinate/Glu and Fe, leading to increased isomer energetics (Figures and and Supporting Information Figure ). For both metals, the bidentate RCs for oxo isomerization show bidentate Glu to be favorable when oxo is closer to the axial position (Figure ). However, when oxo is closer to the equatorial plane, bidentate succinate and bidentate Glu RCs show comparable energies for Fe whereas bidentate succinate is more favorable than bidentate Glu for V (Figure ). This suggests that the Fe-oxo species maintains a bidentate Glu in either isomer orientation, while the V-oxo species likely has either bidentate Glu or succinate coordination depending on the isomer orientation (Figure ). The significant differences between Fe and V RCs with bidentate succinate/Glu demonstrate that V does not accurately capture the oxo isomerization of the ferryl intermediate. In a prior study, the bidentate succinate RCs for halogenases revealed that isomerization between axial and equatorial oxo could take place with bidentate succinate for the most part except for structures with NHis-M=O angles between 130° and 140° where succinate must bind in a monodentate fashion. Although this is largely consistent with what we observe for bidentate succinate/Glu RCs of hydroxylases, monodentate isomers are found to be energetically more favorable for structures with a larger range of NHis-M=O angles (Figure ). For example, structures with NHis-M=O angles: 120°-160° (120°-140°) are energetically more favorable with monodentate succinate/Glu rather than bidentate succinate (Glu) (Figure ). This is indeed consistent with isomer energetics of Fe(IV)=O in hydroxylase models where bidentate Glu that binds through axial and equatorial sites is energetically less favorable than monodentate Glu (Figure ). Overall, while halogenases tend to prefer oxo isomerization with bidentate succinate, hydroxylases prefer oxo isomerization with primarily bidentate Glu that binds through equatorial sites and with monodentate Glu for a considerable portion of the RC.