We study isomers of M(IV)=O and M(III)-OH intermediates for Fe and V because identifying the most stable isomers could provide valuable structural information about preferred substrate distances and angles from the metal-oxo/hydroxo moiety. We identify stable isomers in the ground spin state for all Fe and V oxo/hydroxo intermediates and find that the most stable M(IV)=O isomers differ between Fe and V (Figure ). Although crystal structures with ferryl intermediates of non-heme iron hydroxylases are, as expected, not available, crystal structures with vanadyl intermediates show an axial oxo isomer with bidentate succinate and monodentate Glu as the most favored isomer. In our computational studies, while Fe(IV)=O prefers an axial oxo isomer with bidentate Glu to an equatorial oxo isomer with bidentate succinate by over 3 kcal/mol, V(IV)=O shows comparable energies for both isomers along with the isomer found in crystal structures of vanadyl mimics (Figure ). Energetic differences between the most stable bidentate and monodentate succinate/Glu Fe(IV)=O isomers is smaller, i.e., ca. 3 kcal/mol, suggesting a possibility for equilibration between bidentate and monodentate Glu (Figure ). However, the monodentate succinate structure of V(IV)=O with an axial oxo and monodentate Glu is extremely unstable and can only be optimized with the use of additional geometric constraints, indicating that this structure may never be observed (Figure ). We also observe that Fe(IV)=O isomers with axial oxo are energetically more favorable than their equatorial oxo counterparts, while both axial and equatorial oxo isomers are comparable in energy for V(IV)=O (Figure ). Among Fe(IV)=O isomers with equatorial oxo, we find that bidentate succinate (i.e., with a monodentate Glu) is strongly preferred over bidentate Glu (i.e., with a monodentate succinate) by ca. 9 kcal/mol (Figure ). This is consistent with prior calculations 127 where, in the equatorial oxo isomer, Glu is found to bind in a monodentate fashion with one of the Fe-OGlu bonds much longer than the other. On the contrary, these isomers are comparable in energy for V(IV)=O, indicating the possibility of experimentally observing an equatorial oxo isomer with bidentate Glu when vanadyl mimics are used, which incorrectly suggests the favorability of this isomer for Fe(IV)=O (Figure ). These differences in energetic preferences of M(IV)=O isomers demonstrate that using vanadyl in place of native non-heme iron hydroxylases may not lead to valid structural mimicry. Study of M(III)-OH isomers that are formed after HAT reveals that the most stable M(III)-OH isomer is same for both Fe and V (Figure ). Contrary to our observations for M(IV)=O isomers, the relative energetic favorability of M(III)-OH isomers is consistent between Fe and V with smaller differences in isomer energetics (Figure ). We also find that the metal-hydroxo moiety forms a hydrogen bond (HB) with Glu in most isomers for both Fe and V (Figure ). Energetic comparison of a given isomer, e.g., axial OH with bidentate succinate and monodentate Glu, with and without an HB between the hydroxo moiety and Glu reveals that the HB stabilizes the Fe(III)-OH isomer by ca. 4 kcal/mol (Figure ). However, no additional energetic stability is observed for the corresponding V(III)-OH isomers, suggesting weaker HBs in vanadium-based mimics (Figure ). Thus, despite the comparable relative energies of minimal models of the Fe and V active sites, differences in HB interactions between Fe and V hydroxo intermediates could imply differences in isomerization of M(III)-OH isomers, which further determine the rebound reaction step in the catalytic cycle. Next, we analyze differences in metal-ligand (M-L) bond lengths to identify if they are sensitive reporters of the differences in bonding between the favored spin states for Fe and V isomers of M(IV)=O and M(III)-OH. M-oxo bonds in HS and IS Fe(IV)=O isomers are longer than those in LS V(IV)=O isomers by ca. 0.04 Å and 0.03 Å, respectively, but LS Fe-oxo bonds are more comparable to V-oxo bonds, i.e., they differ by < 0.01 Å, highlighting that differences in spin state are likely the main source of differences in isomer structure and potentially energetics (Supporting Information Table ). All other M-L bonds of Fe and V intermediates, regardless of spin state, differ by < 0.10 Å except for specific isomers with elongated V-NHis, V-Osuc, or V-OGlu bonds (Supporting Information Tables ). Consistent with observations on halogenases , relative isomer energetics differ significantly between HS Fe(IV)=O and LS V(IV)=O intermediates, while they are comparable between IS Fe(IV)=O and LS V(IV)=O intermediates (Figure and Supporting Information Table and Figure ). Further study of isomer geometries reveals that the orientation of the Glu carboxylate can change isomer energetics by 1-4 kcal/mol depending on the denticity of succinate for both Fe and V intermediates (Supporting Information Figure ). Isomers where the free Glu carboxylate oxygen is pointed away from succinate are more energetically favorable than those with the free Glu carboxylate oxygen oriented towards succinate (Supporting Information Figure ). This energetic preference becomes significant in the presence of bidentate succinate likely due to a combination of steric and electrostatic effects.