## 4 Conclusions and Outlook  
Here we report for the first time a QM/MM computational investigation of the active site geometry of the [NiFeSe] hydrogenase based on the available structural data from X-ray crystallographic data collected at 1.3 A resolution [1]. The IR vibrational frequencies of CN- and CO ligands coordinated to the Fe ion have been computed using a normal-mode analysis based on the QM/MM optimized geometries. Overall, the computed QM/MM optimized geometries are in good agreement with the  
Figure 7: The computed vibrational IR spectra for the QM/MM optimized geometries of conformer I (black line), II (blue line), and III (red line) are shown for [Ni\({}^{2+}\)Fe\({}^{3+}\)Se] hydrogenase.  
experimentally resolved geometries, such that the computed vibrational frequencies may be assumed to reflect accurately the underlying structures. In comparison with experimental FTIR frequencies, the computed frequencies suggest that a mixture of structural isomers, as well as Fe oxidation states, may be present in a given protein sample, thus leading to the pattern of CO and CN-vibrational stretching bands observed. Based on the CO and CN-symmetric stretching frequencies, the Ni-IS\({}_{\text{I}}\) state can be assigned to conformer II in the case of Fe\({}^{2+}\), while the Ni-IS\({}_{\text{II}}\) state shows best agreement with conformers I and III in the case of Fe\({}^{3+}\). This pattern suggests that iron oxidation may be favored in conformers I and III. Independent of the iron oxidation state, conformers I and III show similar vibrational behavior, an interesting correlation that requires additional investigation. A systematic red-shift of 20-40 cm\({}^{-1}\) in the antisymmetric stretching frequencies for both Fe\({}^{2+}\) and Fe\({}^{3+}\) is observed.  
Here, the electronic state of nickel has been treated as 2+ as the oxidized states of [NiFeSe] hydrogenases are known to be EPR silent. Nonetheless, a worthwhile computational investigation would be a comparison of the optimized geometries considering additional ligand oxidization states, such as cysteines or selenocysteine oxidized to sulfenates. In addition, modeling bridging ligands at the Fe-Ni locus would provide some insight into the enzyme's catalytic mechanisms. Furthermore, as the assignment of CN- and CO ligands in the crystal structure may not necessarily be accurate, the present calculations should be extended to test for alternate assignments. Also, in the structure of [PERSON] et al., the total Ni occupancy of the catalytic site could only be refined to 85%, while a minor Ni site could not be clearly resolved. Therefore, future models may also wish to address variable Ni positions.  
Methodology, S.M., N.E.-M., D.T.; Software, N.E.-M., D.T., S.M.; Formal Analysis, S.M., N.E.-M., D.T., M.A.M.; Investigation, S.M. Data Curation, S.M.; Writing--Original Draft Preparation, N.E.-M.; Writing--Review and Editing, N.E.-M., S.M., D.T., M.A.M.; Visualization, S.M., N.E.-M.; and Funding Acquisition, M.A.M. All authors have read and agreed to the published version of the manuscript. Funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy--EXC 2008/1--390540038\". Gefordert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der Lander--EXC 2008/1--390540038\". We acknowledge support by the German Research Foundation and the Open Access Publication Fund of TU Berlin. This work has been supported in part by the European Union's Horizon 2020 research and innovation programme under grant agreement No 810856. The authors thank [PERSON] for insightful discussions. The authors declare no conflict of interest.