## 1 Introduction
In addition, the sulfur and selenium atoms are also directly bound to each other. Figure 1: **(A)** The catalytic site of [NiFeSe] hydrogenase contains the Fe-Ni center (shown as green and blue spheres, respectively) coordinated to the side chains of Cys75, Cys78, and Cys492, and to Sec489. Three nonproteic ligands (two CN-ligands and one CO ligand) are coordinated to Fe. **(B)** Three [Fe\({}_{4}\)S\({}_{4}\)] clusters, **(C)** proximal, **(D)** medial, and **(E)** distal, form an electron transfer path connecting the catalytic center to the protein surface (protein depicted in white ribbon representation). Structure taken from PDB: 5 JSH, resolution 1.3 Å [1]. The structural and mechanistic details of [NiFeSe] hydrogenase have been investigated using a range of spectroscopic methods including X-ray diffraction (XRD) [2], Fourier transform infrared spectroscopy (FTIR) [2, 10], resonance Raman (RR) [2], electron paramagnetic resonance (EPR) [1], circular dichroism (CD), UV/Vis [5], and gas chromatography-mass spectrometry GC-MS [1]. RR experiments indicate various redox dependent states, giving insight into hydrogen production as well as oxygen tolerance [2]. The UV/Vis spectrum of the [NiFeSe] hydrogenase is very similar to that of the [NiFe] hydrogenase, showing the characteristic broad absorption centered at 400 nm due to the oxidized FeS centers [5]. [NiFeSe] hydrogenase is nickel-EPR silent in the oxidized state [1], indicating that the enzyme is in a Ni(II) state that is contrary to most oxidized [NiFe] hydrogenases, in which nickel is in a paramagnetic Ni(III) spin state. GC experiments [1] indicate that H\({}_{2}\) production of [NiFeSe] hydrogenases is higher than that of [NiFeS] hydrogenases [5]. Particularly, FTIR spectroscopy has provided insight into the electronic structure and coordination pattern of the catalytic metal ions of hydrogenases. As the frequencies of the nonproteic ligands (CO, CN-) are highly sensitive to the changes in electronic environment, particularly at the Fe locus, FTIR spectra can help elucidate the electronic states governing the catalytic events. In general, for a given redox state, the frequencies of CO bands of [NiFeSe] are lower than those of equivalent redox states of standard sulfur-Ni-Fe hydrogenases [11]. This behavior is rationalized by the lower electronegativity of the Se atom relative to the S atom present in standard Ni-Fe hydrogenases; an increased \(\pi\)-donating effect from the \(d\) orbitals of Se to the 2p\(\pi^{\mathrm{e}}\) orbital of the CO ligand is also present [12]. Also, the FTIR spectra of [NiFeSe] show CN-bands that are comparable in intensity with CO bands, suggesting a high \(\pi\)-mobility in the CN-ligands due to hydrogen bond coordination with atoms of surrounding amino acids [10]. In addition to a range of experimental studies, computational studies have also attempted to shed light on the structure and chemistry of [NiFe] hydrogenases with FeS active sites [13, 14, 15]. Hybrid quantum mechanical and molecular mechanical (QM/MM) applications have been particularly invaluable for probing the connection between the electronic structure and function in these complex enzymatic systems [16, 17, 18]. The combination of QM methodology, which describes the chemically active region involving charge-transfers or bond-formation, and MM methods to treat the bulk protein and solvent environment is a computationally efficient method for describing large biomolecules [16]. In the case of hydrogenase, this QM/MM approach has been shown to accurately describe experimentally measured vibrational frequencies [13, 14, 15]. The present work reports on QM/MM structural and vibrational frequency computations to analyze the three proposed [NiFeSe] conformers from the structures of [PERSON] et al. [1]. For each of the three conformers, both Fe\({}^{2+}\) and Fe\({}^{3+}\) oxidization states have been investigated, providing for a total of six models.