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[NiFe] hydrogenases are key enzymes in the hydrogen metabolism in many microorganisms and oxidize molecular hydrogen reversibly at a heterobimetallic active site. The released electrons are usually transferred through three iron–sulfur clusters to the physiological redox partner.[1, 2] The catalytic center has two metal ions Ni and Fe, which are bridged by two cysteinyl thiolates. Two further are bound to the Ni while the Fe is additionally coordinated by three diatomic ligands: one CO and two CNÀ.[3] Different redox states of the bimetallic center are largely defined by the oxidation state of the Ni and the chemical nature of an additional ligand at a third bridging position between the two metals, which serves as the cleavage site for hydrogen.[4, 5] Elucidating fundamental processes of microbial energy conversion is essential to promote biotechnological applications of hydrogen as a clean fuel.[6] In this respect, a comprehensive understanding of the catalytic mechanism of hydrogenase requires the characterization of the active site structure of the species involved. Electron paramagnetic resonance (EPR) and infrared (IR) spectroscopy are wellestablished techniques for probing the various states of the catalytic cycle.[4, 5] However, both methods are associated with inherent limitations. EPR spectroscopy is restricted to paramagnetic Ni states, whereas information about the active site Fe (FeII, S= 0) is largely inaccessible. IR spectroscopy probes all states of the active site by exploring the stretching vibrations of the diatomic ligands of the Fe. Thereby, it senses variations of the electron density distribution in the bimetallic complex, albeit without …