The light-induced Ni-L state of [NiFe] hydrogenases is well suited to investigate the identity of the amino acid base that functions as a proton acceptor in the hydrogen turnover cycle in this important class of enzymes. Density functional theory calculations have been performed on large models that include the complete [NiFe] center and parts of the second coordination sphere. Combined with experimental data, in particular from electron paramagnetic resonance and Fourier transform infrared (FTIR) spectroscopy, the calculations indicate that the hydride ion, which is located in the bridging position between nickel and iron in the Ni-C state, dissociates upon illumination as a proton and binds to a nearby thiolate base. Moreover, the formation of a functionally relevant nickel-iron bond upon dissociation of the hydride is unequivocally observed and is in full agreement with the observed g values, ligand hyperfine coupling constants, and FTIR stretching frequencies. This metal-metal bond can be protonated and thus functions like a base. The nickel-iron bond is important for all intermediates with an empty bridge in the catalytic cycle, and the electron pair that constitutes this bond thus plays a crucial role in the hydrogen evolution catalyzed by the enzyme.