We have applied resonance Raman (RR) spectroscopy on single protein crystals of the O2-tolerant membrane-bound [NiFe] hydrogenase (MBH from Ralstonia eutropha) which catalyzes the splitting of H2 into protons and electrons. RR spectra taken from 65 MBH samples in different redox states were analyzed in terms of the respective component spectra of the active site and the unprecedented proximal [4Fe-3S] cluster using a combination of statistical methods and global fitting procedures. These component spectra of the individual cofactors were compared with calculated spectra obtained by quantum mechanics/molecular mechanics (QM/MM) methods. Thus, the recently discovered hydroxyl-coordination of one iron in the [4Fe-3S] cluster was confirmed. Infrared (IR) microscopy of oxidized MBH crystals revealed the [NiFe] active site to be in the Nir-B [Ni(III)] and Nir-S [Ni(II)] states, whereas RR measurements of these crystals uncovered the Nia-S [Ni(II)] state as the main spectral component, suggesting its in situ formation via photodissociation of the assumed bridging hydroxyl or water ligand. On the basis of QM/MM calculations, individual band frequencies could be correlated with structural parameters for the Nia-S state as well as for the Ni-L state, which is formed upon photodissociation of the bridging hydride of H2-reduced active site states.