[Fe]-Hydrogenase II isolated from C. pasteurianum contains 14 Fe which are distributed among the so-called Il cluster (the catalytic center) and two [4Fe-4S] clusters. Insights gained from Mossbauer studies of M-[4Fe-4S](2+) cluster assemblies (M is a paramagnetic center) in sulfite reductase and carbon monoxide dehydrogenase have suggested that the H cluster contains a [4Fe-4S](2+) cluster covalently linked to a smaller Fe-containing cluster. Recent X-ray studies of two [Fe]-hydrogenases, combined with the results of FTIR studies, have revealed that the H cluster contains a novel binuclear Fe cluster, [2Fe](H), that is linked by a cysteinyl sulfur to a [4Fe-4S] cluster; [2Fe](H) was found to have CO, CN-; and thiolate ligands. The analysis of the Mossbauer spectra of Hydrogenase II in the oxidized, reduced, and the CO-inhibited states has enabled us to assign the Fe-57 magnetic hyperfine tensors observed by ENDOR and Mossbauer spectroscopy to the two subclusters. Thus, A(I) = +25.3 MHz and A(II) = -28.4 MHz of H-ox-CO can be assigned to the two delocalized pairs of [4Fe -4S](H)(2+). In our coupling model these A-values result for j approximate to 100 cm(-1) where j describes the exchange interaction between [4Fe - 4S](H)(2+) and [2Fe](H). The 18 MHz A-value of H-ox obtained by ENDOR must result from one Fe site of [2Fe](H), while the 7.5 MHz ENDOR A-value seems to be associated with [4Fe-4S]H. Analysis of the Mossbauer spectra of H-red shows that the 4Fe cluster is in the 2+ state and that [2Fe]H contains presumably two low-spin Fe-II sites with Delta E-Q approximate to 0.85 mm/s and delta approximate to 0.08 mm/s. The observation that the [4Fe-4S] cluster is in the 2+ state in H-ox, H-ox-CO, and H-red suggests that the [2Fe](H) subcluster is in the mixed-valent (FeFeII)-Fe-III state in H-ox and H-ox-CO. Given the environment of strong-field ligands in [2Fe](H), the FeeU site must have low-spin configuration. While such an assignment is compatible with the EPR g-values, low-spin Fe-III sites with g approximate to 2 commonly exhibit very anisotropic Fe-57 A-tensors (due to spin-dipolar interactions) and thus the isotropic A-values of H-ox and H-ox-CO observed by ENDOR are difficult to explain. This point is discussed in some detail.