The active site of the Fe-57-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H-ox) and the CO inhibited form (H-ox-CO) all Six Fe-57 hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six Fe-57 nuclei in the H-cluster of the H-ox-CO state. Four large Fe-57 hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak Fe-57 hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H-ox state only two different Fe-57 hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An 57 Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all Six Fe-57 nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [(FeFeII)-Fe-I] redox configuration where the paramagnetic Fe-I atom is attached to the [4Fe-4S](H) subduster. The Fe-57 hyperfine interactions of the formally diamagnetic [4Fe-4S]H are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Munck [Popescu, C.V.; Monck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.