The active site of the 57Fe-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 (Hox) and the CO inhibited form (H ox-CO) all six 57Fe 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 57Fe nuclei in the H-cluster of the Hox-CO state. Four large 57Fe 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 57Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe]H subcluster. For the Hox state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An 57Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six 57Fe 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 [Fe IFeII] redox configuration where the paramagnetic Fe I atom is attached to the [4Fe-4S]H subcluster. The 57Fe 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 Münck [Popescu, C.V.; Münck, 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.
All Science Journal Classification (ASJC) codes
- Colloid and Surface Chemistry