STRUCTURAL ORGANIZATION OF THE NI AND (4FE-4S) CENTERS IN THE ACTIVE FORM OF DESULFOVIBRIO-GIGAS HYDROGENASE - ANALYSIS OF THE MAGNETIC-INTERACTIONS BY ELECTRON-PARAMAGNETIC-RESONANCE SPECTROSCOPY

The Desulfovibrio gigas hydrogenase is a typical (NiFe) hydrogenase co ntaining a Ni center and three FeS centers, one [3Fe-4S] and two [4Fe- 4S] clusters. When the enzyme is activated under hydrogen gas, the Ni center becomes paramagnetic, giving a characteristic electron paramagn etic resonance (EPR) signal with g values at 2.19, 2.14 and 2.01, the Ni-C signal. Two redox states of the enzyme can be prepared, in which the [4Fe-4S] clusters are either diamagnetic or paramagnetic. In this latter state, the magnetic coupling between metal centers induces both the appearance at low temperature of a complex EPR spectrum, the spli t Ni-C signal, and a significant enhancement of the relaxation rates o f the Ni center. Good simulations of the split Ni-C signal recorded at three different microwave frequencies (X-band, Q-band, and S-band) ar e obtained by using a model based on a point dipole approximation of t he dipolar and exchange interactions between paramagnets. The spectral analysis demonstrates that only one [4Fe-4S](1+) cluster is significa ntly coupled to the Ni site and provides a detailed description of the relative arrangement of the two centers. In addition, the magnetic ch aracteristics of this [4Fe-4S](1+) cluster can be deduced from the sim ulations. Moreover, the spin-spin and spin-lattice relaxation times of the interacting centers were measured in the two redox states of the enzyme, either by power saturation and pulsed EPR experiments at low t emperature or from the broadening of the EPR lines at higher temperatu re. The relaxation behavior of the Ni center is well explained by usin g in the theoretical analysis, the set of structural and magnetic para meters deduced from the spectral simulations. Our structural conclusio ns on the active D. gigas hydrogenase are compared to the preliminary data of a low-resolution crystal structure of the oxidized enzyme [Vol beda, A., Piras, C., Charon, M. H., Hatchikian, E. C., Frey, M., & Fon tecilla-Camps, J. C. (1993) News Lett. Protein Crystallogr. 28, 30-33] .