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
B. Guigliarelli et al., 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, Biochemistry, 34(14), 1995, pp. 4781-4790
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]
.