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Biochemistry. 1997 Jun 24;36(25):7847-54.

Nature and electronic structure of the Ni-X dinuclear center of Desulfovibrio gigas hydrogenase. Implications for the enzymatic mechanism.

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  • 1Unité de Bioénergétique et Ingénierie des Protéines, UPR CNRS 9036Institut de Biologie Structurale et de Microbiologie et Université de Provence, Marseille, France.


The recent determination of the X-ray crystal structure of Desulfovibrio gigas hydrogenase has revealed that the active site is a Ni-X dinuclear center [Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., & Fontecilla-Camps, J. C. (1995) Nature 373, 580-587]. This unexpected result calls for a re-examination of the magnetic and redox properties that have been attributed previously to a mononuclear Ni center. We have used a combination of dosimetric and electron paramagnetic resonance (EPR) techniques to investigate the nature and the electronic structure of the Ni-X center in the redox forms of D. gigas hydrogenase giving EPR signals. The metal atom X was first shown to be Fe by accurate metal content analyses. Next, by determining the EPR characteristics of a polycrystal powder, it was shown that the redox form of the enzyme studied in the X-ray crystal experiments was essentially Ni-A. The temperature dependence of the Ni-A, Ni-B, Ni-C, and Ni-L EPR signals was studied over a large temperature range. No deviation from Curie's law could be detected, which places strong constraints upon the magnitude of the possible magnetic interactions between the Ni and Fe centers. When these results and the other available magnetic data are analyzed in the light of the crystal structure, it is concluded that the Fe center is diamagnetic in all the redox states of the enzyme. On the basis of these results, a mechanistic scheme consistent with a large body of experimental data can be proposed for Ni-containing hydrogenases.

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