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Crystallographic data on the NiFe hydrogenase from Desulfovibrio gigas are presented that provide new information on the structure and mode of action of its dihydrogen activating metal center. Recently we found this center to contain, besides Ni, a second metal ion which was tentatively assigned to Fe (Volbeda, A.; Charon, M. H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587). This assignment is now unambiguously confirmed by a crystallographic analysis using 3 Å resolution X-ray data collected at wavelengths close to either side of the Fe absorption edge. Moreover, we report the structure of another crystal form of the as-purified D. gigas hydrogenase refined at 2.54 Å resolution, showing that the active site Fe binds three diatomic ligands. The electron density map shows an additional small peak at a position bridging the two active site metal ions, which may be assigned to some form of oxygen. This bridging oxygen species is proposed to be the signature of the inactive form of the enzyme. An infrared analysis similar to the one reported for Chromatium vinosum hydrogenase (Bagley, K. A.; Duin, E. C.; Roseboom, W.; Albracht, S. P. J.; Woodruff, W. H. Biochemistry 1995, 34, 5527−5535) shows the existence of three bands at exceptionally high frequencies, that shift their position in a concerted fashion depending on the redox state of the enzyme. Based on these high frequencies, the diatomic Fe ligands may be assigned to nonexchangeable triply bonded molecules, possible candidates being CO, CN- and NO. The frequency shifts of the infrared bands suggest a redox role for the Fe center during catalysis. Based on the new crystal structure and a number of spectroscopic results, possible modes of hydrogen binding to the active site are discussed.
Volbeda et al. (Mon,) studied this question.