UNUSUAL LIGAND STRUCTURE IN NI-FE ACTIVE-CENTER AND AN ADDITIONAL MG SITE IN HYDROGENASE REVEALED BY HIGH-RESOLUTION X-RAY STRUCTURE-ANALYSIS

Background: The hydrogenase of Desulfovibrio sp, catalyzes the reversi ble oxidoreduction of molecular hydrogen, in conjunction with a specif ic electron acceptor, cytochrome c(3). The Ni-Fe active center of Desu lfovibrio hydrogenase has an unusual ligand structure with non-protein ligands. An atomic model at high resolution is required to make concr ete assignment of the ligands which coordinate the Ni-Fe center, These in turn will provide insight into the mechanism of electron transfer, during the reaction catalysed by hydrogenase. Results: The X-ray stru cture of the hydrogenase from Desulfovibrio vulgaris Miyazaki has been solved at 1.8 Angstrom resolution and refined to a crystallographic R factor of 0.229, The overall folding pattern and the spatial arrangem ent of the metal centers are very similar to those found in Desulfovib rio gigas hydrogenase. This high resolution crystal structure enabled us to assign the non-protein ligands to the Fe atom in the Ni-Fe site and revealed the presence of a Mg center, located approximately 13 Ang strom from the Ni-Fe active center. Conclusions: From the nature of th e electron-density map, stereochemical geometry and atomic parameters of the refined structure, the most probable candidates for the four li gands, coordinating the Ni-Fe center, have been proposed to be diatomi c S=O, C=O and C=N molecules and one sulfur atom, The assignment was s upported by pyrolysis mass spectrometry measurements, These ligands ma y have a role as an electron sink during the electron transfer reactio n between the hydrogenase and its biological counterparts, and they co uld stabilize the redox state of Fe(II), which may not change during t he catalytic cycle and is independent of the redox transition of the N i. The hydrogen-bonding system between the Ni-Fe and the Mg centers su ggests the possible involvement of the Mg center in the reaction cycle s of hydrogen metabolism.