@article {2414,
	title = {17O ENDOR detection of a solvent-derived Ni-(OH(x))-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas},
	journal = {J Am Chem Soc},
	volume = {124},
	number = {2},
	year = {2002},
	note = {<p>0002-7863 (Print)0002-7863 (Linking)Journal ArticleResearch Support, Non-U.S. Gov{\textquoteright}tResearch Support, U.S. Gov{\textquoteright}t, Non-P.H.S.</p>
},
	month = {Jan 16},
	pages = {281-6},
	abstract = {<p>Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --\&gt; Ni-C --\&gt; Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme{\textquoteright}s activity.</p>
},
	keywords = {Binding Sites, Desulfovibrio/*enzymology, Electron Spin Resonance Spectroscopy/methods, Hydrogenase/*chemistry/*metabolism, Iron/chemistry/metabolism, Models, Molecular, Nickel/chemistry/metabolism, Oxygen Isotopes},
	url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve\&db=PubMed\&dopt=Citation\&list_uids=11782180 },
	author = {Carepo, M. and Tierney, D. L. and Brondino, C. D. and Yang, T. C. and Pamplona, A. and Telser, J. and Moura, I. and Moura, J. J. and Hoffman, B. M.}
}
