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# XAS studies of metal site structures of hydrogenases

Abstract (Summary)
Structural information was obtained from the analysis of nickel K-edge X-ray Absorption Spectroscopic (XAS) data of the NiFe hydrogenase from Chromatium vinosum. A series of redox poised C. vinosum enzyme samples have been studied, with structural comparisons of the nickel site in the hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC27774), Escherichia coli (Hydrogenase-1), and Alcaligenes eutrophus H16 (NADH- and H$\sb2$-reducing, soluble hydrogenase). The data indicate that the nickel K-edge energies shift 0.9-1.6 eV to lower energy upon reduction from oxidized (Forms A and B) to fully reduced forms. XANES analysis indicates there may be changes in the geometry of the nickel site in the hydrogenases. The oxidized forms, Form A and Form B, are likely to be five-coordinate, but Form C probably has a six-coordinate geometry. The electron density of the nickel changes little upon redox poised. The ligands around the nickel site are predominantly sulfur donor atoms, with Ni-S distances of 2.2 A. The present study is in agreement with the result of the crystallographic data on Desulfovibrio gigas hydrogenase (Volbeda, A., etc. Nature 1995, 373, 580-7). Except for the oxidized A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at $\sim$2.2 A. The enzyme obtained from T. roseopersicina shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase show a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized A. eutrophus enzyme indicate that the scattering is dominated by 3 $\pm$ 1 (N,O)-donor atoms at 2.06(2) A, with contributions from 3 $\pm$ 1 S-donor ligands at 2.35(2) A. This changes upon reduction composed of $\sim$4 S-donor ligands at a Ni-S distance of 2.19(2) A. The role of nickel as a substrate or inhibitor binding site was studied by using XAS to examine the structural changes during the binding of hydrogen and carbon monoxide onto the enzyme. XANES study shows that no substantial electron density or geometry changes in the nickel site of the enzyme upon photolysis. In addition, the EXAFS results show that the ligand environment of the nickel site remains essentially unchanged in the presence of hydrogen or carbon monoxide. This suggests the nickel site is probably not a binding site for the substrate or for carbon monoxide.
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