The roles that nickel plays in biological systems appear to be largely dependent on the ligand environment. Non-redox active Ni-containing proteins have a predominantly O/N ligand donor environment. In contrast, Ni-containing redox active enzymes contain S-donor ligands. This study was undertaken to investigate the roles of the Ni ligands in the redox active enzyme, hydrogenase, and the isomerase, glyoxalase-I. An X-ray absorption spectroscopic (XAS) study of the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. The results indicate that Forms A, B, and C are formally Ni(III), whereas Forms SIu and SIr are formally Ni(II). In addition, the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in Forms A, B, and SIu; distorted four-coordinate sites in SIr and R; and a six-coordinate Ni site in Form C. The loss of a short Ni-O bond, and a shortening of the Ni-Fe distance, accounts for the change in coordination number from five to four that accompanies formation of SIr. Comparison of Forms C and L rules out the presence of H2 or H− binding in Form C, whereas analysis of the SI-CO complex reveals the presence of Ni-CO ligation. A hydrogenase modeling study of complexes containing thiolate ligands indicate the importance of S-donor ligands. Modification of the thiolates by protonation and alkylation leads to changes in the redox behavior of the model complexes without inducing large structural changes. This is similar to the behavior exhibited in hydrogenases. Escherichia coli glyoxalase-I is maximally activated by Ni2+, unlike other known glyoxalase-I enzymes that are active with Zn2+. An XAS study of the active site Ni and Zn-substituted glyoxalase-I indicated metal sites consistent with Ni(Glu)2(His) 2(OH2)2 and Zn(Glu)2(His)2(OH 2) structures, respectively. A similar study using Ni-glyoxalase-I and complexes formed with the product and various inhibitors was conducted. The results indicated interaction of S-D-lactoylglutathione (product) or octylglutathione with the enzyme did not change the structure of the Ni site nor did incorporation of SeMet for Met. However, the addition of an hydroxamate inhibitor results in a Ni site where the hydroxamate substitutes for both water molecules and a Glu ligand. In contrast, the SeMet-substituted enzyme hydroxamate complex loses both water molecules, but retains both histidine and glutamate ligands. The results are suggestive of a mechanism that involves both water molecules.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-3612 |
| Date | 01 January 2002 |
| Creators | Davidson, Gerard |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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