An XAS investigation of product and inhibitor complexes of Ni-containing GlxI from Escherichia coli: Mechanistic implications

Escherichia coli glyoxalase I (GlxI) is a metalloisomerase that is maximall y activated by Ni2+ unlike other known GlxI enzymes which al-e active with Zn2+. The metal is coordinated by two aqua ligands, two histidines (5 and 7 4), and two glutamates (56 and 122), The mechanism of E, coli Ni-GlxI was i nvestigated by analyling Ni K-edge X-ray absorption spectroscopic (XAS) dat a obtained from the enzyme and complexes formed with the product, S-D-lacto ylglutathione, and various inhibitors. The analysis of X-ray absorption nea r edge structure (XANES) was used to determine the coordination number and geometry of the Ni site in the various Ni-GlxI complexes, Metric details of the Ni site structure were obtained from the analysis of extended X-ray ab sorption fine structure (EXAFS). Interaction of S-D-lactoylglutathione (pro duct) or octylglutathione with the enzyme did not change the structure of t he Ni site. However, analysis of XAS data obtained from a complex formed wi th a peptide hydroxamate bound to Ni-GlxI is consistent with this inhibitor binding to the Ni center by displacement of both water molecules. XANES an alysis of this complex is best fit with a five-coordinate metal and, given the fact that both histidine ligands are retained, suggests the loss of a g lutamate ligand, The loss of a glutamate ligand would preserve the neutral charge on the Ni complex and is consistent with the lack of a significant s hift in the Ni K-edge energy in this complex. These data are compared with data obtained from the E, coli Ni-GlxI selenomethionine-substituted enzyme. The replacement of three methionine residues in the native enzyme with sel enomethionine does not affect the structure of the Ni site. However, additi on of the peptide hydroxamate inhibitor leads to the formation of a complex whose structure as determined by XAS analysis is consistent with inhibitor binding via displacement of both water molecules but retention of both his tidine and glutamate ligands, This leads to an anionic complex, which is co nsistent with an observed 1.7 eV decrease in the Ni K-edge energy. Plausibl e reaction mechanisms for Ni-GlxI are discussed in light of the structural information available.