X-ray Absorption Studies of the Zn₂⁺ Site of Glyoxalase I

X-ray edge and extended absorption fine structure spectra of Zn2+ at the active site of glyoxalase I have been measured. The edge spectrum reveals a simple set of transitions consistent with a 7-coordinate or distorted octahedral Zn2+ model complex. Analysis of the fine structure rules out sulfur ligands to Zn2+ and yields a best fit complex with Zn2+-N (or Zn2+-O) distances of 2.04 and 2.10 Å, which are too great for tetrahedral Zn2+ coordination but are appropriate for an octahedral or more highly coordinated complex. Peaks of electron density in the Fourier-transformed region of the higher order shells at distances of 3-4 Å from the Zn2+ are similar to those found with known Zn2+-imidazole model complexes, including carbonic anhydrase [Yachandra, V., Powers, L., & Spiro, T. G. (1983) J. Am. Chem. Soc. 105, 6596-6604], indicating at least two imidazole ligands to Zn2+on glyoxalase I. Binding of the heavy atom substrate analogue S-(p-bromobenzyl)glutathione did not significantly alter the number of atoms directly bonded to Zn2+ or their distances. No evidence for coordination of the cysteine sulfur of glutathione by the Zn2+ was obtained, and no heavy atom signal from bromine was detected, indicating this atom to be ≥4 Å from the Zn2+. However, conformational changes of the imidazole ligands of Zn2+ upon binding of the substrate analogue were suggested by changes in the relative intensity of the doublet peaks at 3-4 Å from the Zn2+ and assignable to imidazole. Thus, binding of the substrate analogue in the second coordination sphere may induce a small conformation change in the inner coordination sphere of Zn2+, possibly a rotation of the imidazole ligands.