The Catalytic Mn~(2+) Sites in the Enolase-lnhibitor Complex: Crystallography, Single-Crystal EPR, and DFT Calculations

摘要

Crystals of Zn~(2+)/Mn~(2+) yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn~(2+) at the higher affinity site, site Ⅰ. The structure of the Zn~(2+)/Mn~(2+)-PhAH complex was solved at a resolution of 1.54 A, and the two catalytic metal binding sites, Ⅰ and Ⅱ, show only subtle displacement compared to that of the corresponding complex with the native Mg~(2+) ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electron paramagnetic resonance) and ~1H ENDOR(electron-nuclear double resonance) were carried out on a single crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotation patterns resolved a total of six Mn~(2+) sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn~(2+) sites shows that Mn~(2+) ions populate both sites Ⅰ and Ⅱ and the zero-field splitting (ZFS) tensors of the Mn~(2+) in the two sites were determined. The Mn~(2+) site with the larger D value was assigned to site Ⅰ based on the ~1H ENDOR spectra, which identified the relevant water ligands. This assignment is consistent with the seemingly larger deviation of site Ⅰ from octahedral symmetry, compared to that of site Ⅱ. The ENDOR results gave the coordinates of the protons of two water ligands, and adding them to the crystal structure revealed their involvement in a network of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfine interactions with the inhibitor were not determined, the spectroscopic properties of the Mn~(2+) in the two sites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out on a cluster representing the catalytic site, with Mn~(2+) in site Ⅰ and Zn~(2+) in site Ⅱ, and vice versa, gave overestimated D values on the order of the experimental ones, although the larger D value was found for Mn~(2+) in site Ⅱ rather than in site Ⅰ. This discrepancy was attributed to the high sensitivity of the ZFS parameters to the Mn-O bond lengths and orientations, such that small, but significant, differences between the optimized and crystal structures alter the ZFS considerably, well above the difference between the two sites.