The Catalytic Mn2+ Sites in the Enolase-Inhibitor Complex: Crystallography, Single-Crystal EPR, and DFT Calculations

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• 作者：Raanan Carmieli ; Todd M. Larsen ; George H. Reed ; Samir Zein ; Frank Neese ; Daniella Goldfarb
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：April 11, 2007
• 年：2007
• 卷：129
• 期：14
• 页码：4240 - 4252
• 全文大小：643K
• 年卷期：v.129,no.14(April 11, 2007)
• ISSN：1520-5126

文摘

Crystals of Zn²⁺/Mn²⁺ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) weregrown under conditions with a slight preference for binding of Zn²⁺ at the higher affinity site, site I. Thestructure of the Zn²⁺/Mn²⁺-PhAH complex was solved at a resolution of 1.54 Å, and the two catalyticmetal binding sites, I and II, show only subtle displacement compared to that of the corresponding complexwith the native Mg²⁺ ions. Low-temperature echo-detected high-field (W-band, 95 GHz) EPR (electronparamagnetic resonance) and ¹H ENDOR (electron-nuclear double resonance) were carried out on asingle crystal, and rotation patterns were acquired in two perpendicular planes. Analysis of the rotationpatterns resolved a total of six Mn²⁺ sites, four symmetry-related sites of one type and two out of the fourof the other type. The observation of two chemically inequivalent Mn²⁺ sites shows that Mn²⁺ ions populateboth sites I and II and the zero-field splitting (ZFS) tensors of the Mn²⁺ in the two sites were determined.The Mn²⁺ site with the larger D value was assigned to site I based on the ¹H ENDOR spectra, whichidentified the relevant water ligands. This assignment is consistent with the seemingly larger deviation ofsite I from octahedral symmetry, compared to that of site II. The ENDOR results gave the coordinates ofthe protons of two water ligands, and adding them to the crystal structure revealed their involvement in anetwork of H bonds stabilizing the binding of the metal ions and PhAH. Although specific hyperfineinteractions with the inhibitor were not determined, the spectroscopic properties of the Mn²⁺ in the twosites were consistent with the crystal structure. Density functional theory (DFT) calculations carried out ona cluster representing the catalytic site, with Mn²⁺ in site I and Zn²⁺ in site II, and vice versa, gaveoverestimated D values on the order of the experimental ones, although the larger D value was found forMn²⁺ in site II rather than in site I. This discrepancy was attributed to the high sensitivity of the ZFSparameters to the Mn-O bond lengths and orientations, such that small, but significant, differences betweenthe optimized and crystal structures alter the ZFS considerably, well above the difference between the twosites.