Carmieli Raanan, Larsen Todd M, Reed George H, Zein Samir, Neese Frank, Goldfarb Daniella
Department of Chemical Physics, The Weizmann Institute of Science, Rehovot, Israel.
J Am Chem Soc. 2007 Apr 11;129(14):4240-52. doi: 10.1021/ja066124e. Epub 2007 Mar 17.
Crystals of Zn2+/Mn2+ yeast enolase with the inhibitor PhAH (phosphonoacetohydroxamate) were grown under conditions with a slight preference for binding of Zn2+ at the higher affinity site, site I. The structure of the Zn2+/Mn2+-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 Mg2+ 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 Mn2+ sites, four symmetry-related sites of one type and two out of the four of the other type. The observation of two chemically inequivalent Mn2+ sites shows that Mn2+ ions populate both 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, 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 Mn2+ 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 Mn2+ in site I and Zn2+ 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 Mn2+ 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.
在对高亲和力位点I上的Zn²⁺结合稍有偏好的条件下，生长出了含有抑制剂PhAH（膦酰基乙酰氧肟酸）的Zn²⁺/Mn²⁺酵母烯醇化酶晶体。Zn²⁺/Mn²⁺-PhAH复合物的结构在1.54 Å的分辨率下得到解析，与相应的天然Mg²⁺离子复合物相比，两个催化金属结合位点I和II仅显示出细微的位移。在单晶上进行了低温回波检测高场（W波段，95 GHz）电子顺磁共振（EPR）和¹H电子-核双共振（ENDOR），并在两个相互垂直的平面上获取了旋转模式。对旋转模式的分析总共解析出六个Mn²⁺位点，一种类型的四个对称相关位点和另一种类型的四个位点中的两个。观察到两个化学不等价的Mn²⁺位点表明Mn²⁺离子占据了位点I和II，并且确定了两个位点中Mn²⁺的零场分裂（ZFS）张量。基于¹H ENDOR光谱，将具有较大D值的Mn²⁺位点指定为位点I，该光谱确定了相关的水配体。该指定与位点I与八面体对称性相比似乎更大的偏差一致，与位点II相比。ENDOR结果给出了两个水配体质子的坐标，并将它们添加到晶体结构中揭示了它们参与稳定金属离子和PhAH结合的氢键网络。尽管未确定与抑制剂的特定超精细相互作用，但两个位点中Mn²⁺的光谱性质与晶体结构一致。对代表催化位点的簇进行密度泛函理论（DFT）计算，位点I中为Mn²⁺，位点II中为Zn²⁺，反之亦然，得到的D值高估了约为实验值的数量级，尽管在位点II中而不是位点I中发现Mn²⁺的D值更大。这种差异归因于ZFS参数对Mn-O键长度和取向的高敏感性，使得优化结构与晶体结构之间虽小但显著的差异会使ZFS发生相当大的变化，远高于两个位点之间的差异。
