Iranzo Olga, Jakusch Tamas, Lee Kyung-Hoon, Hemmingsen Lars, Pecoraro Vincent L
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA.
Chemistry. 2009;15(15):3761-72. doi: 10.1002/chem.200802105.
Cd(II) has been used as a probe of zinc metalloenzymes and proteins because of the spectroscopic silence of Zn(II). One of the most commonly used spectroscopic techniques is (113)Cd NMR; however, in recent years (111m)Cd Perturbed Angular Correlation spectroscopy ((111m)Cd PAC) has also been shown to provide useful structural, speciation and dynamics information on Cd(II) complexes and biomolecules. In this article, we show how the joint use of (113)Cd NMR and (111m)Cd PAC spectroscopies can provide detailed information about the Cd(II) environment in thiolate-rich proteins. Specifically we show that the (113)Cd NMR chemical shifts observed for Cd(II) in the designed TRI series (TRI = Ac-G(LKALEEK)(4)G-NH(2)) of peptides vary depending on the proportion of trigonal planar CdS(3) and pseudotetrahedral CdS(3)O species present in the equilibrium mixture. PAC spectra are able to quantify these mixtures. When one compares the chemical shift range for these peptides (from delta = 570 to 700 ppm), it is observed that CdS(3) species have delta 675-700 ppm, CdS(3)O complexes fall in the range delta 570-600 ppm and mixtures of these forms fall linearly between these extremes. If one then determines the pK(a2) values for Cd(II) complexation [pK(a2) is for the reaction Cd(peptide-H)(2)(peptide)-->Cd(peptide)(3)(-) + 2H(+)] and compares these to the observed chemical shift for the Cd(peptide)(3)(-) complexes, one finds that there is also a direct linear correlation. Thus, by determining the chemical shift value of these species, one can directly assess the metal-binding affinity of the construct. This illustrates how proteins may be able to fine tune metal-binding affinity by destabilizing one metallospecies with respect to another. More important, these studies demonstrate that one may have a broad (113)Cd NMR chemical shift range for a chemical species (e.g., CdS(3)O) which is not necessarily a reflection of the structural diversity within such a four-coordinate species, but rather a consequence of a fast exchange equilibrium between two related species (e.g., CdS(3)O and CdS(3)). This could lead to reinterpretation of the assignments of cadmium-protein complexes and may impact the application of Cd(II) as a probe of Zn(II) sites in biology.
由于锌（II）的光谱沉默特性，镉（II）已被用作锌金属酶和蛋白质的探针。最常用的光谱技术之一是（113）Cd核磁共振；然而，近年来（111m）Cd扰动角关联光谱法（（111m）Cd PAC）也已被证明能提供有关Cd（II）配合物和生物分子的有用结构、形态和动力学信息。在本文中，我们展示了如何联合使用（113）Cd核磁共振和（111m）Cd PAC光谱来提供有关富含硫醇盐蛋白质中Cd（II）环境的详细信息。具体而言，我们表明在设计的肽TRI系列（TRI = Ac - G（LKALEEK）（4）G - NH2）中观察到的Cd（II）的（113）Cd核磁共振化学位移取决于平衡混合物中三角平面CdS（3）和假四面体CdS（3）O物种的比例。PAC光谱能够对这些混合物进行定量分析。当比较这些肽的化学位移范围（从δ = 570至700 ppm）时，可以观察到CdS（3）物种的化学位移为δ 675 - 700 ppm，CdS（3）O配合物的化学位移在δ 570 - 600 ppm范围内，而这些形式的混合物的化学位移则在这两个极端值之间呈线性变化。如果接着确定Cd（II）络合的pK（a2）值[pK（a2）是针对反应Cd[（肽 - H）（2）（肽）]+→Cd（肽）（3）- + 2H +]，并将其与观察到的Cd（肽）（3）-配合物的化学位移进行比较，会发现也存在直接的线性相关性。因此，通过确定这些物种的化学位移值，可以直接评估构建体的金属结合亲和力。这说明了蛋白质如何能够通过使一种金属物种相对于另一种金属物种不稳定来微调金属结合亲和力。更重要的是，这些研究表明，对于一种化学物种（例如CdS（3）O），可能有一个宽泛的（113）Cd核磁共振化学位移范围，这不一定反映这种四配位物种内部的结构多样性，而是两种相关物种（例如CdS（3）O和CdS（3））之间快速交换平衡的结果。这可能导致对镉 - 蛋白质配合物归属的重新解释，并可能影响Cd（II）作为生物学中锌（II）位点探针的应用。
