Musgrave Kristin B, Laplaza Catalina E, Holm R H, Hedman Britt, Hodgson Keith O
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
J Am Chem Soc. 2002 Mar 27;124(12):3083-92. doi: 10.1021/ja011861q.
In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the [Ni(II)-X-Fe(4)S(4)] bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase. The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe(4)S(4) cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X. The metallopeptides HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS. All peptides bind an Fe(4)S(4) cubane-type cluster. Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as M(OH(2))(6). Best fits of the data were obtained with ca. 20% M(OH(2))(6) and ca. 80% M(II) with mixed N/S coordination. The collective XAS results for HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M demonstrate the presence of an Fe(4)S(4) cluster and support the existence of the distorted square-planar coordination units [Ni(II)(S.Cys)(N.His)(3)] and [Ni(II)(S.Cys)(2)(N.His)(2)] in the HC(4)H(2) and HC(5)H metallopeptides, respectively. In the HC(5)H metallopeptide, tetrahedral [Co(II)(S.Cys)(2)(N.His)(2)] is present. We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-S.Cys)-Fe bridges, have been achieved. This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides. The scaffolding concept should be extendable to other bridged metal assemblies.
在早期工作中，已合成了具有螺旋-环-螺旋基序和63个残基的从头设计肽，作为稳定[Ni(II)-X-Fe(4)S(4)]桥连组装体的潜在支架，该组装体是梭菌一氧化碳脱氢酶中A簇的光谱推导结构。63聚体在环中包含一个共有三半胱氨酸铁氧化还原蛋白结构域，用于结合Fe(4)S(4)簇，并且在环附近有半胱氨酸和组氨酸残基用于结合Ni(II)，其中一个半胱氨酸残基被设计为桥连X。已通过Fe、Ni和Co的K边光谱和扩展X射线吸收精细结构（EXAFS）对金属肽HC(4)H(2)-[Fe(4)S(4)]-Ni和HC(5)H-[Fe(4)S(4)]-M进行了研究，这两种金属肽分别含有三个用于Ni(II)配位的组氨酸和一个半胱氨酸残基，以及两个用于结合M = Ni(II)和Co(II)的组氨酸和两个半胱氨酸残基。所有肽都结合一个Fe(4)S(4)立方烷型簇。由于存在少量具有低Z配体的六配位物种（为简单起见指定为M(OH(2))(6)），使得对Ni和Co数据的解释变得复杂。数据的最佳拟合结果是约20% 的M(OH(2))(6)和约80% 的具有混合N/S配位的M(II)。HC(4)H(2)-[Fe(4)S(4)]-Ni和HC(5)H-[Fe(4)S(4)]-M的综合X射线吸收光谱（XAS）结果证明了Fe(4)S(4)簇的存在，并分别支持了HC(4)H(2)和HC(5)H金属肽中扭曲的平面正方形配位单元[Ni(II)(S.Cys)(N.His)(3)]和[Ni(II)(S.Cys)(2)(N.His)(2)]的存在。在HC(5)H金属肽中，存在四面体[Co(II)(S.Cys)(2)(N.His)(2)]。我们得出结论，已实现了包括Ni-(μ(2)-S.Cys)-Fe桥在内的设计支架结合位点。这是对旨在稳定桥连生物组装体的从头设计金属肽的首次XAS研究，也是对设计肽的金属衍生物的少数XAS分析之一。支架概念应可扩展到其他桥连金属组装体。
