The primary and secondary coordination spheres of metal binding sites in metalloproteins have been investigated extensively, leading to the creation of high-performing functional metalloproteins; however, the impact of the overall structure of the protein scaffold on the unique properties of metalloproteins has rarely been studied. A primary example is the binuclear CuA center, an electron transfer cupredoxin domain of photosynthetic and respiratory complexes and, recently, a protein co-regulated with particulate methane and ammonia monooxygenases. The redox potential, Cu-Cu spectroscopic features, and a valence delocalized state of CuA are difficult to reproduce in synthetic models, and every artificial protein CuA center to-date has used a modified cupredoxin. Here we present a fully functional CuA center designed in a structurally non-homologous protein, cytochrome c peroxidase (CcP), by only two mutations (CuACcP). We demonstrate with UV-visible absorption, resonance Raman, and MCD spectroscopy that CuACcP is valence delocalized. CW and pulsed (HYSCORE) X-band EPR show it has a highly compact gz area and small Az hyperfine principal value with g and A tensors that resemble axially perturbed CuA. Stopped-flow kinetics found that CuA formation proceeds through a single T2Cu intermediate. The reduction potential of CuACcP is comparable to native CuA and can transfer electrons to a physiological redox partner. We built a structural model of the designed Cu binding site from EXAFS and validated it by mutation of coordinating Cys and His residues, revealing that a triad of residues (R48C, W51C, and His52) rigidly arranged on one α-helix is responsible for chelating the first Cu atom and that His175 stabilizes the binuclear complex by rearrangement of the CcP heme-coordinating helix. This design is a demonstration that a highly conserved protein fold is not uniquely necessary to induce certain characteristic physical and chemical properties to a metal redox center.

中文翻译：

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在全α-螺旋蛋白支架中设计的双核 CuA 中心

金属蛋白中金属结合位点的一级和二级配位球已被广泛研究，从而产生了高性能的功能性金属蛋白；然而，很少研究蛋白质支架的整体结构对金属蛋白独特性质的影响。一个主要的例子是双核 CuA 中心，光合作用和呼吸复合物的电子转移铜氧还蛋白结构域，以及最近与微粒甲烷和氨单加氧酶共同调节的蛋白质。CuA 的氧化还原电位、Cu-Cu 光谱特征和价离域状态很难在合成模型中重现，迄今为止，每个人工蛋白质 CuA 中心都使用了修饰的铜氧还蛋白。在这里，我们展示了一个功能齐全的 CuA 中心，该中心设计在结构上非同源的蛋白质、细胞色素 c 过氧化物酶 (CcP) 中，只有两个突变 (CuACcP)。我们用紫外可见吸收、共振拉曼和 MCD 光谱证明了 CuACcP 是价离域的。CW 和脉冲 (HYSCORE) X 波段 EPR 显示它具有高度紧凑的 gz 区域和小的 Az 超精细主值，g 和 A 张量类似于轴向扰动的 CuA。停流动力学发现 CuA 的形成是通过单个 T2Cu 中间体进行的。CuAccP 的还原电位与天然 CuA 相当，可以将电子转移到生理氧化还原伙伴。我们从 EXAFS 建立了设计的 Cu 结合位点的结构模型，并通过协调 Cys 和 His 残基的突变对其进行了验证，揭示了三联体残基（R48C、W51C、和 His52) 刚性排列在一个 α-螺旋上负责螯合第一个 Cu 原子，并且 His175 通过重排 CcP 血红素配位螺旋来稳定双核复合物。这种设计证明了高度保守的蛋白质折叠并不是将某些特征性物理和化学性质诱导到金属氧化还原中心所必需的。