Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils
Designing a functional type 2 copper center that has nitrite reductase activity within α-helical coiled coils
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DOI：
10.1073/pnas.1212893110
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发表时间：
2012-12-26
影响因子：
11.1
通讯作者：
Pecoraro, Vincent L.
中科院分区：
文献类型：
--
作者：
Tegoni, Matteo;Yu, Fangting;Pecoraro, Vincent L.
从头蛋白质设计的最终目标之一是实现能够催化底物上的氧化还原反应的系统。这一目标是具有挑战性的，因为氧化还原活性蛋白质需要对蛋白质的还原和氧化状态进行设计考虑。在本文中，我们描述了从头设计的金属肽Cu（I/II）（TRIL 23 H）（3）（+/2+）的光谱表征和催化活性，其中Cu（I/II）嵌入在α-螺旋卷曲的线圈中，作为亚硝酸铜还原酶的Cu-T2中心的模型。在Cu（I/II）（TRIL 23 H）（3）（+/2+）中，Cu（I）与三个组氨酸配位，如X射线吸收数据所示，Cu（II）与三个组氨酸和一个或两个水分子配位。两种离子都结合在三股卷曲螺旋的内部，亲和力范围从纳摩尔到微摩尔[Cu（II）]和皮摩尔[Cu（I）]。Cu（His）（3）活性位点的特征在于两种氧化态，揭示了与天然酶中Cu-T2位点的相似性。水溶液中的Cu（II）（TRIL 23 H）（3）（2+）可被抗坏血酸还原为Cu（I）（TRIL 23 H）（3）（+），再被亚硝酸盐氧化生成一氧化氮。在pH 5.8下，在还原剂（抗坏血酸盐）和底物（亚硝酸盐）均过量的情况下，铜肽Cu（II）（TRIL 23 H）（3）（2+）充当还原亚硝酸盐的催化剂，具有至少5次周转，并且在3.7小时后没有催化效率的损失。的催化活性，这是在肽的浓度的第一顺序，也显示了pH值的依赖性，描述和讨论。
One of the ultimate objectives of de novo protein design is to realize systems capable of catalyzing redox reactions on substrates. This goal is challenging as redox-active proteins require design considerations for both the reduced and oxidized states of the protein. In this paper, we describe the spectroscopic characterization and catalytic activity of a de novo designed metallopeptide Cu(I/II)(TRIL23H)(3)(+/2+), where Cu(I/II) is embeded in alpha-helical coiled coils, as a model for the Cu-T2 center of copper nitrite reductase. In Cu(I/II)(TRIL23H)(3)(+/2+), Cu(I) is coordinated to three histidines, as indicated by X-ray absorption data, and Cu(II) to three histidines and one or two water molecules. Both ions are bound in the interior of the three-stranded coiled coils with affinities that range from nano-to micromolar [Cu(II)], and picomolar [Cu(I)]. The Cu (His)(3) active site is characterized in both oxidation states, revealing similarities to the Cu-T2 site in the natural enzyme. The species Cu(II) (TRIL23H)(3)(2+) in aqueous solution can be reduced to Cu(I)(TRIL23H)(3)(+) using ascorbate, and reoxidized by nitrite with production of nitric oxide. At pH 5.8, with an excess of both the reductant (ascorbate) and the substrate (nitrite), the copper peptide Cu(II)(TRIL23H)(3)(2+) acts as a catalyst for the reduction of nitrite with at least five turnovers and no loss of catalytic efficiency after 3.7 h. The catalytic activity, which is first order in the concentration of the peptide, also shows a pH dependence that is described and discussed.