De novo design of binuclear metalloproteins

细胞色素C氧化酶(COX)中的CuA位含有一个双核铜中心，它由两个桥连的半胱氨酸残基组成，形成一个Cu2S2钻石簇。我们在从头设计的4-螺旋束蛋白中设计了这种铜构型。所设计的蛋白质具有与COX相同的紫外-可见光谱和电子自旋共振光谱，这表明我们成功地在4-螺旋束蛋白中构建了Cu2S2钻石簇。接下来，我们设计了锌铜超氧化物歧化酶或血蓝蛋白的金属构型。我们在设计的蛋白质中放置了六个His残基。设计的蛋白质结合了一个铜离子。然后，我们加入半胱氨酸残基来监测金属结合。我们获得的证据表明，该突变蛋白可能同时与两个铜离子或铜和锌离子结合。半胱氨酸残基可能决定了铜离子在蛋白质中的位置。提出了一种利用一个半胱氨酸残基设计多金属键合的新策略。需要进行详细的分析，我们才能按照新的设计方法构建金属装订。

The CuA site in cytochrome C oxidase (COX) contains a binuclear copper center, which is composed of two bridging Cys residues forming a Cu_2S_2 diamond cluster. We designed this copper configuration in the de novo designed 4-helix bundled protein. The designed protein exhibited the same UV vis and ESR spectra as those of COX, indicating that we successfully constructed the Cu_2S_2 diamond cluster in the 4-helix bundled protein. Next, we designed a metal configuration from Zn-Cu superoxide dismutase or hemocyanin. We placed six His residues in the designed protein. The designed protein bound one Cu ion. Then, we added Cys residue to monitor the metal binding. We obtained an evidence that shows this mutant protein might bind two Cu ions or Cu and Zn ions simultaneously. The Cys residue might determine the position of Cu ion in the protein. A new strategy to design the plural metal bindings can be proposed using one Cys residue. The detail analyses are required, we are able to construct the metal binding following the new design method.