Copper is a ubiquitous metal in biology that, among other functions, is implicated in enzymatic redox catalysis and in protein electron transfer (ET). When it comes to ET, copper sites are found in two main forms, mononuclear type 1 (T1) and binuclear Cu_A sites, which share a common cupredoxin fold. Other relevant copper sites are the so-called type 2 (T2), which are more resilient to undergo direct electrochemistry and are usually involved in catalysis. Here we report the electrochemical and spectroscopic characterization of a novel T2-like copper site engineered following the loop swapping strategy. The ligand loop sequence of the newly discovered T1 copper site from Nitrosopumilus maritimus was introduced into the Cu_A scaffold from Thermus thermophilus yielding a chimeric protein that shows spectroscopic features different from both parental proteins_, and resemble those of red T2 copper sites, albeit with a shorter Cu-S(Cys) bond length. The novel T2 site undergoes efficient direct electrochemistry, which allows performing temperature-dependent cyclic voltammetry studies. The obtained results reveal that this chimera constitutes the first example of a copper protein with entropically controlled reduction potential, thereby contrasting the enthalpic supremacy observed for all other copper sites reported so far. The underlying bases for this entropic control are critically discussed.

铜是生物学中普遍存在的金属，除其他功能外，它还参与酶促氧化还原催化和蛋白质电子转移 (ET)。在 ET 方面，铜位点以两种主要形式存在，单核 1 型 (T1) 和双核 Cu _A位点，它们共享一个共同的铜氧还蛋白折叠。其他相关的铜位点是所谓的 2 型 (T2)，它们更容易发生直接电化学反应，通常参与催化。在这里，我们报告了按照循环交换策略设计的新型 T2 样铜位点的电化学和光谱表征。Nitrosopumilus maritimus新发现的 T1 铜位点的配体环序列被引入到 Cu _A支架中嗜热栖热菌产生的嵌合蛋白显示出与两种亲本蛋白不同的光谱特征_，并且类似于红色 T2 铜位点，尽管具有较短的 Cu-S(Cys) 键长。新的 T2 位点经过有效的直接电化学反应，可以进行温度相关的循环伏安法研究。获得的结果表明，这种嵌合体构成了具有熵控制还原电位的铜蛋白的第一个例子，从而与迄今为止报道的所有其他铜位点观察到的焓至上形成对比。这种熵控制的基本基础进行了批判性讨论。