Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis₃ binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis₃ binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis₃ binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

亚硝酸铜还原酶 (CuNiR) 是一种将亚硝酸盐转化为一氧化氮的铜酶，是细菌中全球氮循环的重要组成部分。CuNiR 活性位点相对简单的 CuHis ₃结合位点使其成为小分子建模和从头蛋白质设计研究的诱人目标。我们之前曾报道过平行三股盘绕线圈系统中的对称 CuNiR 模型，其活动范围跨越三个数量级。在本报告中，我们研究了反平行三螺旋束支架内的相同 CuHis ₃结合位点，该支架允许设计不对称结构。我们确定一个简单的 CuHis ₃结合位点可以设计在这个支架内，与平行盘绕线圈中的类似构建体相比具有增强的活性。结合更复杂的设计或重新定位该结合位点可以将该活性降低多达 15 倍。比较这些结构，我们重申了先前的结果，其中由 Cu (I) X 射线吸收光谱确定的 1s 到 4p 跃迁能量的蓝移与基于咪唑的结构内的增强活性相关。通过这一步骤和最近在该支架内成功的电子转移位点设计，我们离功能齐全的从头设计的亚硝酸还原酶又近了一步。