Department of Chemistry, University of Michigan, Ann Arbor, MI, USA.
Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA.
J Biol Inorg Chem. 2021 Oct;26(7):855-862. doi: 10.1007/s00775-021-01889-1. Epub 2021 Sep 6.
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 CuHis 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 CuHis binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis 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.
亚硝酰基化铜还原酶（CuNiR）是一种将亚硝酸盐转化为一氧化氮的铜酶，是细菌全球氮循环的重要组成部分。CuNiR 活性部位相对简单的 CuHis 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螺旋卷曲螺旋系统内的对称 CuNiR 模型，其活性跨越三个数量级的范围。在本报告中，我们研究了反平行三螺旋束支架内相同的 CuHis 结合位点，该支架允许设计不对称结构。我们确定可以在该支架内设计一个简单的 CuHis 结合位点，其活性相对于平行卷曲螺旋中的可比结构增强。结合更复杂的设计或重新定位该结合位点会使这种活性降低多达 15 倍。通过比较这些构建体，我们再次证实了之前的结果，即 Cu(I) X 射线吸收光谱测定的 1s 到 4p 跃迁能的蓝移与基于咪唑的构建体中活性的增强相关。通过这一步骤和最近在该支架内成功设计的电子转移位点，我们离完全功能性的从头设计的亚硝酸盐还原酶又近了一步。
