Pitts Winston C, Deb Aniruddha, Penner-Hahn James E, Pecoraro Vincent L
Department of Chemistry, University of Michigan, Ann Arbor, MI 48108, USA United States.
Department of Biophysics, University of Michigan, Ann Arbor, MI 48108, United States.
ACS Catal. 2024 Mar 15;14(6):4362-4368. doi: 10.1021/acscatal.3c06159. Epub 2024 Mar 7.
Herein, we report a three stranded coiled-coil (3SCC) protein containing a type II copper center (CuT2) composed of 6-membered ring N-heterocycles. This design yields the most active homogenous copper nitrite reductase (CuNiR) mimic in water. We achieved this result by controlling three factors. First, previous studies with N and N -Methyl Histidine had indicated that a ligand providing pyridine-like electronic character to the copper site was superior to the more donating N for nitrite reduction. By substitution of the parent histidine with the non-coded amino acids pyridyl alanine (3'-Pyridine [3'Py] vs 4'-Pyridine [4'Py]), an authentic pyridine donor was employed without the complications of the coupling of both electronic and tautomeric effects of histidine or methylated histidine. Second, by changing the position of the nitrogen atom within the active site (4'-Pyridine vs. 3'Pyridine) a doubling of the enzyme's catalytic efficiency resulted. This effect was driven exclusivity by substrate binding to the copper site. Third, we replaced the leucine layer adjacent to the active site with an alanine, and the disparity between the 3'Py and 4'Py became more apparent. The decreased steric bulk minimally impacted the 3'Py derivative; however, the 4'Py decreased by an order of magnitude (600 mM to 50 mM), resulting in a 40-fold enhancement in the / compared to the analogues histidine site and a 1500-fold improvement compared with the initially reported CuNiR catalyst of this family, TRIW-H. When combined with XANES/EXAFS data, the relaxing of the Cu(I) site to a more 2-coordinate Cu(I) like structure in the resting state increases the overall catalytic efficiency of nitrite reduction via the lowering of . This study illustrates how by combining advanced spectroscopic methods, detailed kinetic analysis, and a broad toolbox of amino acid side chain functionality, one can rationally design systems that optimize biomimetic catalysis.
在此，我们报道了一种三链卷曲螺旋（3SCC）蛋白，其包含由六元环N - 杂环组成的II型铜中心（CuT2）。这种设计产生了水中活性最高的均相亚硝酸铜还原酶（CuNiR）模拟物。我们通过控制三个因素实现了这一结果。首先，先前对N - 甲基组氨酸的研究表明，为铜位点提供吡啶样电子特性的配体在亚硝酸盐还原方面优于供电子能力更强的N。通过用非编码氨基酸吡啶基丙氨酸取代母体组氨酸（3'-吡啶[3'Py]与4'-吡啶[4'Py]），使用了一种纯正的吡啶供体，而没有组氨酸或甲基化组氨酸的电子效应和互变异构效应耦合的复杂性。其次，通过改变活性位点内氮原子的位置（4'-吡啶与3'-吡啶），酶的催化效率提高了一倍。这种效应完全是由底物与铜位点的结合驱动的。第三，我们用丙氨酸取代了活性位点相邻的亮氨酸层，3'Py和4'Py之间的差异变得更加明显。空间位阻的减小对3'Py衍生物的影响最小；然而，4'Py降低了一个数量级（从600 mM降至50 mM），与类似的组氨酸位点相比， / 提高了40倍，与该家族最初报道的CuNiR催化剂TRIW - H相比提高了1500倍。当与XANES/EXAFS数据相结合时，静止状态下Cu（I）位点向更类似二配位Cu（I）结构的松弛通过降低 提高了亚硝酸盐还原的整体催化效率。这项研究说明了如何通过结合先进的光谱方法、详细的动力学分析和广泛的氨基酸侧链功能工具箱，合理设计优化仿生催化的系统。
