Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
Department of Chemistry, Northwestern University, Evanston, Illinois 60201, United States.
Biochemistry. 2021 Feb 2;60(4):346-355. doi: 10.1021/acs.biochem.0c00852. Epub 2021 Jan 19.
Efficiently carrying out the oxygen reduction reaction (ORR) is critical for many applications in biology and chemistry, such as bioenergetics and fuel cells, respectively. In biology, this reaction is carried out by large, transmembrane oxidases such as heme-copper oxidases (HCOs) and cytochrome oxidases. Common to these oxidases is the presence of a glutamate residue next to the active site, but its precise role in regulating the oxidase activity remains unclear. To gain insight into its role, we herein report that incorporation of glutamate next to a designed heme-copper center in two biosynthetic models of HCOs improves O binding affinity, facilitates protonation of reaction intermediates, and eliminates release of reactive oxygen species. High-resolution crystal structures of the models revealed extended, water-mediated hydrogen-bonding networks involving the glutamate. Electron paramagnetic resonance of the cryoreduced oxy-ferrous centers at cryogenic temperature followed by thermal annealing allowed observation of the key hydroperoxo intermediate that can be attributed to the hydrogen-bonding network. By demonstrating these important roles of glutamate in oxygen reduction biochemistry, this work offers deeper insights into its role in native oxidases, which may guide the design of more efficient artificial ORR enzymes or catalysts for applications such as fuel cells.
高效进行氧还原反应（ORR）对于生物学和化学中的许多应用都至关重要，例如生物能量学和燃料电池。在生物学中，该反应由大型跨膜氧化酶如血红素铜氧化酶（HCOs）和细胞色素氧化酶来执行。这些氧化酶的共同点是在活性位点旁边存在一个谷氨酸残基，但它在调节氧化酶活性方面的确切作用仍不清楚。为了深入了解其作用，我们在此报告，在两个血红素铜中心的生物合成模型中，在设计的血红素铜中心旁边掺入谷氨酸可提高 O 结合亲和力、促进反应中间体的质子化，并消除活性氧物质的释放。模型的高分辨率晶体结构揭示了涉及谷氨酸的扩展的、水介导的氢键网络。在低温下对还原态氧合亚铁中心进行电子顺磁共振，并随后进行热退火，允许观察到关键的过氧氢中间物，该中间物可归因于氢键网络。通过证明谷氨酸在氧还原生物化学中的这些重要作用，这项工作深入了解了其在天然氧化酶中的作用，这可能为设计更高效的人工 ORR 酶或用于燃料电池等应用的催化剂提供指导。
