HYSCORE Evidence That Endogenous Mena- and Ubisemiquinone Bind at the Same Q Site (QD) of Escherichia coli Nitrate Reductase A

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• 作者：Rodrigo Arias-Cartin ; Sevdalina Lyubenova ; Pierre Ceccaldi ; Thomas Prisner ; Axel Magalon ; Bruno Guigliarelli ; Stphane Grimaldi
• 刊名：Journal of the American Chemical Society
• 出版年：2010
• 出版时间：May 5, 2010
• 年：2010
• 卷：132
• 期：17
• 页码：5942-5943
• 全文大小：145K
• 年卷期：v.132,no.17(May 5, 2010)
• ISSN：1520-5126

文摘

Through the use of an Escherichia coli strain deficient in menaquinone biosynthesis, purified nitrate reductase A (NarGHI)-enriched inner membrane vesicles were titrated and monitored by electron paramagnetic resonance (EPR) spectroscopy, revealing the formation of protein-bound ubisemiquinone (USQ) species. Two-dimensional ESEEM (HYSCORE) experiments on these radicals revealed the same magnetic interaction with an ¹⁴N nucleus as found for menasemiquinone stabilized at the Q_D site of E. coli NarGHI and assigned to His66 N_δ, a distal heme axial ligand. Moreover, this signature was lost in the NarGHI_H66Y mutant, which is known to be unable to react with quinols. These findings demonstrate that NarGHI-bound USQ can be formed and detected by EPR. They also provide the first direct experimental evidence for similar binding of natural menasemiquinones and ubisemiquinones within the same protein Q site of NarGHI.