The quinol oxidation site QD in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI QD quinol oxidation site is analyzed in detail using 1,2H hyperfine (hf) spectroscopy in combination with H2O/D2O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the QD site (MSKD) previously studied by us. DMSKD and MSKD are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSKD field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two β-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (Aiso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSKD and (ii) a near in-plane orientation of its isoprenyl chain at Cβ relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.

中文翻译：

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1,2H超精细光谱学和DFT建模揭示了脱甲基menasemiquinone与大肠杆菌硝酸还原酶A（NarGHI）的结合模式。

大肠杆菌呼吸硝酸盐还原酶A（EcNarGHI）中的喹诺醇氧化位点QD与细菌天然合成的三种类异戊二烯醌反应，即泛醌（UQ），甲萘醌（MK）和脱甲基甲萘醌（DMK）。使用1,2H超细（hf）光谱结合H2O / D2O交换实验和DFT建模，详细分析了脱甲基menasemiquinone（DMSK）与EcNarGHI QD quinol氧化位点的结合模式，并将其与结合到其中的menasemiquinone进行了比较我们之前研究过的QD网站（MSKD）。显示DMSKD和MSKD通过短（〜1.7Å）H键以相似且强烈不对称的方式结合。在DMSKD场扫描EPR谱上解析的特定hf模式的起源明确地归因于类异戊二烯侧链的两个β-亚甲基质子的轻微不等价贡献。DFT计算表明，它们较大的各向同性hf耦合常数（Aiso〜12和15 MHz）与（i）DMSKD的特定高度不对称结合模式以及（ii）异戊二烯基链在Cβ相对位置的近平面取向一致与芳环的距离，与游离或与NarGHI结合的MSK的预测值相差约90°。我们的结果为如何在单个Q位点上通过蛋白质环境选择性地微调不同天然醌的构象和氧化还原特性提供了新的见解。