Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of E. coli Ia RNR is composed of two homodimers that form the active α2β2 complex. Catalysis is initiated by long-range radical translocation over a ~32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface. Resolving the PCET pathway at the α/β interface has been a long-standing challenge due to the lack of structural data. Herein molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α2β2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.

中文翻译：

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大肠杆菌核糖核苷酸还原酶α/β界面的构象运动和水网络

核糖核苷酸还原酶 (RNR) 催化所有四种核糖核苷酸转化为脱氧核糖核苷酸，并且对于所有生物体的 DNA 合成至关重要。大肠杆菌 Ia RNR 的活性形式由形成活性 α2β2 复合物的两个同源二聚体组成。催化是由通过约 32 Å 质子耦合电子转移 (PCET) 途径的长程自由基易位引发的，该途径涉及界面处的 Y356β 和 Y731α。由于缺乏结构数据，在 α/β 界面解析 PCET 通路一直是一个长期的挑战。在本文中，基于最近解决的活性 α2β2 复合物的低温电子显微镜结构进行了分子动力学模拟，以检查界面水的结构和波动，以及沿 PCET 途径的界面残基的氢键相互作用和构象运动。我们的自由能模拟表明，Y731 能够采样翻转构象，它指向界面以促进与 Y356 的界面 PCET，以及与 Y730 的堆叠构象，使 PCET 与该残基共线。Y356 和 Y731 表现出与界面水分子的氢键相互作用，并且在某些构象中，共享一个桥接水分子，这表明来自 Y356 和 Y731 的 PCET 的主要质子受体是界面水。Y731 的构象灵活性以及 Y731 和 Y356 与界面水和氢键水链的氢键相互作用对于沿 PCET 途径的有效自由基易位至关重要。