Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase
Mutational analysis of a conserved positive charge in the c-ring of E. coli ATP synthase
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DOI：
10.1016/j.bbabio.2023.148962
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发表时间：
2023-02-24
影响因子：
4.3
通讯作者：
Steed, P. Ryan
中科院分区：
文献类型：
--
作者：
Shrestha, Rashmi K.;Founds, Michael W.;Steed, P. Ryan
F1Fo ATP 合酶是一种普遍存在的分子马达，利用旋转机制合成三磷酸腺苷 (ATP)，这是生命的基本能量货币。膜嵌入的 Fo 马达将质子的电化学梯度转化为旋转，然后用于驱动可溶性 F1 马达中催化 ATP 合成的构象变化。在大肠杆菌中，Fo 马达由 c10 环（转子）和亚基 a（定子）组成，它们共同提供两个促进质子易位的水性半通道。先前的工作表明，每个c亚基胞浆侧的Arg50和Thr51参与质子易位过程，并且在c亚基的该区域保守正电荷。为了进一步研究这些残基的作用以及这些位置活性的化学要求，我们生成了 13 个取代突变体，并测定了它们的体外 ATP 合成、H+ 泵送和被动 H+ 渗透活性，以及突变体在体内进行氧化磷酸化的能力。虽然极性和疏水突变在任一位置通常都是可以容忍的，但引入负电荷或去除极性会导致重大缺陷。我们讨论改变静电对转子和定子之间的相互作用、水通道中的水结构以及转子与心磷脂的相互作用的可能影响。
F1Fo ATP synthase is a ubiquitous molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The membrane-embedded Fo motor converts the electrochemical gradient of protons into rotation, which is then used to drive the conformational changes in the soluble F1 motor that catalyze ATP synthesis. In E. coli, the Fo motor is composed of a c10 ring (rotor) alongside subunit a (stator), which together provide two aqueous half channels that facilitate proton translocation. Previous work has suggested that Arg50 and Thr51 on the cytoplasmic side of each subunit c are involved in the proton translocation process, and positive charge is conserved in this region of subunit c. To further investigate the role of these residues and the chemical requirements for activity at these positions, we generated 13 substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and passive H+ permeability activities, as well as the ability of mutants to carry out oxidative phosphorylation in vivo. While polar and hydrophobic mutations were generally tolerated in either position, introduction of negative charge or removal of polarity caused a substantial defect. We discuss the possible effects of altered electrostatics on the interaction between the rotor and stator, water structure in the aqueous channel, and interaction of the rotor with cardiolipin.