Yamakita Atsuyoshi, Liu YingTao, Futai Masamitsu, Iwamoto-Kihara Atsuko
Department of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan.
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.
Biochim Biophys Acta Bioenerg. 2019 May 1;1860(5):361-368. doi: 10.1016/j.bbabio.2019.03.002. Epub 2019 Mar 12.
The γ subunit located at the center of ATP synthase (FF) plays critical roles in catalysis. Escherichia coli mutant with Pro substitution of the γ subunit residue γLeu218, which are located the rotor shaft near the c subunit ring, decreased NADH-driven ATP synthesis activity and ATP hydrolysis-dependent H transport of membranes to ~60% and ~40% of the wild type, respectively, without affecting FF assembly. Consistently, the mutant was defective in growth by oxidative phosphorylation, indicating that energy coupling is impaired by the mutation. The ε subunit conformations in the γLeu218Pro mutant enzyme were investigated by cross-linking between cysteine residues introduced into both the ε subunit (εCys118 and εCys134, in the second helix and the hook segment, respectively) and the γ subunit (γCys99 and γCys260, located in the globular domain and the carboxyl-terminal helix, respectively). In the presence of ADP, the two γ260 and ε134 cysteine residues formed a disulfide bond in both the γLeu218Pro mutant and the wild type, indicating that the hook segment of ε subunit penetrates into the αβ-ring along with the γ subunits in both enzymes. However, γ260/ε134 cross-linking in the γLeu218Pro mutant decreased significantly in the presence of ATP, whereas this effect was small in the wild type. These results suggested that the γ subunit carboxyl-terminal helix containing γLeu218 is involved in the conformation of the ε subunit hook region during ATP hydrolysis and, therefore, is required for energy coupling in FF.
位于ATP合酶(FF)中心的γ亚基在催化过程中发挥着关键作用。大肠杆菌突变体中γ亚基残基γLeu218被脯氨酸取代,该残基位于靠近c亚基环的转子轴上,其NADH驱动的ATP合成活性以及ATP水解依赖的膜H转运分别降至野生型的约60%和约40%,且不影响FF组装。一致地,该突变体在通过氧化磷酸化进行生长方面存在缺陷,表明该突变损害了能量偶联。通过将分别引入ε亚基(εCys118和εCys134,分别位于第二个螺旋和钩状片段)和γ亚基(γCys99和γCys260,分别位于球状结构域和羧基末端螺旋)的半胱氨酸残基进行交联,研究了γLeu218Pro突变酶中ε亚基的构象。在存在ADP的情况下,γLeu218Pro突变体和野生型中两个γ260和ε134半胱氨酸残基均形成了二硫键,表明ε亚基的钩状片段在两种酶中均与γ亚基一起穿透到αβ环中。然而,在存在ATP的情况下,γLeu218Pro突变体中的γ260/ε134交联显著减少,而在野生型中这种影响较小。这些结果表明,包含γLeu218的γ亚基羧基末端螺旋在ATP水解过程中参与ε亚基钩状区域的构象形成,因此是FF中能量偶联所必需的。