F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.

F ₁ F _O -ATP合酶是一种重要的代谢酶，它利用呼吸中的质子动力来再生ATP。为了获得最大的热力学效率，ATP合成应该是完全可逆的，但是反硝化副球菌的酶催化ATP水解的速率远低于催化ATP合成的速率，这通常归因于其独特的ζ亚基。最近，我们发现删除ζ仅略微增加了水解作用，表明其他常见的抑制机制，例如ε亚基（ε-CTD）或Mg-ADP的C末端结构域的抑制可能更为重要。在这里，我们创建了缺少ε-CTD的突变体和缺少ε-CTD和ζ亚基的双重突变体。在这些菌株中的任何菌株中均未观察到ATP水解的实质活化。取而代之的是，即使是双重突变菌株中的水解也只能被氧阴离子，去污剂月桂基二甲基氧化胺或质子原动力活化，它们都被认为可释放Mg-ADP抑制作用。我们的研究结果表明，P。denitrificans ATP合酶受ε和ζ亚基和Mg-ADP抑制作用的共同调节。