Structure of ATP synthase from an early photosynthetic bacterium <i>Chloroflexus aurantiacus</i>

F-type ATP synthase (F₁F_O) catalyzes proton motive force-driven ATP synthesis in mitochondria, chloroplasts, and bacteria. Different from the mitochondrial and bacterial enzymes, F₁F_O from photosynthetic organisms have evolved diverse structural and mechanistic details to adapt to the light-dependent reactions. Although complete structure of chloroplast F₁F_O has been reported, no high-resolution structure of an F₁F_O from photosynthetic bacteria has been available. Here, we report cryo-EM structures of an intact and functionally competent F₁F_O from <i>Chloroflexus aurantiacus</i> (<i>Ca</i>F₁F_O), a filamentous anoxygenic phototrophic bacterium from the earliest branch of photosynthetic organisms. The structures of <i>Ca</i>F₁F_O in its ADP-free and ADP-bound forms for three rotational states reveal a previously unrecognized architecture of ATP synthases. A pair of peripheral stalks connect to the <i>Ca</i>F₁ head through a dimer of δ-subunits, and associate with two membrane-embedded a-subunits that are asymmetrically positioned outside and clamp <i>Ca</i>F_O's c₁₀-ring. The two a-subunits constitute two proton inlets on the periplasmic side and two proton outlets on the cytoplasmic side, endowing <i>Ca</i>F₁F_O with unique proton translocation pathways that allow more protons being translocated relative to single a-subunit F₁F_O. Our findings deepen understanding of the architecture and proton translocation mechanisms of F₁F_O synthases and suggest innovative strategies for modulating their activities by altering the number of a-subunit.