Functional and Structural Characterization of F ₁ -ATPase with common ancestral core domains in stator ring

Extant F ₁ -ATPases exhibit diverse rotational stepping behaviors—3-, 6-, or 9-step cycles—yet the evolutionary origin of these patterns remains unclear. Here, we used ancestral sequence reconstruction to infer the catalytic β and non-catalytic α subunits of a putative ancestral F ₁ -ATPase. We then fused their functionally critical domains into the thermostable F ₁ from Bacillus PS3, yielding a stable chimeric enzyme. Cryo-EM revealed two distinct conformational states—binding and catalytic dwell states—separated by a ~34° rotation of the γ subunit, suggesting a fundamental six-step mechanism akin to that of extant 6-stepping F ₁ -ATPases. Single-molecule rotation assays with ATP and the slowly hydrolyzed ATP analog ATPγS demonstrated that the chimeric motor is intrinsically a 6-stepper, pausing at binding and catalytic dwell positions separated by 32.1°, although the binding dwell is significantly prolonged by an unknown mechanism. These findings indicate that F ₁ -ATPase was originally a 6-stepper and diversified into 3-, 6- and 9-step forms in evolutional adaptation. Based on these results, we discuss plausible features of the entire F _o F ₁ complex, along with potential physiological contexts in last universal common ancestor and related lineages.