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 six‐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 six‐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 six‐stepper and diversified into 3‐, 6‐ and 9‐step forms in evolutionary adaptation. Based on these results, we discuss plausible features of the entire F _o F ₁ complex, along with potential physiological contexts in the last universal common ancestor and related lineages.