Functional and structural characterization of F₁-ATPase with common ancestral core domains in stator ring

Extant F₁-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 F₁-ATPase. We then fused their functionally critical domains into the thermostable F₁ 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 F₁-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 F₁-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_oF₁ complex, along with potential physiological contexts in the last universal common ancestor and related lineages.