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

## TL;DR

This study reconstructs an ancestral F1-ATPase and shows it likely used a six-step rotation mechanism, providing insight into the evolutionary origins of ATP synthase.

## Contribution

The study reconstructs and functionally characterizes an ancestral F1-ATPase, revealing a six-step rotational mechanism as its original function.

## Key findings

- The chimeric F1-ATPase exhibits a six-step rotational mechanism with a 34° γ-subunit rotation between states.
- Single-molecule assays confirm the chimeric motor is a six-stepper with distinct binding and catalytic dwell positions.
- The ancestral F1-ATPase likely evolved into 3-, 6-, and 9-step forms through evolutionary adaptation.

## Abstract

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 FoF1 complex, along with potential physiological contexts in the last universal common ancestor and related lineages.