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Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments

Citation

Kobayashi, Ryohei; Ueno, Hiroshi; Li, Chun-Biu; Noji, Hiroyuki (2021), Rotary catalysis of bovine mitochondrial F1-ATPase studied by single-molecule experiments, Dryad, Dataset, https://doi.org/10.5061/dryad.pg4f4qrjk

Abstract

The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F1 (bMF1) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of single 40 nm gold bead attached to the γ subunit of bMF1 showed two types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATPgS as a slowly hydrolyzing ATP derivative as well as using a functional mutant bE188D with slowed ATP hydrolysis, the two pausing events were distinctively identified. Buffer-exchange experiment with a non-hydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, i.e. the waiting state for hydrolysis, while it remains elusive which catalytic state short pause represents. The angular position of catalytic dwell was determined to be at +80° from ATP-binding angle, mostly consistent with other F1s. The position of short dwell was found at 50-60° from catalytic dwell, i.e. +10-20° from ATP-binding angle. This is a distinct difference from human mitochondrial F1 (hMF1) that also shows the intervening dwell that probably corresponding to short dwell of bMF1, at +65° from binding pause. Furthermore, we conducted ‘stall-and-release’ experiments with magnetic tweezers to reveal how the binding affinity and hydrolysis equilibrium are modulated by the g rotation. Similar to thermophilic F1, bMF1 showed a strong exponential increase in ATP affinity while the hydrolysis equilibrium did not change significantly. This indicates that the ATP binding process generates larger torque than hydrolysis process.