10.5061/DRYAD.PG4F4QRJK
Kobayashi, Ryohei
0000-0001-8364-5354
University of Tokyo
Ueno, Hiroshi
University of Tokyo
Li, Chun-Biu
Stockholm University
Noji, Hiroyuki
University of Tokyo
Rotary catalysis of bovine mitochondrial F1-ATPase studied by
single-molecule experiments
Dryad
dataset
2019
2021-10-13T00:00:00Z
2021-10-13T00:00:00Z
en
https://doi.org/10.1073/pnas.1909407117
8224015 bytes
6
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
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.