Abstract
F1-ATPase is a rotary molecular motor in which the γ-subunit rotates against the α3β3 cylinder. The unitary γ-rotation is a 120° step comprising 80 and 40° substeps, each of these initiated by ATP binding and ADP release and by ATP hydrolysis and inorganic phosphate release, respectively. In our previous study on γ-rotation at low temperatures, a highly temperature-sensitive (TS) reaction step of F1-ATPase from thermophilic Bacillus PS3 was found below 9 °C as an intervening pause before the 80° substep at the same angle for ATP binding and ADP release. However, it remains unclear as to which reaction step the TS reaction corresponds. In this study, we found that the mutant F1(βE190D) from thermophilic Bacillus PS3 showed a clear pause of the TS reaction below 18 °C. In an attempt to identify the catalytic state of the TS reaction, the rotation of the hybrid F1, carrying a single copy of βE190D, was observed at 18 °C. The hybrid F1 showed a pause of the TS reaction at the same angle as for the ATP binding of the incorporated βE190D, although kinetic analysis revealed that the TS reaction is not the ATP binding step. These findings suggest that the TS reaction is a structural rearrangement of β before or after ATP binding.
Highlights
Face of the ␣- and -subunits, predominantly on the -subunit [6]
The hybrid F1 carrying a single copy of (E190D), ␣32(E190D)␥, showed a distinct pause caused by the slow hydrolysis of (E190D) at ϩ200° from the ATP binding angle of the mutant  [18]
The rotational rate of the mutant was in good agreement with the estimated rotational rate, which was one-third of the ATP hydrolysis rate determined in the bulk solution based on the coupling ratio of 3 ATPs/turn; the wild-type F1 showed a large difference between two values around room temperature (Fig. 1A) [21]
Summary
Preparation of F1-ATPase—The ␣(His at N terminus/ C193S)3(His at N terminus)3␥(S108C/I211C) subcomplex of F1-ATPase from thermophilic Bacillus PS3, which was modified for the rotation assay, and the ␣(His at N terminus/ C193S)3(His at N terminus/E190D)3␥(S108C/I211C) subcomplex were expressed in E. coli, purified, and biotinylated as described previously [20]. The former was referred to as wild-type F1 because the effect of the mutations on the ATP hydrolysis activity was minor, and the latter was referred to as F1(E190D).
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