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

<p><span>The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F₁ (<i>b</i>MF₁) 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 <i>b</i>MF₁ showed two types of intervening pauses during the rotation that were discriminated by <i>short</i> <i>dwell</i> and <i>long dwell</i>. <span>Using ATP</span><span>g</span><span>S as a slowly hydrolyzing ATP derivative as well as using a functional mutant </span><span>b</span><span>E188D with slowed ATP hydrolysis, the two pausing events were distinctively identified. </span>Buffer-exchange experiment with a non-hydrolyzable analog (AMP-PNP) revealed that the <i>long</i><i> dwell</i> corresponds to the catalytic dwell, i.e. the waiting state for hydrolysis, while it remains elusive which catalytic state <i>short pause</i> represents. The angular position of <i>catalytic dwell</i> was determined to be at +80° from <i>ATP-binding angle</i>, mostly consistent with other F₁s. The position of <i>short dwell </i>was found at 50-60° from <i>catalytic dwell</i>, <i>i.e.</i> +10-20° from <i>ATP-binding angle</i>. This is a distinct difference from human mitochondrial F₁ (<i>h</i>MF₁) that also shows the intervening dwell that probably corresponding to <i>short dwell</i> of <i>b</i>MF₁, at +65° from <i>binding pause</i>. 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, <i>b</i>MF₁ 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. </span></p>