Rotary catalysis of bovine mitochondrial F-1-ATPase studied by single-molecule experiments

• The reaction scheme of rotary catalysis and the torque generation mechanism of bovine mitochondrial F-1 (bMF(1)) were studied in single-molecule experiments. Under ATP-saturated concentrations, high-speed imaging of a single 40-nm gold bead attached to the gamma subunit of bMF(1) showed 2 types of intervening pauses during the rotation that were discriminated by short dwell and long dwell. Using ATP gamma S as a slowly hydrolyzing ATP derivative as well as using a functional mutant beta E188D with slowed ATP hydrolysis, the 2 pausing events were distinctively identified. Buffer-exchange experiments with a nonhydrolyzable analog (AMP-PNP) revealed that the long dwell corresponds to the catalytic dwell, that is, 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 degrees from the ATP-binding angle, mostly consistent with other F(1)s. The position of short dwell was found at 50 to 60 degrees from catalytic dwell, that is, +10 to 20 degrees from the ATP-binding angle. This is a distinct difference from human mitochondrial F-1, which also shows intervening dwell that probably corresponds to the short dwell of bMF(1), at +65 degrees from the 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 gamma rotation. Similar to thermophilic F-1, bMF(1) 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 the hydrolysis process.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

F-1-ATPase

bovine mitochondrial F-1

single-molecule analysis

molecular motor

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