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Related Experiment Videos

Intersubunit rotation in active F-ATPase

D Sabbert1, S Engelbrecht, W Junge

  • 1Abteilung Biophysik, Fachbereich Biologie/Chemie, Universität Osnabrück, Germany.

Nature
|June 13, 1996
PubMed
Summary
This summary is machine-generated.

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The enzyme ATP synthase (F-ATPase) facilitates ATP synthesis by coupling proton flow with mechanical rotation. This study visualizes the rotation of its gamma subunit within the F1 portion in real-time, confirming its role in ATP hydrolysis.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • ATP synthase (F-ATPase) is a crucial enzyme in energy metabolism, found in bacteria, chloroplasts, and mitochondria.
  • It comprises membrane-bound F0 and peripheral F1 portions, responsible for proton translocation and ATP synthesis, respectively.
  • Evidence suggests ATP release involves mechanical energy transduction via subunit rotation within F1.

Purpose of the Study:

  • To directly observe and quantify the real-time rotation of the gamma subunit within the F1 portion of ATP synthase.
  • To elucidate the mechanical mechanism of ATP release during hydrolysis.
  • To validate the role of gamma subunit rotation in the catalytic cycle of ATP synthase.

Main Methods:

  • Utilized polarized absorption relaxation after photobleaching on immobilized F1 complexes.

Related Experiment Videos

  • Incorporated eosin-labeling on the gamma subunit for real-time tracking.
  • Observed rotation of the gamma subunit relative to the immobilized (alpha beta)3 core.

    • Main Results:

    • Directly recorded the rotation of the gamma subunit relative to the (alpha beta)3 core in functional ATP synthase.
    • Observed rotation occurred within 100 ms, consistent with ATP hydrolysis rates.
    • The observed angular range (≥200 degrees) supports a triple-site catalysis mechanism with gamma acting as a crankshaft.
    • Gamma subunit rotation was inhibited by the non-hydrolyzable ATP analogue, AMP-PNP.

    Conclusions:

    • The study provides direct, real-time evidence of gamma subunit rotation within ATP synthase during ATP hydrolysis.
    • Confirms the mechanical rotation of the gamma subunit as a key component of the enzyme's energy transduction mechanism.
    • Supports a model where the rotating gamma subunit drives ATP synthesis/hydrolysis through a multi-site catalytic mechanism.