Subunit movements in membrane-integrated EF0F1 during ATP synthesis detected by single-molecule spectroscopy

Area of Science:

• Biochemistry
• Molecular Biology
• Bioenergetics

Background:

• ATP synthase is a crucial enzyme responsible for cellular energy production.
• Understanding the rotational mechanism of ATP synthase is key to elucidating energy transduction processes.

Purpose of the Study:

• To investigate the subunit movements within membrane-integrated ATP synthase during catalysis.
• To compare rotational dynamics during ATP synthesis versus ATP hydrolysis.

Main Methods:

• Isolation and fluorescent labeling of E. coli ATP synthase subunits (gamma, epsilon, b).
• Incorporation of labeled enzymes into liposomes.
• Utilizing Fluorescence Resonance Energy Transfer (FRET) to monitor subunit rotation in single molecules.

Main Results:

• Observed stepwise rotation of the gamma-epsilon complex relative to the b-subunits during ATP hydrolysis and synthesis.
• Identified opposite rotation directions for synthesis and hydrolysis.
• Characterized dwell times and rapid movements of the gamma-epsilon complex.
• Found identical FRET levels during proton transport-coupled ATP hydrolysis and synthesis, suggesting shared intermediates.
• Observed distinct FRET levels under non-catalytic conditions, indicating epsilon-subunit movement during active/inactive transitions.

Conclusions:

• The gamma-epsilon complex undergoes stepwise rotation relative to the b-subunits during ATP synthase catalysis.
• Proton transport-coupled ATP hydrolysis and synthesis likely proceed through the same intermediates.
• The epsilon-subunit exhibits movement associated with the transition between active and inactive states.