Angle-dependent rotation velocity consistent with ADP release in bacterial F-ATPase

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• The F1-ATPase enzyme facilitates cellular energy production through rotation.
• Understanding the rotation kinetics of Paracoccus denitrificans F1-ATPase (PdF1) is crucial for elucidating its mechano-chemical coupling.
• Previous studies noted large rotational steps in PdF1, unlike other F-ATPases, hindering kinetic scheme establishment.

Purpose of the Study:

• To investigate the rotation kinetics of single-molecule PdF1.
• To identify and characterize short-lived states within the PdF1 rotation cycle.
• To compare experimental findings with theoretical models to establish a mechano-chemical kinetic scheme.

Main Methods:

• A model-based method was employed to analyze single-molecule trajectories of PdF1.
• Angular velocity was extracted from experimental data and compared with theoretical calculations.
• Kinetic analysis was performed to identify intermediate states and their lifetimes.

Main Results:

• A short-lived state with a 20 μs lifetime was detected 40° after ATP binding.
• This state corresponds to a three-nucleotide occupancy preceding ADP release.
• A similar state was observed in Thermophilic bacillus F1-ATPase, suggesting a conserved mechanism.

Conclusions:

• The identified short-lived state is a conserved intermediate in F1-ATPase rotary mechanism.
• This state plays a role in overcoming nucleotide release bottlenecks.
• The findings provide insights into the mechano-chemical coupling of rotary ATPases.