Free energy coupling between H+-generating and H+-consuming pumps. Ratio between output and input forces

Area of Science:

• Biochemistry
• Cellular Respiration
• Bioenergetics

Background:

• The H+/ATP stoichiometry, representing proton to ATP conversion, is crucial for understanding mitochondrial energy transduction.
• Existing models often assume a fixed stoichiometry, typically four protons per ATP.
• Factors influencing proton motive force (delta mu H) and ATP synthesis (delta Gp) require careful control to accurately assess this ratio.

Purpose of the Study:

• To investigate the delta Gp/delta mu H ratio in mitochondria under varying conditions.
• To determine if the delta Gp/delta mu H ratio can exceed the accepted H+/ATP stoichiometry of 4.
• To evaluate the implications of these findings for the delocalized protonic coupling model.

Main Methods:

• Mitochondria were studied near state 4 respiration with varying uncoupler or K+/valinomycin concentrations.
• Measurements were conducted in media with 1 mM or 50 mM inorganic phosphate (Pi).
• Careful control of factors like ATPase inactivation and adenylate kinase activity was implemented to ensure accurate delta Gp and delta mu H measurements.

Main Results:

• The delta Gp/delta mu H ratio was observed to exceed 4, approaching 6 at lower delta mu H values.
• Both uncouplers and K+ transport significantly decreased delta mu H and the rate of ATP synthesis.
• Despite lower delta mu H, a relatively large delta Gp was observed, particularly at high Pi concentrations.

Conclusions:

• The observed delta Gp/delta mu H ratios above 4 are thermodynamically incompatible with the delocalized protonic coupling model.
• These findings suggest a potential limitation or re-evaluation needed for current models of mitochondrial energy transduction.
• The study highlights the complex relationship between proton motive force, ATP synthesis, and stoichiometry under physiological conditions.