Mechanism of activation of the chloroplast ATP synthase - A kinetic study of the thiol modulation of isolated ATPase and membrane-bound ATP synthase from spinach by Escherichia coli thioredoxin

The mechanism of thiol modulation of the chloroplast ATP synthase by Escher ichia coli thioredoxin was investigated in the isolated ATPase subcomplex a nd in the ATP synthase complex reconstituted in bacteriorhodopsin proteolip osomes. Thiol modulation was resolved kinetically by continuously monitorin g ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteolipo somes, The binding rate constant of reduced thioredoxin to the oxidized ATP ase subcomplex devoid of its epsilon subunit could be determined. It did no t depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of t he epsilon-bearing ATPase subcomplex was slow and favored the release of ep silon. The rate constant of thioredoxin binding to the membrane-bound ATP s ynthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthas e turnover on thioredoxin-gamma subunit interaction. These findings, and mo re especially the comparisons between the isolated ATPase subcomplex and th e ATP synthase complex, can be interpreted in the frame of the rotational c atalysis hypothesis. Finally, thiol modulation changed the catalytic proper ties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synth ase thiol modulation.