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 Eschericia coli thioredoxin.
The mechanism of thiol modulation of the chloroplast ATP synthase by Escherichia coli thioredoxin was investigated in the isolated ATPase subcomplex and in the ATP synthase complex reconstituted in bacteriorhodopsin proteoliposomes. Thiol modulation was resolved kinetically by continuously monitoring ATP hydrolysis by the isolated subcomplex and ATP synthesis by proteoliposomes. The binding rate constant of reduced thioredoxin to the oxidized ATPase subcomplex devoid of its epsilon subunit could be determined. It did not depend on the catalytic turnover. Reciprocically, the catalytic turnover did not seem to depend on thioredoxin binding. Thiol modulation by Trx of the epsilon-bearing ATPase subcomplex was slow and favored the release of epsilon. The rate constant of thioredoxin binding to the membrane-bound ATP synthase increased with the protonmotive force. It was lower in the presence of ADP than in its absence, revealing a specific effect of the ATP synthase turnover on thioredoxin-gamma subunit interaction. These findings, and more especially the comparisons between the isolated ATPase subcomplex and the ATP synthase complex, can be interpreted in the frame of the rotational catalysis hypothesis. Finally, thiol modulation changed the catalytic properties of the ATP synthase, the kinetics of which became non-Michaelian. This questions the common view about the nature of changes induced by ATP synthase thiol modulation.
He
X
X
Section de Bioénergétique, Bâtiment 532, and Protéines Membranaires Transductrices d'Energie, CNRS-URA 2096, CEA Saclay, F91191 Gif-sur-Yvette Cedex, France.
Miginiac-Maslow
M
M
Sigalat
C
C
Keryer
E
E
Haraux
F
F
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D004789:Enzyme Activation; D004926:Escherichia coli; D007700:Kinetics; D010940:Plant Proteins / Q000378:metabolism; D018724:Spinacia oleracea; D013438:Sulfhydryl Compounds; D013879:Thioredoxins / Q000378:metabolism
D010940:Plant Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D000251:Adenosine Triphosphatases