The pHin and pHout dependence of the rate of ATP synthesis catalyzed by the chloroplast H(+)-ATPase, CF0F1, in proteoliposomes.
The H(+)-ATPase from chloroplasts was isolated, purified, and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. The rate of ATP synthesis was measured after energization of the proteoliposomes by an acid-base transition as a function pHout and pHin. At any given pHout, the rate increased sigmoidally with increasing internal H+ concentration with half-maximal rates at about pHin 5.3 for all pHout values between 9.3 and 7.2. At any given pHin, the rate decreased sigmoidally with increasing external H+ concentration with half-maximal rates at about pHout 7.8. The dependence on internal H+ concentration was attributed to the protonation of three monovalent groups with the same pKin, and the dependence on external proton concentration to the deprotonation of two groups with the same pKout. The following scheme is proposed. The first step of the reaction is the binding of three (or four) protons from the inside of the F0 part, followed by ADP and Pi binding at the F1 part. Then, the enzyme conformation is changed, and the proton binding sites are exposed to the outside, followed by proton release from F0 and ATP release from F1. The reaction cycle is closed by changing the enzyme conformation to the form where protons can bind from the inside.
Possmayer
F E
FE
Biologisches Institut, Universität Stuttgart, Germany.
Gräber
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008433:Mathematics; D008962:Models, Theoretical; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011510:Proteolipids / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011522:Protons
D008081:Liposomes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011510:Proteolipids; D011522:Protons; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases