Subunit movements in single membrane-bound H+-ATP synthases from chloroplasts during ATP synthesis.
Subunit movements within the H(+)-ATP synthase from chloroplasts (CF(0)F(1)) are investigated during ATP synthesis. The gamma-subunit (gammaCys-322) is covalently labeled with a fluorescence donor (ATTO532). A fluorescence acceptor (adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP)-ATTO665) is noncovalently bound to a noncatalytic site at one alpha-subunit. The labeled CF(0)F(1) is integrated into liposomes, and a transmembrane pH difference is generated by an acid base transition. Single-pair fluorescence resonance energy transfer is measured in freely diffusing proteoliposomes with a confocal two-channel microscope. The fluorescence time traces reveal a repetitive three-step rotation of the gamma-subunit relative to the alpha-subunit during ATP synthesis. Some traces show splitting into sublevels with fluctuations between the sublevels. During catalysis the central stalk interacts, with equal probability, with each alphabeta-pair. Without catalysis the central stalk interacts with only one specific alphabeta-pair, and no stepping between FRET levels is observed. Two inactive states of the enzyme are identified: one in the presence of AMPPNP and one in the presence of ADP.
Bienert
Roland
R
Department of Physical Chemistry, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany.
Rombach-Riegraf
Verena
V
Department of Physical Chemistry, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany.
Diez
Manuel
M
Department of Physical Chemistry, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany.
Gräber
Peter
P
Department of Physical Chemistry, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany. Electronic address: peter.graeber@physchem.uni-freiburg.de.
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry*; D025222:Chloroplast Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D018613:Microscopy, Confocal; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D010944:Plants
D005456:Fluorescent Dyes; D010940:Plant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D025222:Chloroplast Proton-Translocating ATPases