Abstract
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.
Highlights
Single molecule investigations of the rotational mechanism have been carried out almost exclusively with bacterial F0F1 and its subcomplexes
In this work we investigate subunit rotation in membraneintegrated CF0F1 in single enzymes using spFRET
The procedure reported for binding of ATP to noncatalytic site 4 has been adapted in this work for binding of AMPPNPATTO655
Summary
AMPPNP labeled at the ribose moiety with a fluorescence acceptor (ATTO655) was bound at a noncatalytic nucleotide-binding site on the ␣-subunit. Labeling procedures have been reported for CF0F1 that allow a selective occupation of each catalytic and noncatalytic nucleotide-binding site [17,18,19]. The donor- and acceptor-labeled CF0F1 was reconstituted into liposomes, and the movement of the ␥-subunit relative to the ␣-subunit was measured with spFRET in freely diffusing proteoliposomes during proton transport-coupled ATP synthesis in a confocal microscope. These data show for the first time subunit rotation in eukaryotic Hϩ-ATP synthases during ATP synthesis
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