Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase.
F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of epsilon during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of epsilon. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of epsilon within the central stalk.
Zimmermann
Boris
B
Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.
Diez
Manuel
M
Zarrabi
Nawid
N
Gräber
Peter
P
Börsch
Michael
M
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling; D013997:Time Factors
D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; C496259:F0F1-ATPase epsilon subunit, E coli; D006180:Proton-Translocating ATPases