Figure 6
From: The lateral distance between a proton pump and ATP synthase determines the ATP-synthesis rate
Coupled cyt. bo 3-ATP synthase activity. (A) ATP production by the ATP synthase, driven by an electrochemical gradient generated by cyt bo 3 (as shown in the scheme). ATP synthesis was measured as a change in luminescence from the luciferin-luciferase couple over 3 × 30 s. The reactions were started by the addition of ubiquinol Q1H2 (20 μM final concentration) in the presence of 2 mM DTT and 80 μM ADP. Measurements were done at pH 7.5 in 20 mM Tris-PO4 buffer supplemented with 2.5 mM MgCl2. Rates were calculated from the average slopes, calibrated by addition of well-defined amount of ATP (5 pmol, see mark at 30 s). The trace shown was obtained with 100 nm, 100% DOPC liposomes. (B) ATP-synthesis rates measured in DOPC vesicles with cyt. bo 3 labeled with either ATTO (A) 647N (and unlabeled ATP synthase) or Abberior STAR 635 (and ATP-synthase labeled with ATTO 594). Rates are compared to those obtained with proteoliposomes with unlabeled cyt. bo 3 and ATP-synthase in the presence (2.5 μM DDM, Detergent ctrl., see text for explanation) or absence (No label) of detergent. (C) Normalized ATP-synthesis rates of DOPC:DOPG vesicles with cyt. bo 3 and ATP-synthase labeled with ATTO 647N and ATTO 594 respectively (blue) or vesicles with unlabeled protein (green). The rates are normalized to that obtained with 100% DOPC vesicles to facilitate comparison. At 100% DOPC the activity was a factor of ~5 larger with the labeled than with the unlabeled proteins (c.f. panel B). Error bars is the standard deviation from measurements with four samples (except Detergent ctrl., two samples).
