Figure 4.

Characterization of Pd-CI in liposomes. (a) K_M curve for Q₁₀ in proteoliposomes. The K_M and V_max values were determined to be 1.1 ± 0.2 mM and 27.9 ± 1.2 µmol min^–1 (mg CI)^–1, respectively (± S.E.M. of the fit). (b) Inhibition by piericidin A and rotenone. The measured IC₅₀ values for piericidin A and rotenone were 24 ± 3 nM and 452 ± 48 nM, respectively (± S.E. of the fit). (c) Proton pumping measured by an ACMA fluorescence quench assay. Proton pumping was initiated by addition of 1 mM NADH and proteoliposomes uncoupled by addition of 25 µg mL^–1 alamethicin (AlaM). Liposomes without Pd-CI were added as a control. (d) Coupling Pd-CI catalysis to ATP synthesis. Pd-CI was co-reconstituted in liposomes with E. coli ATP synthase (CI-AOX-F₁F_O) and NADH-coupled ATP production was measured as luminescence using a luciferase-based assay. No ATP was generated by proteoliposomes containing only ATP synthase (F₁F_O only) while addition of 2 μM piericidin A (pierA) or 20 μg mL^–1 gramicidin A (gramA) inhibited ATP synthesis. Alternative oxidase (AOX) was directly added to the assay mixture at 20 µg mL^–1 in panels (a–c) and at 5 µg mL^–1 in panel (d). See “Materials and methods” for the assembly of proteoliposomes and other assay conditions.