Fluorescence Quenching Dependent on ∆pH or ∆ψ.

Fluorescence quenching of the pH-responsive fluorophores 9-amino-6-chloro-2-methoxyacridine (ACMA) (excitation: 430 nm, emission: 470 nm) or acridine orange (excitation: 493 nm, emission: 530 nm) was performed essentially as previously described (13). The following modifications were made: 0.2 mg⋅mL⁻¹ (final concentration) IMVs or 5 μL⋅mL⁻¹ F₁F_o proteoliposomes were added NADH or ATP were used to initiate quenching as indicated. Unless otherwise indicated the concentration of acridine orange was 5 μM. Assays were performed at 37 °C. For cytochrome bo₃ (cbo₃) proteoliposomes fluorescence quenching of the pH responsive fluorophore, ACMA, was measured and is described in SI Appendix. Fluorescence quenching of the ∆ψ responsive fluorophore oxonol VI was performed as previously described (13), except quenching was measured photometrically at 590–630 nm and NADH was simultaneously measured at 340 nm.