FIGURE 2.

NADH- or ATP-dependent proton electrochemical gradient formation by mutant F₀F₁ in membranes. 200 μg of membrane vesicle protein from strain DK8, harboring plasmid pBWU13 wild type or carrying the mutations for βE381C or βD380C, were suspended in 2 ml of buffer containing 10 mm HEPES-KOH, 300 mm KCl, 5 mm MgCl₂, 1 μm valinomycin, and 1 μm acridine orange at pH 7.5, with vigorous mixing. Fluorescence intensity at 530 nm (excitation at 460 nm) was monitored at 25 °C. A, NADH-driven quenching. Proton pumping was initiated at the indicated time (arrow) by addition of 1 mm NADH. B, ATP-driven quenching. Proton pumping was initiated at the indicated time (arrow) by addition of 1 mm ATP. 1 μm carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, as indicated (arrows) to abolish the proton gradient and establish the maximum fluorescence value. The fluorescence traces are depicted as follows: wild type (blue), βD380C (red), βE381C (green). The traces are representative of those from several trials.