Determination of proton flux and conductance at pH 6.8 through single F0 sectors from Escherichia coli

Abstract

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H+) flux and conductance through a single F0 of the F1F0 ATP synthase. Lipid vesicles reconstituted with just a few functional F0 from Escherichia coli were loaded with 250 mM K+ and suspended in a low K+ solution. The pH of the weakly buffered external solution was recorded during sequential treatment with the potassium ionophore valinomycin, the protonophore carbonyl cyanide 3-chlorophenylhydrazone, and HCl. From these pH traces and separate determinations of vesicle size and lipid concentration we calculate the proton conductance through a single F0 sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type F0 has a proton flux of 3100 ± 500 H +/s/F0 at a transmembrane potential of 106 mV (25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS. © 2004 by the Biophysical Society.