| Determination of Proton Flux and Conductance at pH 6.8 through Single Fo 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 Fo of the F1Fo ATP synthase. Lipid vesicles reconstituted with just a few functional Fo 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 Fo sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type Fo has a proton flux of 3100 ± 500 H+/s/Fo at a transmembrane potential of 106 mV (25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS. | |||||||||||||
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