Determination of Proton Flux and Conductance at pH 6.8 through Single Fo Sectors from Escherichia coli

Article, Other literature type English OPEN
Franklin, Michael J. ; Brusilow, William S. A. ; Woodbury, Dixon J. (2004)
  • Publisher: Biophysical Society
  • Journal: Biophysical Journal, volume 87, issue 5, pages 3,594-3,599 (issn: 0006-3495)
  • Related identifiers: doi: 10.1529/biophysj.104.044248
  • Subject: Bioenergetics | Biophysics

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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