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

Michael J. Franklin ^*, William S. A. Brusilow  and Dixon J. Woodbury 

^* Departments of Physiology and Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan; and Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah

Correspondence: Address reprint requests to Dixon J. Woodbury, PhD, Dept. of Physiology and Developmental Biology, 574 WIDB, BYU, Provo, UT 84602. Tel.: 801-422-7562; Fax: 801-422-0700; E-mail: dixon_woodbury{at}byu.edu.

We have developed a mathematical model in concert with an assay that allows us to calculate proton (H⁺) flux and conductance through a single F_o of the F₁F_o ATP synthase. Lipid vesicles reconstituted with just a few functional F_o 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 F_o sector. This methodology is sensitive enough to detect small (15%) conductance changes. We find that wild-type F_o has a proton flux of 3100 ± 500 H⁺/s/F_o at a transmembrane potential of 106 mV (25°C and pH 6.8). This corresponds to a proton conductance of 4.4 fS.