Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence.
FOF1-ATP synthase converts two energetic "currencies" of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the single-enzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (<1 microM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of approximately 0.25 mol ADP per mole of enzyme. There was no detectable detachment of F1 from FO. (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (>1 microM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s(-1) at pH 8. Proton slip probably presents the free-wheeling of the central rotary shaft, subunit gamma, in an open structure of the (alphabeta)3 hexagon with no nucleotides in the catalytic sites.
Feniouk
Boris A
BA
Division of Biophysics, Faculty of Biology/Chemistry, University of Osnabrück, D-49069 Osnabrück, Germany.
Mulkidjanian
Armen Y
AY
Junge
Wolfgang
W
D020130:Bacterial Chromatophores / Q000201:enzymology* / Q000502:physiology*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008027:Light; D008564:Membrane Potentials / Q000502:physiology; D009711:Nucleotides / Q000378:metabolism; D011487:Protein Conformation; D018892:Proton-Motive Force / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013053:Spectrophotometry
D009711:Nucleotides; D006180:Proton-Translocating ATPases