PMC

Functional model of F₀. (A) Schematic illustration of F₀ operation. (B) Simple model for proton transfer; see text for details. (C) Hypothetical energy profile for proton transfer; see text for details. (D) Kinetic scheme.

The rate constant of charge flow through F₀ (see ) as a function of the pH and the isotope composition of the medium (H₂O: ○, D₂O: •). Medium contained 20 mM glycylglycine, 20 mM sodium phosphate, 20 mM 4-morpholinepropanesulfonic acid (MES), 20 mM glycine, 50 mM KCl, 2 mM K₄[Fe(CN)₆], 5 μM DMF, 2 mM KCN, 3 μM myxothiazol. The solid line was calculated by the kinetic model of F₀ conductance as described in the text with the parameters listed in .

Effect of pH on the H/D-isotope effect and on the Arrhenius activation energy of the charge transfer through F₀. (A) H/D isotope effect was calculated as the ratio of the rate constants in H₂O and D₂O (data taken from ). (B) The pH-dependence of the Arrhenius activation energy (E_a). Measurements were done in the presence of 3 μM myxothiazol in the temperature interval from 3°C to 40°C. A single experiment was done at pH 9; other points are the average of at least three experiments.

(A) Size distribution of F₁-depleted chromatophores by transmission electron microscopy (TEM). Because of drying on the microscope grid the originally spherical particles appeared as flat disks (see text). (B) Absorption transients attributable to carotenoid bandshifts of electrochromic origin as function of the K⁺ concentration in the suspending medium. These transients were caused by a diffusion potential that was generated by submitting chromatophores to a K⁺-concentration jump in the presence of the K⁺-ionophore valinomycin. F₁-depleted chromatophores (940 μM bacteriochlorophyll) were preincubated with 45 mM KCl, 205 mM NaCl, 5 mM MgCl₂, 20 mM glycylglycine-NaOH, pH 7.9. The final bacteriochlorophyll concentration in the samples was 12 μM.

Electrochromic absorption transients at a wavelength of 522 nm in F₁-depleted chromatophores as caused by a short flash of light. The upper traces were obtained without and with blocking of F₀ by 3 μM oligomycin, respectively. The difference trace (± oligomycin) represents the charge flow across F₀ (A) without and (B) with 3 μM myxothiazol added to deactivate the electrogenic and proton pumping activity of the cytochrome bc₁ complex. The actinic flash is indicated by an arrow. The bottom traces were obtained in the presence of 1 μM valinomycin to enhance the ion conductance of all chromatophores in the sample. It was apparent that absorption transients not originating from electrochromism as well as any electrochromic responses to localized electric fields were negligible under our conditions. The suspending medium contained: 100 mM KCl, 5 mM magnesium acetate, 2 mM K₄[Fe(CN)₆], 5 μM 1,1′-dimethylferrocene (DMF), 2 mM KCN, 20 mM glycylglycine-KOH, pH 7.9; chromatophores were added to 12 μM bacteriochlorophyll.

Transient charge transfer through F₀ in the presence of 3 μM myxothiazol as inhibitor of cytochrome-bc₁. (A) Two transients of charge flow through F₀, both electrochromic differences (± oligomycin) as the bottom trace in . ○, the transient recorded under excitation with a flash of saturating energy. Noisy line, the transient recorded at attenuated excitation flash energy to produce 33% signal saturation. The extent after the flash was normalized to unity. The insert maps allowed (uncolored) and forbidden (hatched) area in the parameter-field of α and β (see details in the text). (B) The noisy line represents the difference between the original transients shown in A; a histogram of the deviations is shown in the insert. The thick solid line is the same difference calculated according to the model with the parameters α = 0.05, β = 0.71, γ = 10 (other parameters are listed in ; see details in the text). It is consistent with the experimental error of 3%. The dashed curve was calculated with the same set of parameters except that γ = 1, and the dotted curve was calculated for another set of parameters, namely α = 0.1, β = 0.71, and γ = 10.

Proton transfer through F₀ as monitored by two pH indicators reporting pH transients from either side of the chromatophore membrane, (A, B) from the n-side (Cresol red, 90 μM, at a wavelength of 575 nm), and (C, D) from the p-side (Neutral red, 26 μM plus 0.3% w/v BSA, at 545 nm). Traces in parts A and C were recorded in the absence, and traces B and D in the presence of the ionophore valinomycin (2 μM). In the latter case the transmembrane voltage was collapsed by the valinomycin-mediated K⁺ current in <3 ms (see ). The relaxation of the pH difference was then entirely due to the entropic driving force (ΔpH). The proton release from bc₁ was slower in the presence of valinomycin (compare to traces 2 in and ). The medium contained 50 mM KCl, 5 mM MgCl₂, 2 mM K₄[Fe(CN)₆], 2 mM KCN, 5 μM dMF; pH was 7.9. Chromatophore stock was in 5 mM MgCl₂, 50 mM KCl, 10% sucrose. Traces 1 without, and traces 2 with 3 μM oligomycin added; traces 2-1 = difference trace ± oligomycin. Actinic flashes are marked by arrows; black bars correspond to 0.002 pH units.