Klaas J. Hellingwerf

The Effect of 'Probe Binding' on the Quantitative Determination of the Proton-Motive Force in Bacteria

The electrical potential across the cytoplasmic membrane of bacteria can be calculated from the distribution of the lipophilic cation tetraphenylphosphonium between the bulk phases of the medium and the cytoplasm. In order to determine the bulk phase concentrations, information about the binding of the probe to the cellular components is required. In de-energized cells of Rhodopseudomonas sphaeroides the binding appears to be proportional to the free probe concentration. The bulk phase concentrations can only be determined when knowledge is available about the distribution of the binding of the probe over the different cellular components. In this report, models for binding are presented which are based on the assumption that the binding is an energy-independent process. These models allow a proper calculation of the electrical potential when the binding of the probe to the different cellular components is known.

Light-induced generation of a protonmotive force and Ca2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The chemiosmotic hypothesis (Mitchell, 1966,1972) postulates that proton translocation by primary proton pumps generates an electrochemical proton gradient (∆p) across the cytoplasmic membrane. The electrochemical proton gradient or proton motive force (pmf) is composed of electrical and chemical parameters according to the equation:

The relation between electron transfer, proton-motive force and energy-consuming processes in cells of Rhodopseudomonas sphaeroides

\Delta p=\Delta \psi -2.3\text{ RT/F }\!\!\Delta\!\!\text{ pH (mV)}

FUNCTIONAL RECONSTITUTION OF PHOTOSYNTHETIC CYCLIC ELECTRON-TRANSFER IN LIPOSOMES

(1) where ∆ψ represents the electrical potential and ∆pH the chemical potential of protons across the membrane (2.3 RT/F is equal to 58.8 mV at 25°C).

The protein composition of the cytoplasmic membrane of aerobically and anaerobically grown Escherichia coli

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton‐motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of Rhodopseudomonas sphaeroides

Abstract In the phototrophic bacterium Rhodopseudomonas sphaeroides the two components of the proton-motive force (the ΔΨ and the ΔpH) were measured with ion-selective electrodes. Simultaneously, the rate of energy-consuming processes such as alanine transport or ATP-synthase activity was measured at increasing electron-transfer activity. The results indicate that the activity of the electron-transfer system determines the activity of the energy-consuming processes. The proton-motive force can increase or decrease with the activity of the electron-transfer chain depending on the experimental conditions. Consequently, under certain conditions the activity of secondary transport increases while the proton-motive force decreases. A fixed minimal rate of electron transfer is required before secondary solute transport can occur, independent of the magnitude of the proton-motive force. A threshold value of the proton-motive force for secondary solute transport is not required.

Chapter 10 Transport across bacterial membranes

Light-harvesting complex II (B800/850, LHC II complexes) and reaction centers with both light-harvesting complex I and II (B875 and B800/850; i.e.,RC LHC I-LHC II complexes) have been isolated from Rhodobacter sphaeroides . Both complexes have been incorporated into liposomes made from phospholipids purified from Escherichia coli. The electrochromic band shift of carotenoids, present in these complexes, could stillbe observed in these liposomes upon generation of a potassium diffusion potential. For RC LHC I-LHC II complexes the characteristics of the liposomes were studied in more detail: the extent of the absorbance changes increased with the amount of pigment protein complex incorporated and was maximal at a ratio of 70 nmol bacteriochlorophyll per mg lipid. Higher amounts of incorporated complexes led to a decrease in carotenoid signal due to an increasing membrane leakage. The carotenoid absorbance change at 503–487 nm showed a linear dependency on the diffusion potentials both in the negative as well as in the positive potential range. The spectrum of the absorbance changes at a fixed diffusion potential for RC LHC I-LHC II liposomes had asimilar shape as the spectrum found for chromatophores of Rb. Sphaeroides , however, with shifted maxima and minima. The spectrum foundfor LHC II liposomes was the inverted spectrum of the RC LHC I-LHC II liposomes. Electron micrographs of both type of liposomes showed distinct protein particles with a diameter of 15 nmfor RC LHC I-LHC II and 11 nm for RC LHC II complexes.

THE EFFECT OF TRYPSIN TREATMENT ON THE INCORPORATION AND ENERGY-TRANSDUCING PROPERTIES OF BACTERIORHODOPSIN IN LIPOSOMES

The interaction of solubilized reaction centers from the phototrophic bacterium, Rhodobacter sphaeroides , and the solubilized ubiquinol-cytochrome c oxidoreductase of Rhodobacter capsulatus was studied in solution and after coreconstitution in liposomes prepared from Escherichia coli phospholipids. Under both conditions, the ubiquinol-cytochrome c oxidoreductase increased the light-induced cyclic electron transfer, induced by reaction centers with 2,3-dimethoxy-5-methyl-6-(prenyl) 2 -1,4-benzoqu and cytochrome c as redox mediators. This effect was more pronounced at acid pH values. The light-induced cyclic electron transfer in these liposomes resulted in the generation of a protonmotive force. Under conditions where the protonmotive force was composed of a membrane potential only, the highest membrane potential (approx. −200 mV) was generated when 2,3-dimethoxy-5-methyl-6-(prenyl) 10 -1,4-benzo-quinone was used as redox mediator and when both electron transfer proteins were co-reconstituted in a 2:1 molar ratio. At acid pH non-transitent membrane potentials could be generated only in liposomes containing both reaction centers and the ubiquinol-cytochrome c oxidoreductase. These observations show that the pH-dependent direct oxidation of cytochrome c by ubiquinol in the liposomes was indeed catalyzed by the ubiquinol-cytochrome c oxidoreductase and that this oxidoreductase participates in proton pumping. This could also be concluded from the stimulating effect of 2,3-dimethoxy-5-methyl-6-(prenyl) 10 -1,4-benzoquinone on the membrane-potential-generating capacities in liposomes containing both electron transfer complexes. Such a stimulation was not observed in liposomes containing only reaction centers. The presence of cytochrome c in the co-reconstituted system was found to be essential for proton pumping.