Wim Crielaard

RECONSTITUTION OF ELECTROCHROMICALLY ACTIVE PIGMENT-PROTEIN COMPLEXES FROM RHODOBACTER-SPHAEROIDES INTO LIPOSOMES

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 transmembrane electrical potential in intact bacteria: simultaneous measurements of carotenoid absorbance changes and lipophilic cation distribution in intact cells of Rhodobacter sphaeroides

Abstract The electrical potential across the cytoplasmic membrane of Rhodobacter sphaeroides has been measured in intact cells by two independent techniques: the uptake and release of tetraphenyl phosphonium ions and the carotenoid absorbance band-shifts. Simultaneous measurements show that these two procedures give different membrane potentials. Upon energization with either light or during respiration tetraphenylphosphonium-distribution indicates a depolarization of the membrane while the electrochromic carotenoid band-shift indicates a hyperpolarisation. Treatment of the cells with venturicidin resulted in an increased light-induced membrane potential indicated by the carotenoid band-shift and led to a reversal in the polarity of the tetraphenylphosphonium response. The presence of ethylene diaminetetraacetic acid had no effect on the light-induced carotenoid absorbance change, but it decreased the light-induced membrane depolarisation indicated by the tetraphenylphosphonium ions. These results show that at least one of these methods is seriously in error.

FUNCTIONAL RECONSTITUTION OF PHOTOSYNTHETIC CYCLIC ELECTRON-TRANSFER 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.

Spectral identification of the electrochromically active carotenoids of Rhodobacter sphaeroides in chromatophores and reconstituted liposomes

Reaction centers with both light harvesting complexes I and II (B875 and B800/850; i.e., RCLHILHII complexes) have been isolated from Rhodobacter sphaeroides. These complexes have been incorporated into liposomes made from lipids purified from Escherichia coli. The electrochromic bandshift of carotenoids, present in these reconstituted complexes, shows shifted minima and maxima with respect to a similar spectrum in chromatophores of Rb. sphaeroides in a potassium diffusion potential induced difference spectrum (see also Crielaard, W., Hellingwerf, K.J. and Konings, W.N. (1989) Biochim. Biophys. Acta 973, 205-211). The absorbance spectrum, at room temperature or at 77 K, of both membrane preparations did not, however, reveal differences in the carotenoid region. The long-wavelength carotenoid peak in both preparations is located at 513 nm (77 K). A small difference could be observed between the 77 K excitation spectra of the B850 fluorescence. Reconstituted complexes show a carotenoid peak at 513 nm, whereas in chromatophores this peak is located at 514.5 nm. When fluorescence was recorded at 805 nm, to detect B800 excitation, there was a marked difference between both preparations. In liposomes the long wavelength B800-associated carotenoid peak is located at 512.5 nm, whereas in chromatophores this peak is located at 516 nm. These results explain the shifted minima and maxima in a potassium diffusion induced difference spectrum in proteoliposomes. The prediction of two carotenoid pools in chromatophores (De Grooth, B.G. and Amesz, J. (1977) Biochim. Biophys. Acta 462, 247-258) is confirmed, and the field sensitive carotenoids are identified as the pool that is associated with the B800 band (Kramer, H.J.M., Van Grondelle, R., Hunter, C.N., Westerhuis, W.H.J. and Amesz, J. (1984) Biochim. Biophys. Acta 765, 156-165).

Chapter 6 Photoactive yellow protein, a photoreceptor from purple bacteria

Abstract In members of the Archaea positive and negative phototactic responses are mediated via retinal-containing sensory rhodopsin photoreceptors, according to a mechanism that is similar to the mechanism of enterobacterial chemotaxis. In Bacteria the situation is less well resolved, even though the accumulation of photosynthetic bacteria in a light spot is one of the most extensively studied tactic responses of prokaryotes. Only recently, however, has it been reported that in this family of organisms (i.e. in the purple- or proteobacteria) another type of phototactic response occurs: blue light, of physiological intensities, evokes a repellent response. The photoreceptor that presumably mediates this response is the Photoactive Yellow Protein (PYP), a member of the xanthopsins. This family of photoreceptors consists of 4-hydroxy-cinnamate containing proteins, for which rich detail concerning structure and function is available. In this contribution we will review the structure and function of PYP, and the initial molecular genetic studies aimed to further characterize the signal transduction chain responsible for the photoresponses mediated through PYP.

Carotenoid Absorbance Changes in Liposomes Reconstituted with Pigment-Protein Complexes from Rhodobacter Sphaeroides

The electrochromic behaviour of carotenoids has been widely used to determine the electrical potential difference (∆ψ) across photosynthetic membranes, like chromatophores [1] and bacterial cells [2,3]. The carotenoid absorbance change has several advantages over other methods for recording the ∆ψ: (i) the method is non-invasive, (ii) the relationship between the ∆ψ and the absorbance change is linear and (iii) the method shows a rapid response time. The major disadvantage of the carotenoid band shift as a ∆ψ indicator is that a calibration of the ∆ψ dependent bandshifts is not (always) possible in every experimental system [3].

Light Driven Amino Acid Uptake in Membrane Vesicles of Streptococcus Cremoris Fused with Liposomes Containing Bacterial Reaction Centers

The incorporation of primary proton pumps in biological membranes has opened attractive possibilities for studies of proton motive force (Δp)-dependent processes in isolated membrane vesicles from bacterial1, and eukaryotic2 origin. Fused membranes obtained from liposomes containing a Ap-generating system and membrane vesicles of fermentative bacteria lacking an accessible proton pump have been shown to be excellent model systems for studies on the role of the Δp in solute transport1,3,5,6. Cytochrome c oxidase1 and bacteriorhodopsin3 have been used extensively as Δp-generating systems in these fused membranes.

Functional Reconstitution of Photosynthetic Reaction Centre Complexes from Rhodopseudomonas Palustris

Reconstitution is a powerful technique in the elucidation of the mechanisms involved in membrane associated biological energy transduction (see for example Racker & Stoeckenius, 1974). For proton motive force (transmembrane electrochemical proton gradient) generation in reconstituted systems several pumps are available (Driessen et al., 1987). Light-driven pumps could have important experimental advantages over chemically driven pumps, since pumping rates, and thereby electrochemical forces, can be simply controlled with light intensity. However until recently the only light-driven pump available, bacteriorhodopsin, reconstitutes in a scrambled orientation and generally generates an inside positive proton motive force.