Dichroism of bacteriochlorophyll in chromatophores of photosynthetic bacteria.

The dichroism was measured in films of air-dried and, consequently, flattened chromatophores of Chromatium vinosum, Rhodopseudomonas sphaeroides and Rhodospirillum rubrum. The values (deltaA/A) of dichroism in C. vinosum were found to be -1.05 at 590 nm and 0.75 in the near infrared region. The values of dichroism in R. sphaeroides were -0.70 at 590 nm and 0.80 at 870 nm. The values of dichroism in R. rubrum were -1.45 at 590 nm and 0.97 at 870 nm.     

Some effects of o-phenanthroline on electron transport in chromatophores from photosynthetic bacteria

The midpoint potentials of the primary electron acceptors in chromatophores from Rhodopseudomonas spheroides and Chromatium have been studied by titrating the laser-induced P605 and cytochrome c oxidations, respectively. Both midpoint potentials are pH dependent (60 mV/pH unit).o-Phenanthroline shifts the midpoint potentials of the primary acceptors, by +40 mV in Rps spheroides and +135 mV in Chromatium. A similar though less extensive change in midpoint potential was observed in the presence of batho-phenanthroline, but not with 8-hydroxyquinoline. The shifted midpoints retain the same dependence on pH.Some of the effects of o-phenanthroline can be explained by assuming that it chelates the reduced form of the primary electron acceptor. This suggests the presence in the primary electron acceptor of a metal chelated by o- and batho-phenanthroline.In Rps spheroides chromatophores o-phenanthroline inhibits the laser- and flash-induced carotenoid shift at all redox potentials, stimulates the laser-induced P605 oxidation at redox potentials between +350 and +420 mV and slows the decay of the laser-induced cytochrome c oxidation below +180 mV. These effects show that o-phenanthroline may have more than one site of action.     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

Kinetics of the generation of a photo-induced electric potential in chromatophores of photosynthetizing bacteria

Flash-induced formation of an electric potential difference (delta psi) was monitored by a direct method in chromatophores associated with the collodion phospholipid membrane. In Rhodospirillum rubrum and Rhodopseudomonas sphaeriodes chromatophores, the kinetics of delta psi generation exhibit fast (tau less than or equal to 0.3 microseconds) and slow (tau congruent to 200 microseconds) phases, the latter observed in the presence of exogenous quinones. Comparison of the kinetic and potentiometric characteristics of the process with those of electron transport reactions suggests that the fast phase of delta psi rise is due to charge separation between the primary electron donor, P870, and primary electron acceptor QIFe; the slow phase, which is inhibited by o-phenanthroline, is due to electron donation from QIFe to the secondary acceptor, quinone QII. The kinetics of delta psi decay include components arising form the recombination of primary separated charges (tau congruent to 30 ms) and from the passive discharge of the membrane (tau congruent to 400 ms; tau congruent to 1400 ms). From a redox titration of the photo-induced electric signal and the photo-induced absorption changes of P870 at different pH meanings, the value of pK for the primary acceptor FeQI was found to be 7.4 in Rps. sphaeroides chromatophores. In Chromatium minutissimum, a phase ( tau congruent to 20 microseconds) was observed in addition to those seen in Rps. sphaeroids and R. rubrum which was explained by the reduction of P890+ from the high potential cytochrome c555. Possible distribution of the electron transport components in the chromatophore membrane are discussed.     

Fluorescence of bacteriochlorophyll as related to the photochemistry of chromatophores of photosynthetic bacteria

Time courses and the emission spectra of fluorescence and light-induced absorption changes of P890 in chromatophores of the photosynthetic bacteria Chromatium D, Rhodopseudomonas spheroides and Rhodospirillum rubrum were investigated.

The time course of fluorescence in chromatophores was separated into two phases, i.e. an initial rapid rise (ƒ_i) and a subsequent slow increase towards a steady level of emission (ƒ_v). The ƒ_i and the ƒ_v components showed different emission spectra having different peak position. The ƒ_v component was emitted from the longest wavelength-absorbing form of bulk bacteriochlorophyll (B890), the ƒ_i component from both B890 and B850.

The magnitude of the ƒ_v component depended on experimental conditions controlling the states of the cyclic electron transport in chromatophores, including changes in levels of redox potential of the medium, additions of electron donors and inhibitors. The magnitude of the ƒ_i component was not affected by these experimental conditions. It was, therefore, concluded that only the ƒ_v component is related to the cyclic electron transport, and that the magnitude of ƒ_v is controlled by the oxidation-reduction state of the primary electron acceptor for the photochemical reaction center in chromatophores.     

Fusion of chromatophores derived from Rhodopseudomonas sphaeroides

Fusion of chromatophores, the photosynthetic membrane vesicles isolated from the intracytoplasmic membranes of Rhodopseudomonas sphaeroides, was achieved by the use of poly(ethylene glycol) 6000 as fusogen. Ultracentrifugation, electron microscopy, intrinsic density and isotope labeling were used to demonstrate chromatophore fusion. Although studies of the flash-induced shift in the carotenoid absorbance spectrum indicated that the membrane was rendered leaky to ions by either the fusion procedure or the increased size of the fused products, the orientation and integrity of fused chromatophores were otherwise demonstrated to be identical to control chromatophores by freeze-fracture electron microscopy, proteolytic enzyme digestion, enzymatic radioiodination, and transfer of chromatophore phospholipids mediated by phospholipid exchange protein extracted from Rps. sphaeroides.     

Isolation and characterization of glycolipids from some photosynthetic bacteria

Glycosyl glycerides have been found in substantial amounts in Chloropseudomonas ethylicum but could not be detected in two strains of Rhodopseudomonas palustris. Rhodospirillum molischianum possibly contains small amounts of monoglycosyl diglyceride. The glycolipids of C. ethylicum have been separated into two components. One of these, glycolipid I, is a monogalactosyl diglyceride. Glycolipid II, upon acid hydrolysis, yields galactose, rhamnose, and a third, unidentified sugar. The glycolipids or total lipids of the photosynthetic bacteria examined contained saturated and monounsaturated, but none of the more highly unsaturated, fatty acids.     

The dependence of the rate of transhydrogenase on the value of the protonmotive force in chromatophores from photosynthetic bacteria

In conditions where the pH gradient is negligible, the rate of the pyridine nucleotide transhydrogenase in chromatophores of Rhodobacter capsulatus has a threshold dependence on membrane potential. The relationship is similar when either antimycin or myxothiazal or carbonyl cyanide p-trifluoromethoxyphenylhydrazone is used to depress the membrane potential. The relationship is distorted when membrane potential is reduced by lowering the photosynthetic light intensity.     

Distribution of phosphatidylethanolamine in the lipid bilayer of chromatophores of the photosynthetic bacterium rhodospirillum rubrum

The distribution of phosphatidylethanolamine in the two lipid layers of chromatophores ofRhodospirillum rubrum has been analysed by chemical modification of phosphatidylethanolamine (PE) with trinitrobenzenesulfonic acid (TNBA) at low temperatures. Around 45±1% of the total phosphatidylethanolamine is labelled by this procedure independent on chromatophore purity, vesicle size, action of proteases and growth state of the cells. This demonstrates a complete modification of the accessible phosphatidylethanolamine and an asymmetric distribution of phosphatidylethanolamine, with 45% of the phosphatidylethanolamine in the outer part of the bilayer.Abbreviations TNBA 2,4,6 trinitrobenzenesulfonic acid - PE phosphatidylethanolamine - PMS phenazinmethosulfate     

The structure of chromatophores from purple photosynthetic bacteria fused with lipid-impregnated collodion films determined by near-field scanning optical microscopy.

Lipid-impregnated collodion (nitrocellulose) films have been frequently used as a fusion substrate in the measurement and analysis of electrogenic activity in biological membranes and proteoliposomes. While the method of fusion of biological membranes or proteoliposomes with such films has found a wide application, little is known about the structures formed after the fusion. Yet, knowledge of this structure is important for the interpretation of the measured electric potential. To characterize structures formed after fusion of membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides with lipid-impregnated collodion films, we used near-field scanning optical microscopy. It is shown here that structures formed from chromatophores on the collodion film can be distinguished from the lipid-impregnated background by measuring the fluorescence originating either from endogenous fluorophores of the chromatophores or from fluorescent dyes trapped inside the chromatophores. The structures formed after fusion of chromatophores to the collodion film look like isolated (or sometimes aggregated, depending on the conditions) blisters, with diameters ranging from 0.3 to 10 microm (average approximately 1 microm) and heights from 0.01 to 1 microm (average approximately 0.03 microm). These large sizes indicate that the blisters are formed by the fusion of many chromatophores. Results with dyes trapped inside chromatophores reveal that chromatophores fused with lipid-impregnated films retain a distinct internal water phase.     

Nature of photochemical reactions in chromatophores ofChromatium D III. Heterogeneity of the photosynthetic units

The effect of isooctane extraction on photooxidation ofc-type cytochromes was investigated inChromatium chromatophores.Photooxidation of cytochromec-555 was not affected by isooctane-extraction except that the dark recovery was accelerated. Photooxidation of cytochromec-552 was abolished by thorough extraction of ubiquinone-7, but the quantum yield of the cytochrome photooxidation remained unchanged until 90|X% of the total ubiquinone was extracted. The photooxidation of cytochromec-552 was recovered by the addition of ubiquinone-7 but not by menaquinone. A dark incubation of sufficient length was needed for maximal quantum yield of cytochromec-555 photooxidation in the presence of 30 mM ascorbate.It is proposed that there are two types of photosynthetic units (or associations of molecules involved in the primary redox reactions) inChromatium chromatophores. The combinations of primary electron donor-reaction center chlorophyll-primary electron acceptor may be cytochromec-552-P890-ubiquinone in one type and cytochromec-555-P890-X in another.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

Kinetic measurements of electron transfer in coupled chromatophores from photosynthetic bacteria. A method of correction for the electrochromic effects

A quantitative study of the kinetics of electron transfer under coupled conditions in photosynthetic bacteria has so far been prevented by overlap of the electrochromic signals of carotenoids and bacteriochlorophyll with the absorbance changes of cytochromes and reaction centers. In this paper a method is presented by which the electrochromic contribution at any wavelength can be calculated from the electrochromic signal recorded at 505 nm, using a set of empirically determined polynomial functions. The electrochromic contribution to kinetic changes at any wavelength can then be subtracted to leave the true kinetics of the redox changes. The corrected redox changes of the reaction center measured at 542 and 605 nm mutually agree, thus providing an excellent test of self-consistency of the method. The corrected traces for reaction center and of cytochrome b-566 demonstrate large effects of the membrane potential on the rate and poise of electron transfer. It will be possible to study the interrelation between proton gradient and individual electron reactions under flash or steady-state illumination.     

Changes in the acyl lipid composition of photosynthetic bacteria grown under photosynthetic and non-photosynthetic conditions

The acyl lipids and their constituent fatty acids were studied in the photosynthetic bacteria Rhodospirillum rubrum, Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides, which were grown under photosynthetic and non-photosynthetic conditions. The major lipids were found to be phosphatidylethanolamine, phosphatidylglycerol and cardiolipin in each bacterium. The two Rhodopseudomonas species also contained significant quantities of phosphatidylcholine. Other acyl lipids accounted for less than 10% of the total. On changing growth conditions from non-photosynthetic to photosynthetic a large increase in the relative proportion of phosphatidylglycerol was seen at the expense of phosphatidyl-ethanolamine. In Rhodospirillum rubrum the fatty acids of the major phospholipids showed an increase in the proportion of palmitate and stearate and a decrease in palmitoleate and vaccenate on changing growth conditions to photosynthetic. In contrast, the exceptionally high levels (>80%) of vaccenate in individual phospholipids of Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides were unaffected by changing growth conditions to photosynthetic. Analysis of the lipids of chromatophores, isolated from the three bacteria, showed that these preparations were enriched in phosphatidylglycerol. The large increase in this phospholipid, seen during growth under photosynthetic conditions, appeared, therefore, to be due to a proliferation of chromatophore membranes. Possible roles for acyl lipids in the formation and function of the photosynthetic apparatus of bacteria are discussed.     

Linear dichroism of electric field oriented bacteriochlorophyll a-protein from green photosynthetic bacteria

Bacteriochlorophyll a-protein from Prosthecochloris aestuarii strain 2K was oriented in a pulsed electric field. The room temperature linear dichroism spectrum of the oriented protein in the Q_y region of the bacteriochlorophyll a absorption exhibits a single asymmetrical peak at 813 nm with a shoulder extending to the blue. The ≈12 nm fullwidth of the linear dichroism peak is only about half that of the 300 K absorption spectrum. The linear dichroism at 813 nm was not saturated at field strengths of up to 15 kV/cm. The time dependence of the linear dichroism suggests that the orienting particles are aggregates of at least some tens of bacteriochlorophyll a-protein trimers. The linear dichroism peak coincides in wavelength with the 813-nm peak of the 300 K, 4th derivative absorption spectrum of the protein and is therefore attributed to the bacteriochlorophyll a Q_y exciton transition observed in absorption at the same wavelength.     

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.