The size of the photosynthetic unit in purple bacteria

Pigment analysis was performed by means of normal phase HPLC on a number of bacteriochlorophyll a and b containing species of purple bacteria that contain a core antenna only. At least 99% of the bacteriochlorophyll in Rhodobacter sphaeroides R26, Rhodopseudomonas viridis and Thiocapsa pfennigii was esterified with phytol (BChl a _p and BChl b _p, respectively). Rhodospirillum rubrum contained only BChl a esterified with geranyl-geraniol (BChl a _GG). Rhodospirillum sodomense and Rhodopseudomonas marina contained, in addition to BChl a _p, small amounts of BChl a _GG, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol (2,10,14-phytatrienol and probably 2,14-phytadienol). In all species bacteriopheophytin (BPhe) esterified with phytol was present. The BChl/BPhe ratio indicated that in these species a constant number of 25 ± 3 antenna BChls is present per reaction centre. This number supports a model in which the core antenna consists of 12 - heterodimers surrounding the reaction centre. Determination of the in vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of ca. 140 mM^–1 cm^–1 for BChl a containing species and of 130 mM^–1 cm^–1 for Rhodopseudomonas viridis.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - GG geranyl-geraniol - LHI and LHII core and peripheral antenna complexes - P phytol - RC reaction centre Dedicated to the memory of Professor D.I. Arnon.     

Triplet energy transfer between bacteriochlorophyll and carotenoids in B850 light-harvesting complexes ofRhodobacter sphaeroides R-26.1

The build-up and decay of bacteriochlorophyll (BChl) and carotenoid triplet states were studied by flash absorption spectroscopy in (a) the B800-850 antenna complex ofRhodobacter (Rb.)sphaeroides wild type strain 2.4.1, (b) theRb. sphaeroides R-26.1 B850 light-harvesting complex incorporated with spheroidene, (c) the B850 complex incorporated with 3,4-dihydrospheroidene, (d) the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene and (e) theRb. sphaeroides R-26.1 B850 complex lacking carotenoids. Steady state absorption and circular dichroism spectroscopy were used to evaluate the structural integrity of the complexes. The transient data were fit according to either single or double exponential rate expressions. The triplet lifetimes of the carotenoids were observed to be 7.0±0.1 s for the B800-850 complex, 14±2 s for the B850 complex incorporated with spheroidene, and 19±2 s for the B850 complex incorporated with 3,4-dihydrospheroidene. The BChl triplet lifetime in the B850 complex was 80±5 s. No quenching of BChl triplet states was seen in the B850 complex incorporated with 3,4,5,6-tetrahydrospheroidene. For the B850 complex incorporated with spheroidene and with 3,4-dihydrospheroidene, the percentage of BChl quenched by carotenoids was found to be related to the percentage of carotenoid incorporation. The triplet energy transfer efficiencies are compared to the values for singlet energy transfer measured previously (Frank et al. (1993) Photochem. Photobiol. 57: 49–55) on the same samples. These studies provide a systematic approach to exploring the effects of state energies and lifetimes on energy transfer between BChls and carotenoids in vivo.     

Bacteriochlorophyllgehalt und Proteinmuster der Thylakoide von Rhodospirillum rubrum während der Morphogenese des Photosynthese-Apparates

Zusammenfassung Der Zusammenhang zwischen dem spezifischen Bacteriochlorophyllgehalt der Zellen und der Thylakoidmorphogenese wird an Rhodospirillum rubrum untersucht. Bei der Induktion des Photosynthese-Apparates wird zunächst Bacteriochlorophyll synthetisiert, obgleich noch keine Thylakoide gebildet werden (1. Phase). Werden Thylakoide ausgebildet, so steigt der spezifische Bacteriochlorophyllgehalt der Thylakoide in Abhängigkeit vom spezifischen Bacteriochlorophyllgehalt der Zellen an, bis ein Wert von 12–13 g Bacteriochlorophyll je mg Zellprotein erreicht ist (2. Phase). Während der Wert für die Zellen darüber hinaus weiter erhöht werden kann, bleibt der Wert der Thylakoide konstant bei 100 g Bacteriochlorophyll je mg Thylakoid-Protein (3. Phase). Isolierte Thylakoide aus Zellen mit niedrigem Bacteriochlorophyllgehalt besitzen geringere Durchmesser als Thylakoide aus Zellen mit höheren Werten. Auch hinsichtlich der Zusammensetzung der Thylakoide in Abhängigkeit von den steigenden Bacteriochlorophyllgehalten bei induzierten Zellen konnten Unterschiede festgestellt werden. Ähnlich wie die spezifischen Bacteriochlorophyllgehalte der Thylakoide, nähern sich die verschiedenen Proteinbausteine der Thylakoide einem festen Verhältnis zueinander, das sich oberhalb von 10–14 g Bacteriochlorophyll je mg Zellprotein nicht mehr ändert. Mit Zunahme des Bacteriochlorophyllgehaltes der Zellen steigt der Gehalt an thylakoidspezifischen Proteinen in den Membranen an und der Anteil der für die Cytoplasmamembran spezifischen Komponenten nimmt ab.

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The bacteriochlorophyll content and protein composition of chromatophores (=thylakoids) of Rhodospirillum rubrum during morphogenesis of the photosynthetic apparatus

Summary The correlation between the specific bacteriochlorophyll content of whole cells and the morphogenesis of chromatophores was investigated in Rhodospirillum rubrum. During the first phase after induction of the photosynthetic apparatus bacteriochlorophyll is synthesized without formation of chromatophores. In a second phase chromatophores increases in a linear correlation to the specific bacterichlorophyll content of the cells. In a third phase, when the specific bacteriochlorophyll content of the cells has reached 12–13 g/mg protein, the specific bacteriochlorophyll content of the chromatophores remains constant (100 g/mg protein). Isolated chromatophores from the second phase have smaller diameters, than chromatophores from the third phase. The composition of the protein compounds of isolated chromatophores changes during the development of the chromatophores in a similar fashion as the specific bacteriochlorophyll content of chromatophores does change. With increasing bacteriochlorophyll content of the cells the chromatophore specific proteins in the membranes increase whereas proteins specific for the cytoplasmic membrane decrease until both reach a constant level.

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Verwendete Abkürzungen BChl. Bacteriochlorophyll     

Manipulation of the bacteriochlorophyll c homolog distribution in the green sulfur bacterium Chlorobium tepidum

We have shown that the green sulfur bacterium Chlorobium tepidum can be grown in batch culture supplemented with potentially toxic fatty alcohols without a major effect on the growth rate if the concentration of the alcohols is kept low either by programmed addition or by adding the alcohol as an inclusion complex with -cyclodextrin. HPLC and GC analysis of pigment extracts from the supplemented cells showed that the fatty alcohols were incorporated into bacteriochlorophyll c as the esterifying alcohol. It was possible to change up to 43% of the naturally occurring farnesyl ester of bacteriochlorophyll c with the added alcohol. This change in the homolog composition had no effect on the spectral properties of the cells when farnesol was partially replaced by stearol, phytol or geranylgeraniol. However, with dodecanol we obtained a blue-shift of 6 nm of the Q_y band of the bacteriochlorophyll c and a concomitant change in the fluorescence emission was observed. The possible significance of these findings is discussed in the light of current ideas about bacteriochlorophyll organization in the chlorosomes.Abbreviations -CD -cyclodextrin - BChl bacteriochlorophyll - BChl c _H bacteriochlorophyllide c - [E,M] BChl c _F 8-ethyl, 12-methyl, farnesyl BChl c - [E,E] BChl c _F 8-ethyl, 12-ethyl, farnesyl BChl c - [P,E] BChl c _F 8-propyl, 12-ethyl, farnesyl BChl c - [I,E] BChl c _F 8-isobutyl, 12-ethyl, farnesyl BChl c - Car carotenoids     

The role of enoyl-CoA hydratase in the metabolism of isoleucine byPseudomonas putida

Chromatium vinosum, strain D, exhibits two extreme modifications of near infra-red absorption spectra when growing heterotrophically at temperatures either above or below 36.5° C. Chromatophores isolated from cells grown either at 33° C (33° C chromatophores) or 39° C (39° C chromatophores) were analyzed for structural and functional parameters. For this the following chromatophore subunits were solubilized and characterized; (i) a fraction absorbing maximally at 800 nm with shoulders at 820 and 850 nm when derived from 33° C chromatophores or absorbing at 800 nm and 850 nm when derived from 39° C chromatophores; (ii) reaction center-light harvesting bacteriochlorophyll complexes with identical spectra and ratios of reaction center to light harvesting bacteriochlorophyll (1:45); (iii) complexes containing cytochromes, (IV) reaction center bacteriochlorophyll complexes. Irrespective of their origins the fractions exhibited qualitatively identical protein patterns as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Protein patterns of 33° C and 39° C chromatophores revealed an identical ratio of proteins of reaction centers to proteins of cytochrome preparations. But the ratio of proteins of reaction centers to proteins of light harvesting moieties was 1.9 times higher in 39° C chromatophores than in 33° C chromatophores. Correspondingly, the ratio of reaction center per total bacteriochlorophyll was 1.7 times higher in 39° C chromatophores (1:110) then in 33° C chromatophores (1:190). Activities of photophosphorylation were 0.73 and 0.56 moles of ATP per moles of total bacteriochlorophyll per min for 33° C and 39° C chromatophores, respectively. Activities of sulfide oxidation in the light by whole cells were 2.37 and 1.96 moles of sulfide per mole of total bacteriochlorophyll per min for 33° C and 39° C cells. Accordingly, on a reaction center basis activities are significantly lower after growth of the organisms at 39° C than at 33° C. The data indicate that spectral changes in Chromatium vinosum represent changes in the ratio of reaction center to light harvesting bacteriochlorophyll accompanied by a variation of the absorption spectra of the latter bacteriochlorophyll moiety. Concomitantly, activities coupled to the photochemical apparatus were subjected to variations.Abbreviations Bchl Bacteriochlorophyll - LDAO lauryl dimethylamine oxide - SDS sodium dodecyl sulfate     

Resolution of electron and proton transfer events in the electrochromism associated with quinone reduction in bacterial reaction centers

We have measured the electrochromic response of the bacteriopheophytin, BPh, and bacteriochlorophyll, BChl, cofactors during the Q_A ^–Q_B Q_AQ_B ^– electron transfer in chromatophores of Rhodobacter (Rb.) capsulatus and Rb. sphaeroides. The electrochromic response rises faster in chromatophores and is more clearly biexponential than it is in isolated reaction centers. The chromatophore spectra can be interpreted in terms of a clear kinetic separation between fast electron transfer and slower non-electron transfer events such as proton transfer or protein relaxation. The electrochromic response to electron transfer exhibits rise times of about 4 µs (70%) and 40 µs (30%) in Rb. capsulatus and 4 µs (60%) and 80 µs (40%) in Rb. sphaeroides. The BPh absorption band is shifted to nearly equivalent positions in the Q_A ^– and nascent Q_B ^– states, indicating that the electrochromic perturbation of BPh absorption from the newly formed Q_B ^– state is comparable to that of Q_A ^– . Subsequently, partial attenuation of the Q_B ^– electrochromism occurs with a time constant on the order of 200 µs. This can be attributed to partial charge compensation by H⁺ (or other counter ion) movement into the Q_B pocket. Electron transfer events were found to be slower in detergent isolated RCs than in chromatophores, more nearly monoexponential, and overlap H⁺ transfer, suggesting that a change in rate-limiting step has occurred upon detergent solubilization.     

Excitation energy transfer in chlorosomes of Chlorobium phaeobacteroides strain CL1401: the role of carotenoids

The role of carotenoids in chlorosomes of the green sulfur bacterium Chlorobium phaeobacteroides, containing bacteriochlorophyll (BChl) e and the carotenoid (Car) isorenieratene as main pigments, was studied by steady-state fluorescence excitation, picosecond single-photon timing and femtosecond transient absorption (TA) spectroscopy. In order to obtain information about energy transfer from Cars in this photosynthetic light-harvesting antenna with high spectral overlap between Cars and BChls, Car-depleted chlorosomes, obtained by inhibition of Car biosynthesis by 2-hydroxybiphenyl, were employed in a comparative study with control chlorosomes. Excitation spectra measured at room temperature give an efficiency of 60–70% for the excitation energy transfer from Cars to BChls in control chlorosomes. Femtosecond TA measurements enabled an identification of the excited state absorption band of Cars and the lifetime of their S₁ state was determined to be 10 ps. Based on this lifetime, we concluded that the involvement of this state in energy transfer is unlikely. Furthermore, evidence was obtained for the presence of an ultrafast (>100 fs) energy transfer process from the S₂ state of Cars to BChls in control chlorosomes. Using two time-resolved techniques, we further found that the absence of Cars leads to overall slower decay kinetics probed within the Q_y band of BChl e aggregates, and that two time constants are generally required to describe energy transfer from aggregated BChl e to baseplate BChl a.This revised version was published online in October 2005 with corrections to the Cover Date.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30–35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808–866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Q_y transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Q_y transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808–866 complex of C. aurantiacus and the B800–850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 was derived.Abbreviations BChl bacteriochlorophyll - BPheo bacteriopheophytin - CD circular dichroism - LD linear dichroism - Tris Tris(hydroxymethyl)aminomethane     

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis - and -polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the -polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6–10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The -polypeptide of Rps. viridis differed from the other -polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the - and -polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the -polypeptide was tested in reconstitution with BChl a and BChl b with the - and -polypeptides, it had no effect; its role remains undetermined.Abbreviations B820 the subunit form of the core light-harvesting complex in BChl-a-containing bacteria which has an absorption maximum at or near 820 nm - B875 the core light-harvesting complex of Rhodobacter sphaeroides which has an absorption maximum at 875 nm - B881 the core light-harvesting complex of wild-type Rhodospirillum rubrum which has an absorption maximum at 881 nm - B895 the subunit form of the core light-harvesting complex in Rps. viridis which has an absorption maximum near 888–895 nm - B1015 the core light-harvesting complex of Rps. viridis which has an absorption maximum at 1015 nm - CD circular dichroism - LH1 the core light-harvesting complex - OG n-octyl -d-glucopyranoside     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

The Model of Pigment Aggregation in the Chlorosomal Antenna of the Green Bacterium Chloroflexus aurantiacus

Independent experimental and theoretical evaluation was performed for the adequacy of our previously proposed general molecular model of the structural organization of light-harvesting pigments in chlorosomal bacteriochlorophyll (BChl) /d/e-containing superantennae of different green bacteria. Measurement of the temperature dependence of steady-state fluorescence spectra of BChl c was accomplished in intact cells of a photosynthetic green bacterium Chloroflexus aurantiacus; this allows in vivodetermination of the structure of exciton levels of BChl c oligomers in this natural antenna. Experimental data confirm our model of organization of oligomeric pigments in chlorosomal BChl c antenna of green bacterium Chloroflexus aurantiacus. This model implies that the unit building block of the antenna is a cylindrical assembly containing six excitonically coupled linear pigment chains, whose exciton structure with intense upper levels provides for the optimal spectral properties of the light-harvesting antenna.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Q_y transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Q_y transition of BChl c and the -carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Q_y transition is close to the magic angle (55°).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - LD linear dichroism Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium tepidum

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 ± 17 nm × 66 ± 13 nm × 4.3 ± 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA–BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.     

Acid denaturation of the B875 light-harvesting complex in membranes of Rhodobacter sphaeroides

The structural basis for the spectral red shift in the near-IR absorption band of the B875 light-harvesting complex was examined by treatment of membranes from Rhodobacter sphaeroides M21 with acid. This mutant strain lacks the overlapping spectral bands of the B800–850 light-harvesting antenna and gives rise to membrane fragments with both surfaces accessible to protons. At pH 2.2, about half the absorption at 876 nm was converted within 10 min to a free pigment band; the remaining absorption appeared at 880 nm and shifted to 845 nm over the next three hours. These spectral shifts could not be reversed by alkali. Approximately one-third of the characteristic near-IR CD signal of B875 was also lost initially and replaced by a broad trough centered near 854 nm. Thereafter, the CD spectrum was dominated by the strong conservative signal of the 845 nm absorbing component which was attributed to an oligomeric bacteriopheophytin a species, probably a dimer. A kinetic analysis of the acid-induced absorption changes suggested a multi-step model with rate constants of 0.37 min^-1 for the initial rapid change and 0.05 and 0.11 min^-1 for the respective subsequent steps. The non-conservative nature of the near-IR CD spectrum of the intact complex, together with the spectral changes observed after the initial loss of near-IR absorption and CD, suggest that pigment-pigment interactions are not solely responsible for the red shift in this complex.Abbreviations BChl bacteriochlorophyll a - BPheo bacteriopheophytin a     

Electrostatic effect of surfactant molecules on bacteriochlorophyll a and carotenoid binding sites in the LH1 complex isolated from Rhodospirillum rubrum S1 probed by Stark spectroscopy

The LH1 complexes were isolated from the purple photosynthetic bacterium Rhodospirillum rubrum strain S1. They were initially solubilized using LDAO and then purified in the presence of Triton X-100. The purified complexes were then either used directly or following an exchange into LDAO. Stark spectroscopy was applied to probe the electrostatic field around the bacteriochlorophyll a (BChl a) and carotenoid binding sites in the LH1 complexes surrounded by these two different surfactant molecules. Polarizabilty change () and dipole moment change () upon photoexcitation were determined for the BChl a Q_y band. Both of these parameters show smaller values in the presence of LDAO than in Triton X-100. This indicates that polar detergent molecules, like LDAO, affect the electrostatic environment around BChl a, and modify the nonlinear optical parameters ( and values). The electrostatic field around the BChl a binding site, which is generated by the presence of LDAO, was determined to be |E _L | = ∼3.9 × 10⁵ [V/cm]. Interestingly, this kind of electrostatic effect was not observed for the carotenoid-binding site. The present study demonstrates a unique electrostatic interaction between the polar detergent molecules surrounding the LH1 complex and the Q_y absorption band of BChl a that is bound to the LH1 complex.     

Differentiation of the membrane system in cells of Rhodopseudomonas capsulata after transition from chemotrophic to phototrophic growth conditions

Aerobically in the dark grown cultures of Rhodopseudomonas capsulata were shifted to low oxygen partial pressure for 30 min and afterwards to phototrophic conditions (anaerobic, light). During 210 min of adaptation to a phototrophic mode of life the bacteriochlorophyll (BChl) concentration increased 53-fold (doubling time 40 min) and the carotenoid content six fold. Growth was delayed. The light membrane fraction from chemotrophic and induced phototrophic cells contained low concentrations of small photosynthetic units (reaction center+light harvesting BChl B870), and low respiratory activities, especially of succinatecytochrome c oxidase. The heavy membrane fraction, i.e. the intracytoplasmic chromatophore fraction, increased during adaptation approximately 9-fold in surface area per cell, 42-fold in BChl content, 7-fold in reaction center content and 6-fold in the size of the photosynthetic unit.Phospholipid and fatty acid content and patterns changed slightly during adaptation.Non-standard Abbreviations BChl bacteriochlorphyll - R. Rhodopseudomonas     

Evaluation of the electrical capacitance in biological membranes at different phospholipid to protein ratios

Photosynthetic chromatophores of Rhodobacter capsulatus were differently enriched in phospholipid content by freezing, thawing and sonicating in the presence of phospholipid vesicles. Closed vesicles, characterized by different phospholipid to protein molar ratios and increasing average radius at increasing phospholipid enrichment, were collected after sucrose density gradient sedimentation. The electrical capacitance of these systems was evaluated from the ratio of reaction center content, photooxidized by single turnover flash in the presence of antimycin, to the corresponding membrane potential difference, measured from the electrochromic red shift of the endogenous carotenoid band. The values obtained, normalized per protein content, increased at increasing phospholipid enrichment, and correlated linearly with the increasing phospholipid to protein molar ratios. The charging capacitance of chromatophores was evaluated to be 3–6×10^-17 F and was found to increase at increasing average radius of the phospholipid enriched vesicles, as predicted by the equation of the spherical shell dielectric. The carotenoid signal, elicited in the dark by imposing diffusion potentials of known extent with K⁺-valinomycin pulses, significantly decreased at high phospholipid enrichment, indicating that in the presence of large phospholipid excess, a partial displacement of the carotenoid molecules sensing the induced electric field is produced. Concomitantly, the energy transfer efficiency from carotenoids to core light harvesting complexes (B-875) was also partially affected, particularly at high phospholipid to protein molar ratio. All together, these results suggest that the reaction center complexes are dispersed within the lipid bilayer upon fusion and that carotenoids sense a delocalized light-induced transmembrane field.Abbreviations BChl bacteriochlorophyll - [BChl]₂ reaction center - PL phospholipid - cyt cytochrome - transmembrane electrical potential difference - TES 2-2-Hydroxy-1,1-bis-(hydroxymethyl)ethyl-amino-ethanosulfonic acid - mg_p mg protein     

Probing protein structural requirements for formation of the core light-harvesting complex of photosynthetic bacteria using hybrid reconstitution methodology

The - and -polypeptides of LH1 isolated from four different photosynthetic bacteria (Rhodospirillum rubrum, Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas viridis) were used for homologous and hybrid reconstitution experiments with bacteriochlorophyll a. Formation of B820-type subunit complexes and LH1-type complexes were evaluated. The -polypeptides of R. rubrum, Rb. sphaeroides and Rb. capsulatus behaved similarly and formed B820-type subunit complexes in the absence of an -polypeptide. The - and -polypeptides were both required to form a LH1-type complex with each of these three homologous systems. In hybrid experiments where the -polypeptides were tested for reconstitution with -polypeptides other than their homologous partners, half of the twelve possible combinations resulted in formation of both B820- and LH1-type complexes. Three of the combinations that did not result in formation of a LH1-type complex involved the -polypeptide of R. rubrum. It is suggested that these latter results can be explained by charge repulsion between the Lys at position-17 (assigning the conserved His located nearest to the C-terminus as position 0) in the -polypeptide of R. rubrum and each of the heterologous -polypeptides tested, all of which have an Arg at this location. Conclusions that can be derived from these experimental results include: (1) the experimental data support the idea that a central core region of approximately 40 amino acids exists in each of the polypeptides, which contains sufficient information to allow formation of both the B820- and LH1-type complexes and (2) a specific portion of the N-terminal hydrophilic region of each polypeptide was found in which ion pairs between oppositely charged groups on the - and -polypeptides seem to stabilize complex formation.Abbreviations BChl a bacteriochlorophyll a - BChl BChl a is implied - BChl a _P BChl a containing phytol as the esterifying alcohol - BChl a _gg BChl a containing geranylgeraniol as the esterifying alcohol - LH1 the core light-harvesting complex - B873 the core light-harvesting complex of the G-9 mutant (carotenoidless) of R. rubrum or of the wild-type light-harvesting complex after benzene extraction (both with absorption maxima at 873 nm) - B820 the subunit form of B873 consisting of native - and -polypeptides with the same stoichiometry of ₁₁·2BChl as LH1 - B820-type complex a complex exhibiting absorption and CD spectra indistinguishable from B820 but composed of either the -polypeptide only, or of a heterologous mixture of - and -polypeptides - RC reaction center - PRC photoreceptor complex consisting of the RC and LH1 - CD circular dichroism - OG n-octyl -d-glucopyranoside - HFA hexafluoroacetone trihydrate     

Noncyclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus

Chromatophores isolated from the marine phototrophic bacterium Rhodobacter sulfidophilus were found to photoreduce NAD with sulfide as the electron donor. The apparent K _m for sulfide was 370 M and the optimal pH was 7.0. The rate of NAD photoreduction in chromatophore suspensions with sulfide as the electron donor (about 7–12 M/h·mol Bchl) was approximately onetenth the rate of sulfide oxidation in whole cell suspensions. NAD photoreduction was inhibited by rotenone, carbonyl cyanide-m-chlorophenylhydrazone, and antimycin A. Sulfide reduced ubiquinone in the dark when added to anaerobic chromatophore suspensions. These results suggest that electron transport from sulfide to NAD involves an initial dark reduction of ubiquinone followed by reverse electron transport from ubiquinol to NAD mediated by NADH dehydrogenase.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide-m-chlorophenylhydrazone - MOPS 3(N-morpholino)-propane sulfonate - Uq ubiquinone