Linear dichroism of light harvesting bacteriochlorophyll proteins from Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films.

Light-harvesting bacteriochlorophyll-protein complexes from Rhodopseudomonas sphaeroides 2.4.1 and R-26 mutant are solubilized in sodium dodecyl sulfate and imbedded in polyvinyl alcohol. Stretching induces orientation, and the linear dichroism of visible and near infrared absorption is analyzed. Based on a simple model, angles between the particle axis and the transition dipole moments are found. In the near infrared absorption band of the R-26 light-harvesting protein the dichroic ratio varies from 1.30 to 1.57. Using the absorption curves the band is resolved into two exciton components. In the visible absorption band the dichroic ratio has a constant value of 0.43 for the R-26 protein but varies with wavelength for the wild type light-harvesting protein. This variation is attributed to an additional bacteriochlorophyll not present in the R-26 protein.

Structural studies on reconstituted reaction center-phosphatidylcholine membranes.

Reaction center protein, isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R26 mutant, was incorporated into phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. Cytochrome c, added to preformed reaction center-phosphatidylcholine vesicles, rapidly reduced up to 90% of the laser-generated (BChl)2+ of the reaction center (with kinetics of electron transfer similar to those in the chromatophore membrane) which suggests that the portion of the reaction center which accommodates functional cytochrome c binding sites is exposed predominantly on the exterior of the vesicles. Unit cell electron density profiles were derived from lamellar X-ray diffraction from oriented reaction center-phosphatidylcholine membrane multilayers at varying lipid/protein ratios. The analysis of these profiles showed that the reaction center protein incorporates into the phosphatidylcholine membrane with unique sidedness and that the profile of the reaction center protein itself is asymmetric and spans the membrane.

Transient optical spectroscopy of single crystals of the reaction center from Rhodobacter sphaeroides wild-type 2.4.1.

The photoactivity of the crystallized reaction centers from Rhodobacter sphaeroides wild-type strain 2.4.1 has been examined by light-induced absorption spectral changes associated with charge separation and triplet state formation in the reaction center. Upon excitation of a crystal at ambient redox potential, the primary donor 865 nm band bleaches reversibly. The kinetics of its recovery were found to be biphasic with rate constants 11.5 +/- 1.3 s-1 and 0.9 +/- 0.4 s-1 which correspond to lifetimes of 87.0 +/- 9.0 ms and 1.0 +/- 0.7 s, respectively. The ratio of the fast-to-slow component preexponential terms was 3.5 +/- 1.1 suggesting that the majority (78.9 +/- 13.0%) of the reaction centers in the crystals lack the secondary quinone, QB. The addition of sodium ascorbate to the crystals attenuates the 865 nm absorption change, and gives rise to strong carotenoid triplet-triplet absorption changes at 547 nm. These data indicate that the reaction center-bound carotenoid in the crystals is capable of accepting triplet energy from the primary donor triplet.

Analysis of the pigment content of an antenna pigment-protein complex from three strains of Rhodopseudomonas sphaeroides.

The pigment content of a B800-850 light-harvesting pigment-protein complex isolated from three different stains of Rhodopseudomonas sphaeroides has been determined. In each case the ratio of carotenoid to bacteriochlorophyll present is very nearly 1 : 3 an no specificity with regard to carotenoid type was observed. The fourth derivative of the infra-red absorption bands of the complex was determined and it is concluded that the minimal functional unit of B800-850 complex consists of 1 carotenoid molecule and three bacteriochlorophyll molecules. The data presented here, together with the previous study of Austin, (Austin, L.A. (1976) Ph.D. Thesis, University of California at Berkeley, Lawrence Berkeley Laboratory Report No. LBL 5512) suggest that the 800 nm absorption band represents one of these bacteriochlorophyll molecules while the remaining two bacteriochlorophylls are responsible for the 850 nm band. The absorption spectra and circular dichroism spectra of the complexes suggests that their structure has not been greatly altered during the purification.

Magnetic field-induced increase of the yield of (bacterio)chlorophyll emission of some photosynthetic bacteria and of Chlorella vulgaris.

In photosynthetic bacteria, in which the iron-ubiquinone complex X is prereduced, a magnetic field induces an increase of the emmission yield, which is correlated with the decrease in reaction center triplet yield reported previously (Hoff, A.J., Rademaker, H., van Grondelle, R. and Duysens, L.N.M. (1977) Biochim. Biophys. Acta 460, 547--554). Our results support the hypothesis that under these conditions charge recombination of the oxidized primary donor and the reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll. In Chlorella vulgaris and spinach chloroplasts, at 120 K, the magnetic field has an effect similar to that found in bacteria, which suggests that an intermediary electron acceptor between P-680 and Q is present in Photosystem II also.

The resonance Raman spectrum of carotenoids as an intrinsic probe for membrane potential. Oscillatory changes in the spectrum of neurosporene in the chromatophores of Rhodopseudomonas sphaeroides.

The resonance Raman spectrum of the carotenoid neurosporene is shown to be a sensitive monitor of absorption shifts, and thus changes in membrane potential, in chromatophores of the GlC mutant of Rhodopseudomonas sphaeroides. For a Raman excitation wavelength at 472.7 nm, the intensities of the two most prominent resonance Raman features (v1 and v2) respond very differently to small shifts in the absorption maxima. Thus, the ratio intensity v1/intensity v2 is a sensitive probe for absorption shifts. Changes in this ratio of approximately 20% were observed during a valinomycin induced diffusion potential. At 5 degrees C changes in the average intensity ratio of +6, -4 and -14% were brought about by oligomycin, FCCP and sodium deoxycholate, respectively. The changes in intensity ratio were temperature dependent and, in addition, effects due to the laser beam acting as an actinic light could be detected. Oscillatory changes were observed in absolute Raman and Rayleigh scattering intensities for chromatophores at 5 degrees C and for intact cells under growing conditions.

Ubiquinone in Rhodopseudomonas sphaeroides. Some thermodynamic properties.

In Rhodopseudomonas sphaeroides chromatophores there are 25 +/- 3 ubiquinone (Q) molecules/reaction center protein. They comprise several thermodynamically and functionally different ubiquinone complements. There are approx. 19 ubiquinones (Em7 = 90 mV) in the main ubiquinone complement which, within experimental resolution, appears thermodynamically homogenous and follows the redox reaction Q + 2e + 2H+ in equilibrium with QH2 from pH 5--9. A method which takes advantage of the 2H+ bound/molecule of Q reduced is described for measuring the time course of light-activated reaction center-driven reduction and oxidation of the 19 Q complement. No stable semiquinones were detected in the constitutents of the 19 Q complement. There are approx. 6 ubiquinones of lower Em which are currently unaccounted for, although one or possibly two of these can be assigned to the quinones of the reaction center protein. The remainder may be associated with the NADH-ubiquinone oxidoreductase.

Soluble cytochrome composition of the purple phototrophic bacterium, Rhodopseudomonas sphaeroides ATCC 17023.

A detailed study of the soluble cytochrome composition of Rhodopseudomonas sphaeroides (ATCC 17023) indicates that there are five c-type cytochromes and one b-type cytochrome present. The molecular weights, heme contents, amino acid compositions, isoelectric points, and oxidation-reduction potentials were determined and the proteins were compared with those from other bacterial sources. Cytochromes c2 and c' have previously been well characterized. Cytochrome c-551.5 is a diheme protein which has a very low redox potential, similar to certain purple bacterial and algal cytochromes. Cytochrome c-554 is an oligomer, which is spectrally similar to the low-spin isozyme of cytochrome c' found in other purple bacteria (e.g., Rhodopseudomonas palustris cytochrome c-556). An unusual high-spin c-type heme protein has also been isolated. It is spectrally distinguishable from cytochrome c' and binds a variety of heme ligands including oxygen. A large molecular-weight cytochrome b-558 is also present which appears related to a similar protein from Rhodospirillum rubrum, and the bacterioferritin from Escherichia coli. None of the soluble proteins appear to be related to the abundant membrane-bound c-type cytochrome in Rps. sphaeroides which has a larger subunit molecular weight similar to mitochondrial cytochrome c1 and chloroplast cytochrome f.

Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes.

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.

Crystallization and preliminary X-ray and optical spectroscopic characterization of the photochemical reaction center from Rhodobacter sphaeroides strain 2.4.1.

The photochemical reaction center from Rhodobacter sphaeroides 2.4.1 has been crystallized. The crystals were obtained in a solution of beta-octylglucoside by the vapor diffusion technique using polyethylene glycol 4000 as the precipitant at 22 degrees C. The orthorhombic crystals (space group P2(1)2(1)2(1)) have cell constants a = 142.5 A, b = 136.1 A, c = 78.5 A, and diffract to 3.7 A. The crystals display pronounced linear dichroism in the carotenoid absorption spectral region.

Structure of spheroidene in the photosynthetic reaction center from Y Rhodobacter sphaeroides.

The structure of the reaction center of Y Rhodobacter sphaeroides has been solved at 3 A resolution, using the atomic coordinates of the reaction center from the carotenoidless mutant R26 Rhodobacter sphaeroides. The structure has been refined by a stimulated annealing with the computer program X-PLOR, leading to a crystallographic R factor of 0.22 using reflections between 8 and 3 A. The spheroidene molecule which is bound to the Y reaction center has been fitted in the electron density map as a 15-cis isomer with a highly asymmetric structure. The cis-bond is located at proximity from ring 1 of the accessory bacteriochlorophyll on the inactive M side. The nature of the cis-bond was confirmed by resonance Raman spectra obtained from Y reaction center crystals. The structure of spheroidene in Y reaction center is compared to that proposed for 1,2-dihydroneurosporene in Rhodopseudomonas viridis reaction center crystals.

Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy.

The photoreduction of the secondary electron acceptor, QB, has been characterized by light-induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB- state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB-. Assignments are proposed for the C...O vibration of QB- and tentatively for the C = O and C = C vibrations of QB.

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.

Procedures and conditions for application of the pyridine hemochrome method to photosynthetically grown cells of Rhodopseudomonas sphaeroides.

The conditions and pretreatments required in the conventional pyridine hemochrome method were re-examined for application of the method to the measurement of protoheme and heme c contents in photosynthetically grown cells of Rhodopseudomonas sphaeroides. The amounts of hemes were calculated from two kinds of difference absorbances between two wavelengths of the redox difference spectrum of the pyridine hemochromes prepared from the disrupted cells. Extraction of photosynthetic pigments from the cells with organic solvents and separation of hemes in hemoproteins into the protoheme and the heme c fractions by the differential extraction of hemes with an acidified organic solvent were omitted.

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides.

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.

[Study of ferredoxins in membranes of Rhodopseudomonas spheroides by means of Mossbauer spectroscopy]. / Issledovanie membrannykh ferredoksinov Rhodopseudomonas sphaeroides metodom messbauérovskoi spektroskopii.

Mössbauer spectra were investigated in membranes (chromatophores) of Rhodopseudomonas sphaeroides, enriched in 57Fe, over a temperature range from 4.2 to 300 K. The spectrum of isolated chromatophores is a symmetric doublet characterized by an isomeric shift delta=0.60+/-0.03 mm/s, quadrupole splitting delta=0.54+/-0.03 mm/s and a width gamma delta of 1.42+/-0.04 mm/3 at half maximum. These parameters, which are in fact characteristic of the Mössbauer spectra of bacterial ferredoxins, appeared practically invariable over a wide range of temperatures. The spectrum of dithionite-treated chromatophores, measured immediately after dithionite treatment, exhibits, in addition, a doublet having parameters characteristic of high-spin bivalent iron. The doublet linewidth of the Fe2+ (S=2) iron is equal, at room temperature, to the emission spectrum linewidth. At 4K, some broadening of the spectrum is observed, which is of magnetic origin. The intensity of the Fe2+(S=2) doublet from a dithionite-treated sample shows a pronounced drop after several days of storage, with the intensity of the initial doublet rising. The overall area under the spectra, the linewidth and shape are not changed. Based on experimental data obtained, possible models of the active center composed of most frequently encountered membrane-bound ferredoxins of the photosynthetic bacterium Rhodopseudomonas sphaeroides are discussed.

[Analysis of the linear dichroism of reaction centers oriented in polyacrylamide gel]. / Analiz lineinogo dikhroizma reaktsionnykh tsentrov, orientirovannykh v poliakrilamidnom gele.

A model for orientation of rigid disc-shaped and rod-shaped macromolecules in polymer is analysed. Analytical expressions are given for the dependence of linear dichroism value on the angle between the transition dipole vector and the axis of the macromolecules, for certain deformation of the sample. The reaction centers from Rhodopseudomonas sphaeroides R-26 are oriented as rod-shaped particles. The angles between the long axis of the reaction center and transition dipoles for bacteriochlorophyll 31.8 +/- 2,4 degrees (870 nm), 42,3 +/- 0.9 degrees (796 nm), 68.0 +/- 0.6 degrees (600 nm) and for bacteriopheophytin 76.5 +/- 1.9 degrees (760 nm), 39.8 +/- 1.5 degrees (540 nm) are determined from absorption spectra.

[Spectral position of the principal absorption band of pigment complex P870 and the kinetics of photo-induced oxidoreductions in the reaction centers and chromatophores of purple bacteria with preparations at different temperatures and having different degrees of hydration]. / Spektral'naia pozitsiia osnovnoi polosy pogloshcheniia pigmentnogo kompleksa P870 i kinetika fotoindutsirovannykh oksidoreduktsii v reaktsionnykh tsentrakh i khromatoforakh purpurnykh bakterii pri razlichnoi temperature i gidratsii preparatov.

In isolated photosynthetic reaction centres of Rps. spheroides and chromatophores R. rubrum the spectral position of the longest wavelength absorption band of P870, effectiveness of electron removal from the photochemical pair (P870 -- primary electron acceptor, A1) and the rate constant for recombination of photooxidized P870 with photoreduced A1 undergo marked and fully reversible changes over the temperature interval from +20 to -70 degrees. Dehydration of the samples has the effect similar to that induced by temperature lowering. The data suggest that the spectral position of the main maximum of pigment complex P870 absorption band may be regarded as a sensitive inner probe of the structure-functional state of the investigated preparations.

[Possible role of macromolecular components in the functioning of photosynthetic reaction centers of purple bacteria]. / O vozmozhnoi roli makromolekuliarnykh komponentov v funktsionirovanii fotosinteticheskikh reaktsionnykh tsentrov purpurnykh bakterii.

The temperature dependencies of the photoconversion of pigments P870--P890 were studied using isolated chromatophores and photosynthetic reaction centres (RC's) of purple bacteria. The samples were prepared by extraction with organic solvents (light petroleum and a combination of light petroleum and methanol) and modified through cross-linking the functional groups of proteins by treatment with glutaraldehyde or denatured by various physical and chemical treatments. The data provide further evidence that the pool of RC secondary acceptors is formed by the compounds of quinone nature located in the hydrophobic surrounding. Similar molecules localized in a more polar medium act as primary acceptors. The findings indicate on the essential role of macromolecular components in the RC's functioning and also suggest that the photochemical charge separation is conformation-controlled.

[Effect of deuteration on the kinetics of photoinduced electron transport in the reaction centers of purple bacteria]. / Vliianie deiterirovaniia na kinetiku fotoindutsirovannogo perenosa élektrona v reaktsionnykh tsentrakh purpurnykh bakterii.

Substitution of H2O for D2O in the preparations of reaction centres from chromatophore membranes of photosynthesizing bacteria Rhodopseudomonas sphaeroides, 1760-1 results in a decrease of the efficiency of direct electron transfer from photoreduced primary quinone X1- to the secondary ones and in rising possibilities for reversible reaction X1- with photooxidized bacteriochlorophyll. The value of isotope effect which does not usually exceed 1.2 depends on temperature; it is not found in the temperature range below -60 degrees divided by -80 degrees C.