Blue and red shifts of bacteriochlorophyll absorption band around 880 nm in Rhodospirillum rubrum.

The redox potential dependence of the light-induced absorption changes of bacteriochlorophyll in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum has been examined. The highest values of the absorption changes due to the bleaching of P-870 and the blue shift of P-800 in chromatophores and subchromatophore complexes are observed in the 360-410mV redox potential range. At potentials below 300 mV (pH 7.0), the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in subchromatophore complexes and to longer wavelengths in chromatophores. The data on redox titration show that the red and blue shifts of 880-nm bacteriochlorophyll band represent the action of a non-identified component (C340) which has an oxidation-reduction midpoint potential close to 340 mV (n=1) at pH 6.0--7.6. The Em of this component varies by 60 mV/pH unit between pH 7.6 and 9.2. The results suggest that the red shift is due to the transmembrane, and the blue shift to the local intramembrane electrical field. The generation of both the transmembrane and local electrical fields is apparently governed by redox transitions of the component C340.

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.

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.

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.

Identification of the carotenoid present in the B-800-850 antenna complex from Rhodopseudomonas capsulata as that which responds electrochromically to transmembrane electric fields.

Mild proteolysis of Rhodopseudomonas capsulata chromatophores results in a parallel loss of the 800 nm bacteriochlorophyll absorption band a blue shift in the carotenoid absorption bands associated with the B-800-850 light-harvesting complex. Both the light-induced and the salt-induced electrochromic carotenoid band shift disappear in parallel to the loss of the 800 nm bacteriochlorphyll absorption upon pronase treatment of chromatophores. During the time required for the loss of the 800 nm bacteriochlorophyll absorption and the loss of the electrochromic cartenoid band shift photochemistry is not inhibited and the ionic conductance of the membrane remains very low. We conclude that the carotenoid associated with the B-800-850 light-harvesting complex is the one that responds electrochromically to the transmembrane electric field. Analysis of the pigment content of Rps. capsulata chromatophores indicates that all of the carotenoid may be accounted for in the well defined pigment-protein complexes.

2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate, a new carbene generating reagent for probing hydrophobic membrane domains.

A new precursor of a lipophilic photolabel, 2-[8-14C]naphthyl 2-diazo-3,3,3-trifluoropropionate (NADIT) has been synthesized. The suitability of the reagent for labeling the hydrophobic core of membranes is demonstrated by studying its reactivity in chromatophores of Rhodospirillum rubrum G-9+. The label binds preferentially to the phospholipids and intrinsic membrane proteins. In isolated reaction centers treated with NADIT the hydrophobic subunits M and L are more labeled than the H subunit. The high reactivity, dark stability and ease of synthesis favors this very lipophilic reagent to identify the intrinsic hydrophobic sections of membrane proteins.

Steady-state measurements of delta pH and delta psi in Rhodospirillum rubrum chromatophores by two different methods. Comparison with phosphorylation potential.

The pH gradient, delta pH, and the membrane potential, delta psi, formed during light-induced electron transport in Rhodospirillum rubrum chromatophores were measured by two independent methods: (a) using specific electrodes to monitor light-dependent uptake of NH4Cl and SCN- at chromatophore concentrations of about 0.1 mg bacteriochlorophyll/ml and (b) using 9-aminoacridine and 8-anilinonaphthalenesulfonic acid as fluorescent probes for delta pH and delta psi, respectively, at chromatophore concentrations of about 0.01 mg bacteriochlorophyll/ml. The light intensity was measured and set at a level which saturated the highest bacteriochlorophyll concentration used. The steady-state values obtained with each method under phosphorylating conditions were compared with the phosphorylation potential maintained by the chromatophores under identical conditions. The results indicate that under all conditions employed the ratio H+/ATP is greater than 2, and varies between 2.4 and 3.4 depending on the method used for estimation of the electrochemical proton gradient.

A large photoreactive particle from Chromatium vinosum chromatophores.

Large photoreactive particles from Chromatium vinosum are obtained pure and in high yield by using a mixture of detergents at high ionic strength to dissociate the chromatophore membrane. The particles contain all of the secondary electron acceptor of the chromatophores and about half of the cytochrome. Their content of ubiquinone is greatly enridhed as compared with chromatophores. Th individual particles have an estimated molecular weight of between 650,000 and 810,000. Gel electrophoresis of the preparation in sodium dodecylsulfate shows polypeptides with molecular weights of 50-45,000, 30,000, 27,000, 22,000 and 12,000. The 50-45,000 components are cytochromes. The 30,000, 27,000 and 22,000 components may be analogous to the triad of polypeptides present in Rhodopseudomonas spheroides reaction centers. The non-cytochrome components are partly soluble in chloroform/methanol. Aggregates of particles appear in these preparations. Electron microscopy of the aggregates demonstrates rectilinear lattices of isodiametric particles, 120 A in diameter. These sheet-like structures are one unit thick and typically contain 9-16 members. They appear to arise by aggregation during isolation but are probably similar to native aggregates apparent within chromatophores after treatment with detergents at low salt concentration.

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.

Molecular organization of photochemical reaction complex in chromatophore membrane from Rhodospirillum rubrum as detected by immunochemical and proteolytic analyses.

The molecular organization of photochemical reaction (PR) complex in chromatophores from Rhodospirillum rubrum was studied by a combination of proteolytic analysis with proteinase K followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis with rabbit polyclonal antibodies against its five subunits (H, M, L, alpha, and beta). The preparations used for comparison were reaction center complex (RC) (composed of H, M, and L), PR complex, and chromatophores (closed membranous vesicles of polar lipid bilayer having PR complex buried in the membrane). 1. RC was bound with anti-H, anti-M, and anti-L antibodies, whereas PR complex and chromatophores were bound with anti-H and anti-beta antibodies, but not with the other antibodies. 2. With PR complex, H (Mr 31,000 (31K)) was rapidly degraded into two peptides with Mr of 16K and 14.5K (abbreviated as 16K and 14.5K, respectively), M (27K) into 25.5K, and beta (11K) into 10K. Significantly later, the 25.5K of M was degraded into 24K, L (23K) into 19K, and alpha (12K) into 11K. With chromatophores, H and beta were degraded in a manner similar to that with PR complex, whereas M, L, and alpha were not degraded at all. With RC, H, M, and L were rapidly degraded. 3. With RC, the activity for photooxidation of P870 (photochemical activity) was hardly affected till H, M, and L had been degraded into less than 10K, 24K, and 19K, respectively. With PR complex, the absorbance spectrum due to the bacteriochlorophylls of light-harvesting complex-1 composed of alpha and beta (LH1-Bchl) changed in parallel to the degradation of alpha or 10K (a part of beta). 4. Together with the previous results (Ueda et al. (1985) J. Biochem. 98, 1487-1498), the present findings suggest that: 1) RC is directly surrounded by 12 alpha and further by 12 beta; 2) H and beta are mostly and partially exposed, respectively, on the outer surface of the membranous vesicle; 3) a small part of M is exposed on the inner surface of the membranous vesicle.

Isolation of a membrane protein from R rubrum chromatophores and its abnormal behavior in SDS-polyacrylamide gel electrophoresis due to a high binding capacity for SDS.

A membrane protein insoluble in water was isolated by gel chromatography in the presence of 0.1% sodium dodecyl sulfate (SDS) from chromatophores of a photosynthetic bacterium, Rhodospirillum rubrum. This is one of the major membrane proteins of the chromatophore. The protein was found to bind about four grams of SDS per gram, a value which is more than twice the amount generally observed with protein polypeptides derived from water-soluble globular proteins. The electrophoretic behavior of the complex between the membrane protein and SDS is abnormal due to this high capacity for binding SDS. Estimation of the molecular weight of this protein by SDS-polyacrylamide gel electrophoresis was thus impossible. Such an anomaly in SDS binding is unlikely to be restricted to the particular membrane protein described in this paper. The possibility of such a deviation from standard behavior in the interaction with SDS should be taken into consideration in studies of other membrane proteins, since SDS is often used both in analytical and preparative procedures.

Near-infrared absorption spectra of light harvesting bacteriochlorophyll protein complexes from Chromatium vinosum.

Light harvesting (LH) bacteriochlorophyll (Bchl) protein complexes were isolated from chromatophores of Chromatium vinosum, by the combination of detergent solubilization, sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and hydroxyapatite chromatography. Addition of the absorption spectra of these complexes reproduced the absorption spectrum of chromatophores from which these complexes were derived. This result led to the conclusion that these isolated complexes retained the near-infrared absorption spectra which these complexes showed as they existed in chromatophores. Two kinds of spectrally different chromatophores were obtained under different culture conditions. One of them contained two kinds of LH Bchl protein complexes; B890 containing and B850-B800 containing complexes, and the other contained, in addition, B820-B800 containing complex. B890 containing complexes from the two types of chromatophores were spectrally similar, whereas B850-B800 containing complexes were not the same with respect to the location of absorption maxima and the content of B850. It was shown that the variation of the near-infrared absorption spectra of the chromatophores is due to not only the variation of the ratio of complexes but also the variation of the absorption spectrum of a particular complex.

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. I. Diffraction pattern of the photoreaction unit isolated from Rhodospirillum rubrum chromatophore and some characteristics of the structure.

The X-ray diffraction pattern from chromatophore membranes of a photosynthetic bacterium, Rhodospirillum rubrum, indicates that a highly organized protein assembly exists in the membrane. The X-ray scatterer was solubilized from chromatophores by a mixture of cholate and deoxycholate. The basic component was identified as the photoreaction unit, which consists of light-harvesting bacteriochlorophyll proteins and a reaction center. The radial autocorrelation function, calculated directly from the X-ray intensity dats, made it possible to deduce certain structural features of the X-ray scatterer. 1. The maximum dimension of the X-ray scatterer is estimated to be 110-130 A. 2. The arrangement of the units in the chromatophore membrane is random. 3. Protein molecules in the unit form a rigid structure, being arranged mutually in fixed positions to give a distinct X-ray diffraction pattern. 4. The most probable structure is one which has rotational symmetry.

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. III. Basic structure of the photosynthetic unit and its relation to other bacteriochlorophyll forms.

We have performed X-ray diffraction studies on photosynthetic units of Rhodospirillum rubrum and solubilized *B800 + B890 complex from chromatophores of Chromatium vinosum, to investigate the homology of their molecular structures. The native chromatophores of Chromatium vinosum, which contain other bacteriochlorophyll forms, were examined by an X-ray diffraction technique, in order to assess the interactions between the complexes as well as the molecular structures of the bacteriochlorophyll forms. The subchromatophore particles, solubilized by Triton X-100 from cells of Chromatium vinosum, exhibit a major absorption maximum at 881 nm and a minor one at 804 nm, consisting of bacteriochlorophyll form *B800 + B890. The near-IR absorption spectrum of the particle is very similar to that of chromatophores of Rhodospirillum rubrum although the major absorption maximum is shifted slightly. The X-ray diffraction pattern of the subchromatophore particles is very similar to that of chromatophores of Rhodospirillum rubrum. Thus, the subchromatophore particles are considered to be the "photoreaction unit" of Rhodospirillum rubrum. Since the bacteriochlorophyll form, *B800 + B890, is common in the purple bacteria, it is strongly suggested that the photoreaction unit is the basic and common structure existing in the photosynthetic units of purple bacteria. Chromatium vinosum cells exhibit different near-IR absorption spectra, depending on the culture media and also on the intensity of the illumination during culture. The chromatophores from these cells give different equatorial X-ray diffraction patterns. These patterns are much broader than that of solubilized subchromatophore particles, though they have common features. Thus, the molecular structures in the photosynthetic units are different, depending on their constituent bacteriochlorophyll forms.(ABSTRACT TRUNCATED AT 250 WORDS)

Disintegration of Rhodospirillum rubrum chromatophore membrane into photoreaction units, reaction centers, and ubiquinone-10 protein with mixture of cholate and deoxycholate.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).

[Study of the state of intracellular iron in photosynthesizing purple sulfur bacteria using the Mossbauer effect]. / Issledovanie sostoianiia vnutrikletochnogo zheleza fotosinteziruiushcchikh purpurnykh serobakterii metodom éffekta Messbauéra.

The work presents the results of the first stage of the study on the valent and structural state and function of the iron atoms in the donor-acceptor environment of the photosynthetic reaction centres of purple bacteria. 57Fe was introduced by cultivating the microorganisms in a medium enriched in this isotope. At 77 K the maxima observed in the Mössbauer spectra in intact freeze-dried cells at a speed of +2 mm/s and --1 mm/s are attributed to doublets 1.11 with the isomer ahifts of 1.3 and 0.5 mm/s respectively and the constants of the quadrupole splitting (Q.S.) of 2 mm/s and 2.2 mm/s. These are presumed to arise from cytochromes type c. The Mössbauer parameters of the intense assymetric quadrupole-split doublet of a more complex nature in the mid of the spectrum with line widths of 0.5 to 7.0 mm/s and 0.5 to 1.5 mm/s fit to these of bacterial ferredoxin. From the analysis of the control and dithionite-treated samples of the temperature dependency of the observable parameters over a temperature range of 77 to 300 K it can be concluded that in cells the iron atoms are present in various valency and spin states and the relation between the redox states of the iron atoms is dependent, in particular, on the age of the culture. The Mössbauer spectra of the cell fragments indicate that most of the intracellular iron, first of all the heme iron, is bound to the fraction of photosynthetic membranes.

[Molecular organization of the long-wave complexes of purple photosynthesizing bacteria. Effect of pronase on the B890 complex of Chromatium minutissium and Rhodopseudomonas palustris]. / Molekuliarnaia organizatsiia dlinnovolnovykh kompleksov purpurnykh fotosinteziruiushchikh bakterii. Issledovanie deistviia pronazy na kompleksy B890 Chromatium minutissimum i Rhodopseudomonas palustris.

The pronase action on the long-wave complexes B890 from two different purple bacteria has been investigated. Differences in the kinetics of decrease of the reaction center photochemical activity of electron-donor activity of cytochromes and of destruction of Bx890 (875) forms have been discovered. Different rates of the proteolysis of RC proteins were revealed by SDS-gel-electrophoresis. The heavy protein of RC was the first to degrade. The photochemical transformations deltaA890(875) in B890 complexes was observed during formation of peptides with molecular weight about 17 000 from two other RC proteins. On the basis of obtained data the model of molecular organization of B890 complexes from purple bacteria is discussed.