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.

The photoreaction center of Rhodospirillum rubrum mutant strain F24.1.

Rhodospirillum rubrum strain F24.1 is a spontaneous revertant of nonphototrophic mutant F24 derived from wild-type strain S1. Strain F24 shows no detectable photochemical activity and contains, at most, traces of the photoreaction center polypeptides. Strain F24.1 has a phototrophic growth rate close to that of the wild-type strain (Picorel, R., del Valle-Tascón, S. and Ramírez, J.M. (1977) Arch. Biophys. Biochem. 181, 665-670) but shows little photochemical activity. Light-induced absorbance changes in the near-infrared, photoinduced EPR signals and ferricyanide-elicited absorbance changes indicate that strain F24.1 has a photoreaction center content of 7-8% as compared to strain S1. Polyacrylamide gel electrophoresis of isolated F24.1 chromatophores shows the photoreaction center polypeptides to be present in amounts compatible with this value. Photoreaction center was prepared from strain F24.1 and showed no detectable difference with that of strain S1. It is concluded that strain F24.1 photosynthesis is due entirely to its residual 7-8% of typical photoreaction center.

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.

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.

Resonance Raman spectroscopy of cytochrome bc1 complexes from Rhodospirillum rubrum: initial characterization and reductive titrations.

Resonance Raman spectra of bc1 complexes from Rhodospirillum rubrum have been obtained. Various resonance conditions and the stoichiometric redox titration of the complex were used to isolate and identify the contributions of the heme c1 and heme b active sites to the observed spectra. The complex was found to partially photoreduce when exposed to laser excitation.

A new crystal form and preliminary crystallographic data for ferricytochrome c' from Rhodospirillum rubrum.

A new crystal form of ferricytochrome c' from the photosynthetic bacteria, Rhodospirillum rubrum, has been obtained by dialysing protein solution against polyethylene glycol 4000. The crystals belong to the space group P61 (or its enantiomorph P65) with unit cell dimensions: a = b = 51.63 A and c = 155.39 A. The asymmetric unit contains one dimer molecule of 28,000 molecular weight and the solvent content of the unit cell is approximately 38%.

Demonstration of two 3,3'-diaminobenzidine oxidation reactions associated with photosynthetic membranes in anaerobic light-grown Rhodospirillum rubrum.

Photosynthetic membranes of anaerobic light-grown Rhodospirillum rubrum oxidized 3,3'-diaminobenzidine. When glutaraldehyde-treated cells were exposed to 3,3'-diaminobenzidine in the light aerobically, the oxidation appeared to occur by two systems. One reaction was stimulated by white light and the second required molecular oxygen. The O2-dependent activity was inhibited by KCN.

Structure of the extracellular ferredoxin from Rhodospirillum rubrum: close similarity to clostridial ferredoxins.

The amino acid sequence of an [8Fe-8S] ferredoxin isolated from the culture medium of Rhodospirillum rubrum, a photosynthetic purple non-sulfur bacterium, was determined by a combination of various conventional procedures. The sequence was A-Y-K-I-E-E-T-C-I-S-C-G-A-C-A-A-E-C-P-V-N-A-I-E-Q-G-D-T-I-F-V-V-N-A-D-T-C-I-D-C - G-N-C-A-N-V-C-P-V-G-A-P-V-A-E (55 amino acid residues). It lacked methionine, leucine, histidine, arginine, and tryptophan. The molecular weight was calculated to be 5,568 excluding iron and sulfur atoms. The distribution of 8 cysteine residues was exactly the same as that of clostridial-type ferredoxin, suggesting retention of the duplication of the bacterial ancestral ferredoxin gene. The extracellular ferredoxin of R. rubrum was compared with other ferredoxins observed in closely related photosynthetic bacteria and the evolutionary significance of this ferredoxin is discussed.

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.

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.

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).

Isolation, characterization, and comparison of a ubiquitous pigment-protein complex consisting of a reaction center and light-harvesting bacteriochlorophyll proteins present in purple photosynthetic bacteria.

Protein complexes (photochemical reaction complex; PR complex) bound to both light-harvesting bacteriochlorophyll-1 (LH-Bchl-1) and reaction center Bchl (RC-Bchl) were purified from Rhodospirillum rubrum (wild and carotenoid-less), Rhodopseudomonas sphaeroides (wild), and Chromatium vinosum (wild). Another protein complex (LH-2 complex) bound to LH-Bchl-2 was also purified from Rps. sphaeroides. The bacteria were grown in the presence of a [14C]amino acid mixture. The purification procedure included molecular-sieve chromatography in the presence of cholate-deoxycholate, and non-equilibrated isoelectric electrophoresis with 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate. The purified complexes were separated into their constituent proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The molar ratios of the proteins were determined by comparing their radioactivities divided by their molecular weights after consideration of the molecular masses of the complexes. The PR complexes all contained per mol: 1 mol each of RC H-, M-, and L-subunits, 10-13 (probably 12) mol each of two other proteins with molecular weights of 11-12K and 8-11K, 28-32 mol Bchl, 13-15 mol carotenoids (except in the carotenoid-less mutant), 2.6-3.9 mol ubiquinone (or menaquinone in Chr. vinosum), and 53-79 mol phosphate without phospholipid. The LH-2 complex contained per mol: 1 mol 52K protein, about 13 (probably 12) mol each of 9K and 8K proteins, 30 mol Bchl, 10 mol carotenoids, and 38 mol phosphate without phospholipid. The PR complexes and LH-2 complex showed similar X-ray diffraction patterns, implying that they had similar, highly organized molecular structures.

Purification and properties of chlorophyllase from greened rye seedlings.

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.

[Linear dichroism of pigments associated with spherical chromatophores. Models of orientation in polyacrylamide gels]. / Lineinyi dikhroizm pigmentov sfericheskikh khromatoforov. Model' orientatsii v poliakrilamidnom gele.

Linear dichroism and orientation of pigments in chromatophores of photosynthetic bacteria Chromatium minutissimum and Rhodospirillum rubrum using a novel method of orientation in polyacrylamide gel was studied. A model is proposed for orientation of spherical membranes of chromatophores or other similar vesicules. The value of linear dichroism is derived for known deformation of the gel and a certain angle between the transition dipole and a unit vector perpendicular to the membrane plane. The analysis of linear dichroism spectra permits calculation of angles between the normal to the membrane and the transition dipoles in Chr. minutissimum 65 degrees +/- 1.5 degrees (890 nm absorption band), 63 degrees +/- 1 degree (860 nm), 63 degrees +/- 1 degree (800 nm), 45.5 degrees +/- 1 degree (590 nm), 50.5 degrees +/- 0.5 degree (450--550 nm) and in Rsp. rubrum: 71 degrees +/- 1.5 degree (890 nm), 66.5 degrees +/- 1 degree (870 nm), 69 degrees +/- 1.5 degree (800 nm), 37 degrees +/- 0.5 degree (590 nm), 49.5 degrees +/- 0.5 degree (450--550 nm). The 860 nm band shift to shorter wave-lengths observed in Chr. minutissimum chromatophores treated with 0.01 M potassium ferricyanide is not related to reorientation of transition dipoles, but rather to certain changes of lipid-protein environment.

[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.

[Picosecond energy transfer between the spectral forms of pigments from the reaction center of Rhodospirillum rubrum]. / Pikosekundnyi perenos énergii mezhdu spektral'nymi formami pigmentov reaktsionnogo tsentra iz Rhodospirillum rubrum.

Absorption changes of reaction centers from Rhodospirillum rubrum at 748, 796 and 870 nm induced by 532 and 870 nm picosecond light pulses were investigated with a picosecond spectrometer. Kinetics of absorption changes at 748 and 796 had an additional bleaching when induced by the 532 nm pulse, in comparison with those at 870 nm. The additional bleaching was interpreted as a result of the excitation energy transfer via spectral forms of pigments of reaction centers. The experimental results fit the mathematical simulation of the additional bleaching for the following set of rate constant values: intrinsic conversion to the lowest excited singlet state in bacteriopheophytine molecule--10(13) s-1, energy transfer from bacteriopheophytine to P800 3.10(12) s-1, from P800 to P870--2.10(12) s-1.