Mössbauer spectroscopy studies of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26

⁵⁷Fe Mössbauer spectroscopy measurements on reaction centers differing in ubiquinone content, detergent, oxidation state, or the presence of o-phenanthroline all show a single quadrupole doublet of similar splitting (ΔE_Q), center shift (δ) and temperature dependence. The results are indicative of high-spin Fe²⁺ with an approximately invariant first coordination sphere. A crystal field model with strong electron delocalization can account for the temperature dependence of ΔE_Q, but further data are needed to achieve a unique parameterization.     

Orientation and linear dichroism of the reaction centers from Rhodopseudomonas sphaeroides R-26

Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.     

Orientation and linear dichroism of the reaction centers from Rhodopseudomonas sphaeroides R-26.

Linear dicroism of chromatophores and isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides strain R-26 was studied using a novel technique of orientation. The results are discussed in view of the reaction center structure and its position in the membrane. The advantages of the new orientation technique are also outlined.     

Structure of Rhodopseudomonas sphaeroides R-26 reaction center

The molecular replacement method has been successfully used to provide a structure for the photosynthetic reaction center of Rhodopseudomonas sphaeroides at 3.7 A resolution. Atomic coordinates derived from the R. viridis reaction center were used in the search structure. The crystallographic R-factor is 0.39 for reflections between 8 and 3.7 A. Validity of the resulting model is further suggested by the visualization of amino acid side chains not included in the R. viridis search structure, and by the arrangements of the reaction centers in the unit cell. In the initial calculations quinones or pigments were not included; nevertheless, in the resulting electron density map, electron density for both quinones QA and QB appears along with the bacteriochlorophylls and bacteriopheophytins. Kinetic analysis of the charge recombination shows that the secondary quinone is fully functional in the R. sphaeroides crystal.     

Magnetophotoselection of the triplet state of reaction centers from Rhodopseudomonas sphaeroides R-26.

Reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, give rise to large triplet state EPR signals upon illumination at low temperature (11 K). Utilizing monochromatic polarized light to generate the EPR spectra (magnetophotoselection) we have shown that the intensities of the observed triplet signals are strongly dependent upon the wavelength and polarization direction of the excitation. These data can be used to calculate the orientations of the excited transition moments with respect to each other and with respect to the triplet state principal magnetic axes system. Our quantitative approach is to follow the procedure outlined in a previous publication (Frank, H.A., Friesner, R., Nairn, J.A., Dismukes, G.C. and Sauer, K. (1979) Biochim. Biophys. Acta 547, 484-501) where computer simulations of the observed triplet state spectra were employed. The results presented in the present work indicate that the transition moment at 870 nm which is associated with the bacteriochlorophyll 'special pair' lies almost entirely along one of the principal magnetic axes of the triplet state. Aso, the 870 nm transition moment makes an angle of approx. 60 degrees with the 546 nm transition moment which is associated with a bacteriopheophytin. This latter result is in agreement with previous photoselection studies on the same bacterial species (Vermeglio, A., Breton, J., Paillotin, G. and Cogdell, R. (1978) Biochim. Biophys. Acta 501, 514-530).     

A rapid procedure for the isolation and purification of photosynthetic reaction centers from Rhodopseudomonas sphaeroides R-26

A rapid purification procedure has been developed for the isolation of reaction centers From Rhodopseudomonas sphaeroides strain R-26. The procedure takes about 7 h and results in yields of 60–75%. The ratio of the optical absorbances at 280 and 800 nm is between 1.4 and 1.5, and preparations can be made with either one or two quinones per reaction center. EPR spectra show a sharp g 1.83 signal for the ubisemiquinone. The substitution of lauryl maltoside for lauryldimethylamine oxide suppresses reaction-center degradation in solution.     

Characterization of purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26

A highly purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26 was isolated by a procedure involving Triton X-100 solubilization, calcium phosphate column chromatography, and ammonium sulfate fractionation. The purified enzyme complex contains, in nanomoles/mg of protein, cytochrome b, 8.3; cytochrome c1, 8.3; iron-sulfur protein, 15; phospholipids, 182; and ubiquinone, 5. Four major polypeptides with apparent molecular weights of 48,000, 30,000, 24,000, and 12,000 were detected in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr = 48,000 and 30,000 proteins are cytochromes b and c1, respectively. The enzyme complex catalyzes electron transfer from ubiquinol to cytochrome c with a specific activity of 12.6 mumol of cytochrome c reduced per min/mg of protein at 23 degrees C. This is lower than that of the mitochondrial enzyme, although both systems have similar essential redox components and a similar Km for ubiquinol. The activity is fully sensitive to antimycin A and 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole. The enzyme complex is stable at neutral pH and at lower temperatures, but became less stable when the incubation temperature was raised. At 37 degrees C, the half-life is 15 min. The enzymatic activity was insensitive to treatment with N',N'-dicyclohexylcarbodiimide. No p-chloromercuriphenylsulfonate-alkylable sulfhydryl groups were detected. The major phospholipids associated with the purified enzyme complex are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol with molar per cent distributions of 25, 21, and 35, respectively. About 60% of the enzymatic activity was abolished upon treatment with phospholipase A2. The phospholipase A2-inactivated activity can be partially restored by the addition of EDTA followed with phospholipids prepared from either the cytochrome b-c1 complex of the same source or a mixture of phosphatidylglycerol and asolectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure effects on the photochemistry of bacterial photosynthetic reaction centers from Rhodobacter sphaeroides R-26 and Rhodopseudomonas viridis

Electron-transfer reactions and triplet decay rates have been studied at pressures up to 300 MPa. In reaction centers from Rhodobacter sphaeroides R-26, high pressure hastened the electron transfers from both the primary and secondary quinones (Q_A and Q_B) to the primary electron donor bacteriochlorophyll, P. Motion of Q_A between two sites, one nearer to P and the other nearer to Q_B, could account for these pressure effects. In reaction centers from Rhodopseudomonas viridis, charge recombination was slowed by high pressure. Decay rates were also studied for the triplet state, P^R. In Rb. sphaeroides R-26 with Q_A reduced with Na₂S₂O₄, the decay was hastened by pressure. This could be explained if P^R decays through a charge-transfer triplet state, or if the decay kinetics of P^R are sensitive to the distance between P and Q_A⁻. In Rps. viridis reaction centers, and in Rb. sphaeroides reaction centers that were depleted of Q_A, the lifetime of P^R was not altered by pressure.     

Isolation and characterization of cytochrome C1 from photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Cytochrome c1 of photosynthetic bacterium R. sphaeroides R-26 has been purified from isolated cytochrome b-c1 complex to a single polypeptide, using a procedure involving Triton X-100 and urea solubilization, calcium phosphate column chromatography and ammonium sulfate fractionation. The purified protein contains 30 nmoles heme per mg protein and has an apparent molecular weight of 30,000, as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis. Bacterial cytochrome c1 is soluble in aqueous solution in the absence of detergent and has spectral characteristics similar to mammalian cytochrome c1. The amino acid compositions of these two proteins, however, are not comparable.     

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c1 from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150,000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30,000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c1, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c1 is more exposed than is that of mammalian cytochrome c1, but less exposed than that of cytochrome c2. Ferricytochrome c1 undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c1, suggesting that the sulfhydryl groups of cytochrome c1 are the electron donors for photoreduction. Purified cytochrome c1 contains 3 +/- 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c1, the bacterial protein does not form a stable complex with cytochrome c2 or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c1 and bacterial ferricytochrome c2, and between bacterial ferrocytochrome c1 and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c1 is calculated to be 228 mV, which is identical to that of mammalian cytochrome c1.     

Characterization of bacterial photosynthetic reaction center crystals from Rhodopseudomonas sphaeroides R-26 by X-ray diffraction

An orthorhombic crystal form (P2(1)2(1)2(1)) of the reaction center from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 has been characterized. The crystals were grown from polyethylene glycol; the unit cell dimensions are a = 142.2 A, b = 139.6 A, and c = 78.7 A; and they contain one reaction center in each crystallographic asymmetric unit. The crystals diffract to at least 3.0 A resolution, and are suitable for detailed structural studies.     

Characterization by EPR spectroscopy of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides R-26

The EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature. The purified cytochrome b-562 gives a high spin signal at g = 6.0. Anaerobic titration of this signal confirmed the presence of two redox components with Em = 40 and -110 mV at pH 7.5. These values are consistent with the published ones, Em = 55 and -100 mV at pH 7.0, that were optically estimated for the same type of preparation (Iba et al. (1985) FEBS Lett. 183, 151-154). The power saturation behavior of the g = 6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers.     

Slow photoinduced changes in reaction centers of Rhodobacter sphaeroides R-26 by holographic interferometry

Using the on-line holographic interferometry, changes in the refraction index of illuminated solutions containing the Rhodobacter sphaeroides R-26 reaction centers isolated from membranes by detergents of ionic and nonionic nature were registered. Factors affecting the refraction index of these solutions were analyzed. It was shown that the photoinduced changes in this parameter are due to structural changes in the molecular complex of reaction centers. The results of measurements of the kinetics of absorption spectra at lambda = 865 nm for two detergent types are presented, which coincide qualitatively with the results of holographic measurements. The change in the volume of the molecular complex of reaction centers was estimated to be 0.1 divided by 1%.     

Amino-terminal sequences of the L, M, and H subunits of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

We have determined the sequence of the 25-28 amino-terminal residues of the three subunits, L, M, and H, of the membrane-bound reaction center protein of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. The sequences are as follows: L, H2N-Ala-Leu-Leu-Ser-Phe-Glu-Arg-Lys-Tyr-Arg- Val-Pro-Gly-Gly-Thr-Leu-Val-Gly-Gly-Asn-Leu-Phe-Asp-Phe-(His)-Val-; M, H2N-Ala-Glu-Tyr-Gln-Asn-Ile-Phe-Ser-Gln-Val-Gln-Val-Arg-Gly-Pro-Ala-Asp-Leu-Gly-Met-Thr-Glu-Asp-Val-Asn-Leu-Ala-Asn-; H, H2N-Met-Val-Gly-Val-Thr-Ala-Phe-Gly-Asn-Phe-Asp-Leu-Ala-Ser-Leu-Ala-Ile-Tyr-Ser-Phe-Trp-Ile-Phe-Leu-Ala-X-Leu-Ile-. The H sequence, especially after the aspartyl residue at position 11, is rich in hydrophobic residues, consistent with the possibility that this section of the polypeptide chain is located within the membrane. The L sequence is hydrophilic near the amino terminus and then becomes moderately hydrophobic. The M sequence is of average polarity.     

Reaction of cytochrome c2 with photosynthetic reaction centers from Rhodopseudomonas viridis.

The reactions of Rhodopseudomonas viridis cytochrome c2 and horse cytochrome c with Rps. viridis photosynthetic reaction centers were studied by using both single- and double-flash excitation. Single-flash excitation of the reaction centers resulted in rapid photooxidation of cytochrome c-556 in the cytochrome subunit of the reaction center. The photooxidized cytochrome c-556 was subsequently reduced by electron transfer from ferrocytochrome c2 present in the solution. The rate constant for this reaction had a hyperbolic dependence on the concentration of cytochrome c2, consistent with the formation of a complex between cytochrome c2 and the reaction center. The dissociation constant of the complex was estimated to be 30 microM, and the rate of electron transfer within the 1:1 complex was 270 s-1. Double-flash experiments revealed that ferricytochrome c2 dissociated from the reaction center with a rate constant of greater than 100 s-1 and allowed another molecule of ferrocytochrome c2 to react. When both cytochrome c-556 and cytochrome c-559 were photooxidized with a double flash, the rate constant for reduction of both components was the same as that observed for cytochrome c-556 alone. The observed rate constant decreased by a factor of 14 as the ionic strength was increased from 5 mM to 1 M, indicating that electrostatic interactions contributed to binding. Molecular modeling studies revealed a possible cytochrome c2 binding site on the cytochrome subunit of the reaction center involving the negatively charged residues Glu-93, Glu-85, Glu-79, and Glu-67 which surround the heme crevice of cytochrome c-554.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linear-dichroic triplet-minus-singlet absorbance difference spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum,Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1

For the first time, linear-dichroic triplet-minus-singlet (LD-(T - S)) spectra of reaction centers of the photosynthetic bacteria Chromatium vinosum, Rhodopseudomonas sphaeroides R-26 and Rhodospirillum rubrum S1 have been measured using an extension of the technique of absorbance-detected magnetic resonance (ADMR) of the triplet state. For all bacteria studied the LD-(T - S) spectra exhibit a bleaching of the long-wavelength absorbance band that is either split or has a clear shoulder to longer wavelengths. The components are approximately parallel-polarized, indicating that they do not form an exciton pair. Around 800 nm a band appears with a width of about 7 nm, which does not form part of a band shift and that may be attributed to an appearing monomer band. Small features in the LD-(T - S) spectra at both sides of this band are well explained by band shifts of the two components of the 800 nm reaction center absorption band. The transition moment of the component at about 818 nm in reaction centers of Rps. sphaeroides R-26 is at an angle larger than 55° with both the x and the y triplet spin axes. In none of the bacteria do we find evidence for the bleaching of an exciton component of P-860 near 810 nm.     

Oxidation of cytochrome c 2 by photosynthetic reaction centers of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in vivo. Effect of viscosity on the rate of reaction

In Rhodospirillum rubrum and Rhodopseudomonas sphaeroides it is shown that the oxidation of cytochrome c ₂ involves a diffusion limited process. From analysis of the results it follows that the electron transfer probability must be very low. This is corroborated by in vitro studies using the isolated components.     

Singlet and triplet absorption spectra of carotenoids bound in the reaction centers of Rhodopseudomonas sphaeroides R26

The singlet and triplet state absorption spectra are reported for two carotenoids, methoxyneurosporene and spheroidene, incorporated into the reaction center protein of the photosynthetic bacterial carotenoidless mutant Rhodopseudomonas sphaeroides R26. The spectra for the two different carotenoid molecules are identical suggesting a strong interaction between the protein and the different chromophores. Combined effects of electrochromic band shifts and carotenoid structural changes are invoked to account for the spectral observations.     

Immunological comparison of the b and c1 cytochromes from bovine heart mitochondria and the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26

Antibodies against cytochromes b and c1 of bovine heart mitochondria and the photosynthetic bacterium, Rhodopseudomonas sphaeroides R-26, were raised in rabbits. The purified antibodies showed high titers against their respective antigens in enzyme-linked immunosorbent assays. Less than 15% cross-reactivity between the mitochondrial and bacterial cytochromes was detected. Although antibodies against mitochondrial cytochrome b did not inhibit the mitochondrial cytochrome b-c1 complex, a 70% inhibition was obtained when these antibodies were incubated with delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids indicating that the catalytic site(s) of mitochondrial cytochrome b are masked by phospholipids. On the other hand, antibodies against bacterial cytochrome b showed significant inhibition of the intact bacterial cytochrome b-c1 complex, indicating that some of the catalytic site epitopes of bacterial cytochrome b are exposed to the hydrophilic environment. Similar to antibodies against mitochondrial cytochrome b, antibodies against bacterial cytochrome b inhibited 50% activity of the mitochondrial cytochrome b-c1 complex only when they were incubated with the delipidated mitochondrial cytochrome b-c1 complex prior to reconstitution with phospholipids, indicating that the common epitopes between the cytochromes b are masked by phospholipids. Antibodies against mitochondrial and bacterial cytochromes c1 completely inhibited their respective cytochrome b-c1 complexes but no cross-immunoinhibition was observed. However, when antibodies against bacterial cytochrome c1 were incubated with the delipidated mitochondrial cytochrome b-c1 complex before reconstitution with phospholipids, a 65% inhibition was observed, indicating that the common epitopes between the cytochromes c1 were also somewhat masked by phospholipids. Antibodies against mitochondrial cytochrome c1 inhibited 70% of the succinate oxidase activity in the intact mitochondria preparation, but no inhibition was observed in submitochondrial particles, indicating that some mitochondrial cytochrome c1 epitopes are exposed to the cytoplasmic side.