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.     

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.     

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.     

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.     

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.     

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.     

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).     

Protein fluorescence of photosynthetic reaction centers from Rhodopseudomonas sphaeroides

Luminescence emitted by tryptophan residues of reaction center (RC) preparations was studied. The RG preparations were isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides by treatment with lauryl dimethyl amine oxide (LDAO). After excitation at lambda 280 nm the quantum yield of luminescence is 0,02. It is shown that 60% of tryptophanyls are located inside the protein globule in the surrounding of relaxating polar groups and the rest approximately 40% on the outer surface of the globule--predominantly in the positively charged region of the LDAO-RC protein--in the surrounding of protein-bound water molecules. There is a correlation between the pH dependencies of the position of the peak of luminescence from tryptophanyls and effectivity of electron transfer from the primary (quinone) to secondary acceptor. The two parameters are invariant at pH from 7 to 9 and vary at pH less than 7 and pH greater than 9. The phenomena responsible for the observed correlation are discussed on the basis of pH-dependent changes in the RC protein which govern electron transport activity at the reaction center.     

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.     

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.     

Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, PF. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to PF, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates PF from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and PF of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of PF with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.     

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.     

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.     

On the depletion and reconstitution of both QA and metal in reaction centers of the photosynthetic bacterium Rb. sphaeroides R-26

Four possible ways to prepare Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs were studied: (1) first depleting the Fe, then depleting Q_A and finally reconstituting Q_A (D-Fe, D-Q, R-Q), (2) first depleting Q_A, then depleting the Fe and finally reconstituting Q_A (D-Q, D-Fe, R-Q), (3) first depleting Q_A, then reconstituting Q_A and finally depleting Fe (D-Q, R-Q, D-Fe), (4) first depleting Q_A, then depleting the Fe and reconstituting Q_A in the same step (D-Q, D-Fe-R-Q). Our results showed that: method (1) results in the irreversible loss of photochemical activity; method (2) and (3) result in low recovery of the photochemical activity and poor yield of Fe-depleted, Q_A-reconstituted RCs; method (4) gives surprisingly good results. This method allows for the first time to prepare the Q_A-depleted, Fe-depleted, Q_A-reconstituted RCs with high recovery of the photochemical activity and good yield. The sample has 98% of photochemical activity (yield of P⁺ Q_A ^-) compared with that of the native RCs and shows strong polarization of the EPR signal of Q_A ^- under continuous illumination at 5K. The decay halftime of I^- is slow (5 ns) compared with that of the native RCs, but it is the same as that measured for the RCs from which only iron is removed. These results indicate that the depletion of iron and the reconstitution of Q_A have been successful. Reconstitution of the Q_A-depleted, Fe-depleted and Q_A-reconstituted RCs with Zn²⁺ gives also the spin-polarized Q_A ^-, and yields the same decay of I^- (halftime 200 ps) as that of the native RCs.Abbreviations LDAO lauryldimethylamine N-oxide - EDTA ethylenediaminetetraacetic acid - BSA albumin bovine - TL buffer 10 mM Tris.HCl, 0.1% LDAO and 0.1 mM EDTA     

Plasmid rearrangements in the photosynthetic bacterium Rhodopseudomonas sphaeroides

Mu d1(Ap lac) was introduced into the photosynthetic bacterium Rhodopseudomonas sphaeroides 2.4.1. via the R-plasmid R751 in an attempt to isolate fusion derivatives involving photosynthetic operons. The selection system is potentially very powerful since R. sphaeroides is normally Lac negative. Among the exconjugants, photosynthesis-deficient mutants were recovered, some of which had elevated beta-galactosidase levels. Among the mutants examined, beta-galactosidase expression was linked exclusively to R751 . Many of the photosynthesis-deficient mutants were found to have alterations in their indigenous plasmids which apparently involved the exchange of DNA from one plasmid to another. Southern blot analysis revealed that there are extensive DNA sequences which are shared by the two plasmids that are involved in the rearrangements and that no exogenous DNA sequences appear to be involved. It was further discovered that plasmid rearrangement is a general phenomenon which can occur spontaneously in R. sphaeroides 2.4.1 and shows a high correlation with a photosynthesis minus phenotype.     

Lipid-protein associations in chromatophores from the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid-protein interactions were examined in chromatophores isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides using lipid spin-labels. The chromatophores contain fluid bilayer and a significant amount of lipid immobilized by membrane proteins. For a typical preparation of cells grown under 600 ft-c illumination, 59% of the spin-labeled fatty acids were bound. Essentially the entire length of the 18-carbon fatty acid chain was immobilized, judging from results obtained with the spin-label at the 7, 12, and 16 positions. The amount immobilized varies directly with the bacteriochlorophyll content of the chromatophore material, suggesting that a significant fraction of the lipid spin-labels is immoblized on the hydrophobic surfaces of the chlorophyll-binding proteins. Changing the lipid spin-label head group from a negatively charged carboxyl group to a positively charged quarternary amine greatly decreased the amount of immobilized lipid. The changes in immobilized lipid with light level and polar head group suggest that the anntenna bacteriochlorophyll-binding proteins preferentially associate with negatively charged lipids.     

Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26

A synthetic medium for growing Rhodobacter sphaeroides R-26 is developed. This medium opened the way to the preparation of photosynthetic reaction centers incorporated with L-[4'-13C]tyrosine or L-[1'-15N]tryptophan. Gas chromatography combined with mass spectroscopy was used to estimate the metabolic incorporation of the labeled amino acid into the protein. Conditions were found for near-quantitative incorporation of labeled aromatic amino acids into the reaction center.