Energy transfer in the B800-850-carotenoid light-harvesting complex of various mutants of Rhodopseudomonas sphaeroides and of Rhodopseudomonas capsulata

Emission and absorption spectra in the temperature range 4–300 K have been obtained for bacteriochlorophyll light-harvesting complexes (B800–850 complexes) from several mutants of Rhodopseudomonas sphaeroides and a nonphotosynthetic mutant of Rhodopseudomonas capsulata. The energy-transfer properties of these complexes were remarkably similar despite differences in carotenoid composition. Between 300 and 200 K the excitation densities in B800 and B850 are in thermal equilibrium, indicating rapid energy transfer from B800 to B850 and vice versa. The temperature dependence of the ratio of the B800 and B850 emission yields allows the determination of the ratio of the number of B800 and B850 molecules in the complex which is close to 0.5. Below 200 K thermal equilibrium no longer exists. At 4–100 K the B800 emission yield increases with decreasing temperature and becomes dependent on the wavelength of excitation. From the B800 emission yield at 4 K the B800–850 dipole-dipole distance was calculated to be equal to or smaller than 21 Å for all B800–850 complexes. Excitation spectra for B800 and B850 emission show that the overall energy-transfer efficiencies from carotenoid and B800 to B850 are greater than 90% at all temperatures. At 4 K the carotenoid transfers its excitation energy preferentially to B850. Experiments with chromatophores indicated that the energy-transfer properties of the B800–850 complexes were not modified by the isolation procedures.     

Resolved difference spectra of redox centers involved in photosynthetic electron flow in Rhodopseudomonas capsulata and rhodopseudomonas sphaeroides

1. In Rhodopseudomonas sphaeroides the Q_x absorption band of the reaction center bacteriochlorophyll dimer which bleaches on photo-oxidation is both blue-shifted and has an increased extinction coefficient on solubilisation of the chromatophore membrane with lauryldimethylamine-N-oxide. These effects may be attributable in part to the particle flattening effect.2. The difference spectrum of photo-oxidisable c type cytochrome in the chromatophore was found to have a slightly variable peak position in the α-band (λ_max at 551–551.25 nm); this position was always red-shifted in comparison to that of isolated cytochrome c₂ (λ_max at 549.5 ± 0.5 nm). The shift in wavelength maximum was not due to association with the reaction center protein. A possible heterogeneity in the c-type cytochromes of chromatophores is discussed.3. Flash-induced difference spectra attributed to cytochrome b were resolved at several different redox potentials and in the presence and absence of antimycin. Under most conditions, one major component, cytochrome b₅₀ appeared to be involved. However, in some circumstances, reduction of a component with the spectral characteristics of cytochrome b_?90 was observed.4. Difference spectra attributed to (BChl)₂, Q^?_II, c type cytochrome and cytochrome b₅₀ were resolved in the Soret region for Rhodopseudomonas capsulata.5. A computer-linked kinetic spectrophotometer for obtaining automatically the difference spectra of components functioning in photosynthetic electron transfer chains is described. The system incorporates a novel method for automatically adjusting and holding the photomultiplier supply voltage.     

The stereoisomerism of bacterial, reaction-center-bound carotenoids revisited: An electronic absorption, resonance Raman and H-NMR study

In order to solve discrepancies between earlier assignments we have reinvestigated the stereoisomerism of the spheroidene molecule bound to reaction centers (RC) of Rhodobacter sphaeroides. A stable cis isomer could be extracted and purified from the reaction centres by working at very low ambient light. Resonance Raman spectroscopy showed that this cis isomer assumed the same configuration as that of the RC-bound molecule. Proton-NMR spectroscopy of the extracted isomer permitted to assign it the 15–15′ mono cis configuration. Comparisons between resonance Raman spectra of the native form and of the 15 cis extract showed that, in the reaction center, 15 cis spheroidene is in addition twisted into a non-planar conformation. Comparisons of extraction-induced changes in relative intensities of Raman bands of the 760–1060 cm⁻¹ regions, which largely correspond to out-of-plane modes, further indicated that the out-of-plane twist of RC-bound spheroidene should predominantly affect C₈–C₁₂ and/or C_8′–C_12′ regions of the molecule rather than the central region. Comparisons between difference electronic absorption spectra of RC-bound spheroidene and of RC-bound methoxyneurosporene showed that the out-of-plane twisting of both these native forms results in a drastic weakening of their ¹C ← ¹A electronic transitions, compared with those of the planar, 15 cis forms. Finally, it is proposed, on the basis of their resonance Raman spectra, that spirilloxanthin bound to RCs of Rhodospirillum rubrum as well as dihydroneurosporene or dihydrolycopene bound to RCs of Rhodopseudomonas viridis shares 15 cis configurations and out-of-plane twisting with carotenoids bound to RCs of various strains of Rb. sphaeroides.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Electrogenic events in the ubiquinone-cytochrome bc2 oxidoreductase of rhodopseudomonas sphaeroides

The reductant of ferricytochrome c₂ in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c₂ by ZH₂ is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c₂ reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH₂ and ferricytochrome c₂, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c₂ reduction at pH 7.0 and at 24°C was 2 · 109 M^?1 · s^?1, but this varied with pH, being 5.1 · 10⁸ M^?1 · s^?1 at pH 5.2 and 4.3 · 10⁹ M^?1 · s^?1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.     

A Study of the Mechanism of Acetate Assimilation in Purple Nonsulfur Bacteria Lacking the Glyoxylate Shunt: Acetate Assimilation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate shunt, has been studied. It has been found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate shunt. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.__________Translated from Mikrobiologiya, Vol. 74, No. 3, 2005, pp. 313–318.Original Russian Text Copyright © 2005 by Filatova, Berg, Krasil’nikova, Tsygankov, Laurinavichene, Ivanovsky.     

Effects of growth conditions on production of methyl selenides in cultures of Rhodobacter sphaeroides

Rhodobacter sphaeroides 2.4.1 exposed to selenate or selenite produced volatile selenium compounds. Total amounts of dimethyl selenide, dimethyl diselenide, dimethyl sulfide and dimethyl disulfide in culture medium and headspace were determined. The highest selenate volatilization occurred in the late stationary phase of growth. However, cultures deprived of light in the stationary phase of growth produced much less of the volatile organo-selenium compounds. Lower culture pHs increased the rate of selenium volatilization. Low sulfate concentration limited biomass production and selenium volatilization; high sulfate concentrations had an enhancing effect on the release of organo-selenium compounds. Cultures of R. sphaeroides reacted very differently to amendments with increasing amounts of selenate and selenite. Only small amounts of selenite were volatilized; meanwhile high amounts of methylated selenides were found in selenate-poisoned cultures. Received 03 February 1997/ Accepted in revised form 16 May 1997     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

丙酮酸对浑球红假单胞菌突变株(GOGAT~-Nif~-)固氮酶活性的调节

固氮无效的浑球红假单胞菌GOGAT突变株经丙酮酸诱导产生固氮酶活性。固氮酶比活随丙酮酸浓度增加而提高,同时依赖于蛋白质合成的菌体生长。丙酮酸产生固氮酶时氮源Glu的浓度高达60 mmol/L。液相色谱分析表明,丙酮酸诱导固氮酶活性的形成与胞内Gln耗竭有关而与Asn无关。用丙酮酸代替苹果酸诱导,突变株向胞外分泌的游离氨大大减少。丙酮酸诱导时变种谷氨酰胺酶比活较高,它可能参与胞内Gln的分解。