Reaction center light harvesting B875 complexes from Rhodocyclus gelatinosus: characterization and identification of quinones

Reaction center-B875 pigment-protein complexes were purified from Rhodocyclus gelatinosus. The proteic components consist of 7–8 polypeptides among which some were identified by their apparent molecular weights: the light harvesting B875 polypeptides and of 8 and 6 kDa, reaction center L (23 kDa), M (28 kDa) and H (34 kDa), cytochrome c (43 kDa). Four c-type hemes were found per reaction center. Flash-induced absorbance changes showed the presence of both Q_A and Q_B in the complex. Charge recombination times were determined to be: 1.16±0.2 (n=30) for P⁺Q_AQ_B ^- and 7–10 ms for P⁺Q_A ^- in presence of herbicides. From quinone analysis on one hand and kinetics of charge recombination on the other hand, we proposed that in the reaction center of Rhodocyclus gelatinosus Q_A is menaquinone 8 and Q_B is ubiquinone 8.     

On the state of carotenoids bound to reaction centers of photosynthetic bacteria: A resonance raman study

The carotenoids bound to reaction centers of wild, Ga and GlC strains of Rhodopseudomonas spheroides, of Rhodospirillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15′-cis-β-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of $\text{P-870}$. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.     

Spirilloxanthin is released by detergent from Rubrivivax gelatinosus reaction center as an aggregate with unusual spectral properties

A preparation containing spirilloxanthin has been isolated from Rubrivivax gelatinosus SC2, a mutant devoid of the reaction center-associated tetraheme cytochrome c, after solubilisation of membranes with lauryl-di-methyl-amine oxide. It was purified by ammonium sulfate precipitation and gel filtration, and analyzed by SDS-gel electrophoresis. Spirilloxanthin was shown to be aggregated in large particles (apparent M_W > 600 kDa) and was not associated with a specific protein. This aggregate was characterized by absorption, circular dichroism and resonance Raman spectroscopies. The absorption spectrum contained two UV bands at 370 and 300 nm, and did not present the visible bands of spirilloxanthin, which however reappeared when spirilloxanthin was extracted from the aggregate with organic solvents. Resonance Raman spectra indicated that at least four different populations of spirilloxanthin were present in the preparation as a mixture of different trans and cis configurations. These properties are similar to those described for a so-called carotenoprotein solubilized with sodium dodecyl sulfate from Rhodospirillum rubrum membranes [Schwenker et al. (1974) Biochim Biophys Acta 351: 246-260; Kito et al. (1983) Photochem Photobiophys 5: 209-217]. We further observed absorption spectra of pure spirilloxanthin dissolved in mixtures of water, polar solvents and detergent, in the absence of protein, resembling those of the.aggregate. We conclude that the aggregate is not a carotenoprotein, but rather an artefact due to the release of spirilloxanthin from the reaction center, leading to the isomerization and association of spirilloxanthin molecules in a detergent particle. We propose the same interpretation for the complex isolated from Rhodospirillum rubrum.     

Resolution of Rhodocyclus gelatinosus photoreceptor unit components by temperature-induced phase separation in the presence of decyltetraoxyethylene

A simple method for dissociating photoreceptor units from Rhodocyclus gelatinosus is described. Incubation of a chromatophore extract (Agalidis, I., Rivas, E. and Reiss-Husson, F. (1990) Photosynth. Res. 23, 249-255) at 4 degrees C with decyltetraoxyethylene and octyl-beta-D-thioglucopyranoside, followed by temperature-induced phase separation at 20 degrees C, led to the formation of three phases: a pellet composed of pure B875 antenna; an oily layer containing cytochrome c and other proteins; a detergent-poor supernatant containing crude reaction centers. This method provided a first step towards further purification of reaction centers and B875 antenna, respectively.     

Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus.

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl beta-D-thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N-terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate-reduced hemes have paramagnetic properties very similar to those of the two low-potential hemes of the reaction center-bound cytochrome (gz = 3.34), but their alpha band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate-reduced heme and the dithionite-reduced heme are assigned to the two high-potential hemes of the bound cytochrome. Their alpha band was blue-shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome's essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low-potential hemes and a pair of high-potential hemes.     

Natural resistance to inhibitors of the ubiquinol cytochrome c oxidoreductase of Rubrivivax gelatinosus: sequence and functional analysis of the cytochrome bc(1) complex

Biochemical analyses of Rubrivivax gelatinosus membranes have revealed that the cytochrome bc(1) complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q(0) and Q(i) sites. Because the resistance to cytochrome bc(1) inhibitors is primarily related to the cytochrome b primary structure, the petABC operon encoding the subunits of the cytochrome bc(1) complex of Rubrivivax gelatinosus was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome b polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome bc(1) inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b(H) and b(L) hemes, respectively. These characteristics imply different functional properties of the cytochrome bc(1) complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of R. gelatinosus cytochrome bc(1) are discussed with reference to the structure and function of the cytochrome bc(1) complexes from other organisms.     

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q_y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q_x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q_x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q_xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q_y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.     

On the state of carotenoids bound to reaction centers of photosynthetic bacteria: a resonance Raman study.

The carotenoids bound to reaction centers of wild, Ga and GIC strains of Rhodopseudomonas spheroides, of Rhodospirrillum rubrum, strain S1 and of Rhodopseudomonas viridis, yield very similar, but unusual resonance Raman spectra. Through a comparison with resonance Raman spectra of 15,15'-cis-beta-carotene, these carotenoids are shown to assume cis conformations, while the corresponding chromatophores contain all-trans forms only. These cis conformations likely are identical for all the carotenoids studied. They remain unaffected by variations of temperature from 20 to 300 K as well as by the redox state of P-870. They are unstable, being rapidly isomerised towards the all-trans forms when extracted from the reaction centers. The possible nature of these conformers is discussed on the basis of their electronic and vibrational spectra.     

Binding of carotenoids on reaction centers from Rhodopseudomonas sphaeroides R 26

The carotenoid-less reaction centers isolated from Rhodopseudomonas sphaeroides (strain R 26) bind pure all-trans spheroidene as well as spheroidenone in a nearly 1:1 molar ratio with respect to P-870. Neither β-carotene nor spirilloxanthin, both absent from wild-type Rps. sphaeroides, could be bound in appreciable amounts. Resonance Raman spectra of the carotenoidreaction center complex indicate that the carotenoid is bound as a cis isomer, its conformation being very close, although probably not identical, to that assumed by the carotenoid in the wild-type reaction centers. The electronic absorption spectra of the carotenoid-reaction center complexes are in good agreement with such a interpretation. When bound to the R 26 reaction centers, spheroidene displays light-induced absorbance changes identical in peak wavelengths and comparable in amplitudes to those observed in the wild-type reaction centers. Thus the binding of the carotenoid to the R 26 reaction centers most likely occurs at the same proteic site as in the wild-type reaction centers. This site shows selectivity towards the nature of carotenoids, and has the same sterical requirement as in the wild type, leading to the observed all-trans to cis isomerisation.