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1.
Fiedor L  Akahane J  Koyama Y 《Biochemistry》2004,43(51):16487-16496
A simple reconstitution technique has been developed and then applied to prepare a series of light-harvesting antenna 1 (LH1) complexes with a programmed carotenoid composition, not available from native photosynthetic membranes. The complexes were reconstituted with different C(40) carotenoids, having two structural parameters variable: the functional side groups and the number of conjugated C-C double bonds, systematically increasing from 9 to 13. The complexes, differing only in the type of carotenoid, bound to an otherwise identical bacteriochlorophyll-polypeptide matrix, can serve as a unique model system in which the relationship between the carotenoid character and the functioning of pigment-protein complexes can be investigated. The reconstituted LH1 complexes resemble the native antenna, isolated from wild-type Rhodospirillum rubrum, but their coloration is entirely determined by carotenoid. Along with the increase in its conjugation size, the carotenoid absorption transitions gradually shift to the red. Thus, the extension of the conjugation size of the antenna carotenoids provides a mechanism for the spectral tuning of light harvesting in the visible part of the spectrum. The carotenoids in the reconstitution system promote the LH1 formation and seem to bind and transfer the excitation energy specifically only to a species with characteristically red-shifted absorption and emission maxima, apparently, due to a cooperative effect. Monitoring the LH1 formation by steady-state absorption and fluorescence spectroscopies reveals that in the presence of carotenoids it proceeds without spectrally resolved intermediates, leading directly to B880. The effect of the carotenoid is enhanced when the pigment contains the hydroxy or methoxy side groups, implying that, in parallel to hydrophobic interactions and pi-pi stacking, other interactions are also involved in the formation and stabilization of LH1.  相似文献   

2.
Recent AFM data demonstrate that mature photosynthetic membranes of R. sphaeroides are composed of rows of dimeric RC-LH1-PufX complexes with some LH2 complexes 'sandwiched' between these rows of core complexes, and others in discrete LH2-only domains which might form the light-responsive complement of the LH2 antenna. The present work applies membrane fractionation, radiolabelling and LDS-PAGE techniques to investigate the response of R. sphaeroides to lowered light intensity. The kinetics underlying this adaptation to low light conditions were revealed by radiolabelling with the bacteriochlorophyll (bchl) biosynthetic precursor, delta-aminolevulinate, which allowed us to measure only the bchls synthesised after the light intensity shift. We show that (1) the increase in LH2 antenna size is mainly restricted to the mature ICM membrane fraction, and the antenna composition of the precursor upper pigmented band (UPB) membrane remains constant, (2) the precursor UPB membrane is enriched in bchl synthase, the terminal enzyme of the bchl biosynthetic pathway, and (3) the LH2 and the complexes of intermediate migration in LDS-PAGE exhibit completely different labelling kinetics. Thus, new photosynthetic complexes, mainly LH2, are synthesised and assembled at the membrane initiation UPB sites, where the LH2 rings pack between the rows of dimeric cores fostering new LH2-LH1 interactions. Mature membranes also assemble new LH2 rings, but in this case the 'sandwich' regions between the rows of core dimers are already fully occupied and the bulk antenna pool is the favoured location for these new LH2 complexes.  相似文献   

3.
The core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 αβ subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The α one was formylated at its N-terminal residue and the N-terminal methionine of β was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the α polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the α polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension.  相似文献   

4.
The core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 alphabeta subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The alpha one was formylated at its N-terminal residue and the N-terminal methionine of beta was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the alpha polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the alpha polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension.  相似文献   

5.
The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported. The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments. We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra. A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus. A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case. However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles. A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound. The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices. An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions. Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations. The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface). The helix is bent, as expected from the amide proton chemical shifts, and it is similar to the polypeptide fold of the previously determined crystal structure of Rhodopseudomonas acidophila Ac10050 LH2 beta (S. M. Prince et al., Journal of Molecular Biology, 1997, Vol. 268, pp. 412-423). It is concluded that the polypeptide conformation of this region may facilitate assembly of the LH complex.  相似文献   

6.
Hole-burned absorption and line-narrowed fluorescence spectra are studied at 5 K in wild type and mutant LH1 and LH2 antenna preparations from the photosynthetic purple bacterium Rhodobacter sphaeroides. Evidence was found in all samples, even in intact membranes, of the presence of a broad distribution of bacteriochlorophyll species that are unable to communicate energy between each other and to the exciton states of functional antenna complexes. The distribution maximum of these localized species determined by zero phonon hole action spectroscopy is at 783.5 nm in purified LH1 complexes and at 786.8 nm in B850-only mutant LH2 complexes. A well-resolved peak at 807 nm in LH1 complexes is assigned to the exciton band structure of functional core antenna complexes. Similar structure in LH2 complexes overlaps with the distribution of localized species. Off-diagonal (structural) disorder may be responsible for this exciton band structure. Our data also imply that pair-wise inter-chlorophyll couplings determine the resonance fluorescence lineshape of excitonic polarons.  相似文献   

7.
The light harvesting 1 antenna (LH1) complex from Rhodobacter sphaeroides funnels excitation energy to the photosynthetic reaction center. Our ultimate goal is to build up the structure of LH1 from structures of its individual subunits, much as the antenna can self-assemble from its components in membrane-mimicking detergent micelles. The beta subunit adopts a nativelike conformation in Zwittergent 3:12 micelles as demonstrated by its ability to take the first step of assembly, binding BChl a. Multidimensional NMR spectroscopy shows that the beta subunit folds as a helix((L12-S25))-hinge((G26-W28))-helix((L29-W44)) structure with the helical regions for the 10 lowest-energy structures having backbone rmsds of 0.26 and 0.24 A, respectively. Mn(2+) relaxation data and the protein-detergent NOE pattern show the C-terminal helix embedded in the micelle and the N-terminal helix lying along the detergent micelle surface with a 60 degrees angle between their long axes. (15)N relaxation data for residues L12-W44 are typical of a well-ordered protein with a correlation time of 8.25 +/- 2.1 ns. The presence of the hinge region placing the N-terminal helix along the membrane surface may be the structural feature responsible for the functional differences observed between the LH1 and LH2 beta subunits.  相似文献   

8.
Strongly bounded associates of B800–850 (LH2) and B800–830 (LH3) complexes from photosynthetic purple bacterium Thiorhodospira sibirica were investigated. It was shown that associates contain 8–10 complexes (LH2:LH3 ≈ 1:1). Absorption spectra of the monomer LH2 and the monomer LH3 complexes were calculated. Excitation of B800 absorption band of associates results in: (i) intracomplex excitation energy transfer from B800 to B830 or B850 with time constant of about 500 fs; (ii) intercomplex excitation energy transfer from B820 band of LH3 complex to B850 band of LH2 complex with time constant of about 2.5 ps; (iii) excitation deactivation in B850 band of LH2 complex with time constant of about 800 ps. Signal polarization at long-wavelength side of associates absorption spectrum near 900 nm was negative (?0.1). The interaction of LH3 and LH2 complexes in associates is, to some extent, analogous to the interaction of LH2 and LH1 complexes in chromatophores. Time constant of excitation energy transfer between LH3 and LH2 complexes in associates may be regarded as a minimal time constant for energy transfer between the peripheral and core antenna complexes.  相似文献   

9.
Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [N?veke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.  相似文献   

10.
The exciton transfer between light-harvesting complex 1(LH1) and photosynthetic reaction center dimer is investigated theoretically. We assume a ring shape structure of the LH1 complex with dimer in the ring centre. The kinetic equations which describe the energy transfer between the antenna complex and reaction center dimer were derived. It was shown that the dimer does not act as a photon trap. There is a weak localization of the exciton on the dimer and there is relatively rapid back exciton transfer from dimer to antenna complex which depends on the number of the pigment molecules in the antenna ring. The relation between the rates of the exciton transfer from the antenna complex to dimer and back transfer from dimer to antenna complex has been derived.  相似文献   

11.
The integral membrane light-harvesting complex B808–866 from the thermophilic green filamentous bacterium Chloroflexus aurantiacus has been isolated and characterized. Reversed-phase HPLC analysis demonstrated that the number of bacteriochlorophyll (BChl) in the B808–866 antenna complex is 36 ± 2 per reaction center. The main carotenoid type is γ-carotene, and the molar ratio of BChl to carotenoid is 3:2. The steady-state absorption and fluorescence spectroscopy of the B808–866 complex are reminiscent of the well-studied LH2 peripheral antenna of purple bacteria, whereas the protein sequence and the circular dichroism spectrum of B808–866 is more similar to the LH1 inner core antenna. The efficiency of excitation transfer from carotenoid to BChl is about 25%. The above results combined with electron microscopy and dynamic light scattering analysis suggest that the B808–866 antenna is more like the LH1, whereas surrounds the reaction center but probably consists of 24 building blocks with a ring diameter of about 20 nm. The above results suggested that there are probably two reaction centers inside the ring of B808–866. The unique properties of this light-harvesting complex may provide insights on the protein–pigment interactions in bacterial photosynthesis.  相似文献   

12.
Most photosynthetic LH1 antennae undergo dissociation into B820 subunits, suggesting their universal character as structural modules. However, dissociation into subunits seems to occur reversibly only in the absence of carotenoids and the subunits were never found to bind carotenoids. The interactions of carotenoids with B820 have been studied in a newly developed reconstitution assay of the LH1 antenna from Rhodospirillum rubrum (Fiedor, L., Akahane, J., and Koyama, Y. (2004) Biochemistry 43, 16487-16496). These model studies show that B820 subunits strongly interact with carotenoids and spontaneously form stable LH1-like complexes with substoichiometric carotenoid content. This is the first experimental evidence that B820 may occur as a short-lived intermediate in the assembly of LH1 in vivo. The resulting complex of B820 subunits with carotenoid, termed iB873, is homogeneous, according to ion exchange chromatography and reproducible pigment composition. The iB873-bound carotenoid is as efficient in energy transfer to bacteriochlorophyll as the one in native antenna. To our knowledge, iB873 is the first complex binding functional carotenoid, with the spectral and biochemical properties intermediate between that of B820 and the fully assembled LH1.  相似文献   

13.
In the bacterium R. sphaeroides, the polypeptide PufX is indispensable for photosynthetic growth. Its deletion is known to have important consequences on the organization of the photosynthetic apparatus. In the wild-type strain, complexes between the reaction center (RC) and the antenna (light-harvesting complex 1 (LH1)) are associated in dimers, and LH1 does not fully encircle the RC. In the absence of PufX, the complexes become monomeric, and the LH1 ring closes around the RC. We analyzed the functional consequences of PufX deletion. Some effects can be ascribed to the monomerization of the RC.LH1 complexes: the number of RCs that share a common antenna for excitation transfer or a common quinone pool become smaller. We examined the kinetic effects of the closed LH1 ring on quinone turnover: diffusion across LH1 entails a delay of approximately 1 ms, and the barrier appears to be located directly against the quinone-binding (secondary quinone acceptor (Q(B))) pocket. The diffusion of ubiquinol from the RC to the cytochrome bc1 complex is approximately 2-fold slower in the mutant, suggesting an increased distance between the two complexes. The properties of the Q(B) pocket (binding of inhibitors, stabilization of Q(B-), and rate of Q(B)-H2 formation) appear to be modified in the mutant. Another specificity of PufX- is the accumulation of closed centers in the Q(A-) (where Q(A) is the primary quinone acceptor) state as the secondary acceptor pool becomes reduced, which is probably the origin of photosynthetic incompetence. We suggest that this is related to the Q(B) pocket alterations. The malfunction of the reaction center is probably due to a faulty association with LH1 that is prevented in the PufX-containing structure.  相似文献   

14.
15.
In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.  相似文献   

16.
We report on the association of the antenna system to the reaction center in Photosystem I. Biochemical analysis of mutants depleted in antenna polypeptides showed that the binding of the antenna moiety is strongly cooperative. The minimal building block for the antenna system was shown to be a dimer. Specific protein-protein interactions play an important role in antenna association, and the gap pigments, bound at the interface between core and antenna, are proposed to mediate these interactions Gap pigments have been characterized by comparing the spectra of the Photosystem I to those of the isolated antenna and core components. CD spectroscopy showed that they are involved in pigment-pigment interactions, supporting their relevance in energy transfer from antenna to the reaction center. Moreover, gap pigments contribute to the red-shifted emission forms of Photosystem I antenna. When compared with Photosystem II, the association of peripheral antenna complexes in PSI appears to be more stable, but far less flexible and functional implications are discussed.  相似文献   

17.
Lucy I. Crouch  Michael R. Jones 《BBA》2012,1817(2):336-352
In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC–LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC–LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC–LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC–LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC–LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC–LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.  相似文献   

18.
The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center  相似文献   

19.
The effect of the light harvesting 1 (LH1) antenna complex on the driving force for light-driven electron transfer in the Rhodobacter sphaeroides reaction center has been examined. Equilibrium redox titrations show that the presence of the LH1 antenna complex influences the free energy change for the primary electron transfer reaction through an effect on the reduction potential of the primary donor. A lowering of the redox potential of the primary donor due to the presence of the core antenna is consistently observed in a series of reaction center mutants in which the reduction potential of the primary donor was varied over a 130 mV range. Estimates of the magnitude of the change in driving force for charge separation from time-resolved delayed fluorescence measurements in the mutant reaction centers suggest that the mutations exert their effect on the driving force largely through an influence on the redox properties of the primary donor. The results demonstrate that the energetics of light-driven electron transfer in reaction centers are sensitive to the environment of the complex, and provide indirect evidence that the kinetics of electron transfer are modulated by the presence of the LH1 antenna complexes that surround the reaction center in the natural membrane.  相似文献   

20.
Two-dimensional crystals of the reaction-centre-light-harvesting complex I (RC-LH1) of the purple non- sulfur bacterium Rhodospirillum rubrum have been formed from detergent-solubilized and purified protein complexes. Unstained samples of this intrinsic membrane protein complex have been analysed by electron cryomicroscopy (cryo EM). Projection maps were calculated to 8.5 A from two different crystal forms, and show a single reaction centre surrounded by 16 LH1 subunits in a ring of approximately 115 A diameter. Within each LH1 subunit, densities for the alpha- and beta-polypeptide chains are clearly resolved. In one crystal form the LH1 forms a circular ring, and in the other form the ring is significantly ellipsoidal. In each case, the reaction centre adopts preferred orientations, suggesting specific interactions between the reaction centre and LH1 subunits rather than a continuum of possible orientations with the antenna ring. This experimentally determined structure shows no evidence of any other protein components in the closed LH1 ring. The demonstration of circular or elliptical forms of LH1 indicates that this complex is likely to be flexible in the bacterial membrane.  相似文献   

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