Formation of a subunit form of the core light-harvesting complex from sulfur purple bacteria <Emphasis Type="Italic">Ectothiorhodospira haloalkaliphila</Emphasis> with different carotenoid composition

B820 subunits from a purple sulfur bacterium Ectothiorhodospira haloalkaliphila strain ATCC 51935^T were obtained by treatment of carotenoid free LH1-RC complexes of this bacterium with ß-octylglucopyranoside (ß-OG). The same complexes with 100% carotenoid content were unable to dißsociate to B820 subunits, but disintegrated to monomeric bacteriochlorophyll (BChl) regardless of their carotenoid composition. The degree of dissociation of the LH1-RC complexes with an intermediate content of carotenoids (the B820 formation) was directly dependent on the quantity of carotenoids in the samples. The resulting B820 subunits did not contain carotenoids. B820 subunits easily aggregated to form a complex with an absorption peak at 880 nm at decreased ß-OG concentration. Analysis of the spectra of the LH1-RC complexes isolated from the cells with different levels of carotenogenesis inhibition led to the conclusion of the heterogeneity of the samples with a predominance of them in (a) the fraction with 100% of carotenoids and (b) the fraction of carotenoid-free complexes.     

Hexacoordination of bacteriochlorophyll in photosynthetic antenna LH1

The ability of chlorophylls to coordinate ligands is of fundamental structural importance for photosynthetic pigment-protein complexes, where in virtually all cases the pigment is thought to be in a pentacoordinated state. In this study, the correlation of the Q(X) transition energy with the coordination state of the central metal in bacteriochlorophyll is applied in investigating the pigment coordination state in bacterial photosynthetic antenna LH1. To facilitate a detailed spectral analysis in the Q(X) region, carotenoid-depleted forms of LH1 are prepared and model LH1 are constructed with non-native carotenoids having blue-shifted absorption. The deconvolution of the Q(X) envelope in LH1 reveals that the band is the sum of two transitions, which peak near 590 and 607 nm, showing that a significant fraction (up to 25%) of hexacoordinated bacteriochlorophyll is present in the complex. The hexacoordination can be seen also in LH1 antennae from other species of purple photosynthetic bacteria. It seems correlated with the LH1 aggregation state and probably is a consequence of the structural flexibility of the assembled complex. The sixth ligand probably originates from the apoprotein and seems not to affect the chromophore core size. These findings show that in light-harvesting complexes a hexacoordinated state of bacteriochlorophyll is not uncommon. Its presence may be relevant to a correct assembly of the antenna and have functional consequences, as it results in a splitting of the pigment S2 excited state (Q(X)), i.e., the carotenoid excitation acceptor state, what might affect intracomplex carotenoid-to-bacteriochlorophyll energy transfer.     

Oligomerization of light-harvesting I antenna peptides of Rhodospirillum rubrum.

We investigated the oligomerization of the core light-harvesting complex (LH1) of Rhodospirillum rubrum from the separated alpha beta BChl(2) subunits (B820) and the oligomerization of the B820 subunit from its monomeric peptides. The full LH1 complex was reversibly associated from B820 subunits by either varying the temperature in the range 277-300 K or by varying the detergent concentration in the buffer from 0.36 to 0.52% n-octyl-beta-D-glucopyranoside. Temperature-induced transition measurements showed hysteresis: raising the temperature induced dissociation of B873 directly into B820 subunits whereas upon recooling an intermediate spectral form was observed with an absorption maximum located around 850 nm. This intermediate form was also observed in detergent-induced transitions. It is speculated that the B850 form is a small aggregate of B820, for instance a dimer. Additionally, during a temperature-mediated transition at low detergent concentration, a set of spectral forms with maxima slightly blue-shifted from 873 nm were observed, possibly due to opened rings with one or only a few alpha beta BChl(2) units missing. The temperature-induced transition of LH1 is discussed in terms of a simple assembly model. It is concluded that a moderately cooperative assembly explains the formation of small aggregates of B820 as well as of incomplete rings. Furthermore, the B820 subunits were reversibly dissociated into the monomeric B777 form by increasing either the temperature or the detergent concentration. Estimations of the enthalpy and entropy changes for the dimeric association reaction of B777 into B820 yielded an enthalpy change of -216 kJ mol(-1) and an entropy change of -0.59 kJ mol(-1)K(-1), at a detergent concentration of 0.8% n-octyl-beta-D-glucopyranoside.     

Carotenoid-induced cooperative formation of bacterial photosynthetic LH1 complex

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.     

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.     

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.     

Embedding carotenoids of spheroidene-branch biosynthesis into antenna complexes of sulfur photosynthetic bacteria

The possibility of embedding the carotenoids of spheroidene-branch biosynthesis (spheroidene and spheroidenone) from non-sulfur bacteria into the diphenylamine antenna complexes (DPA-complexes) from the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila with carotenoid synthesis inhibited by diphenylamine (DPA) was studied for the first time. It was found that spheroidene was embedded into the DPA-complexes from these bacteria at a level of 75–87%, with spheroidene embedding efficiency being 41–68% for the LH1-RC DPA-complexes and 71–89% for the LH2 DPA-complexes. The energy transfer efficiency from carotenoids to bacteriochlorophyll was shown to depend not only on the type of carotenoid but also on the very structure on the antenna complex.     

Spectroscopic characterisation of a tetrameric subunit form of the core antenna protein from Rhodospirillum rubrum

The core light-harvesting complex (LH1) of Rhodospirillum rubrum is constituted of multiple heterodimeric subunits, each containing two transmembrane polypeptides, alpha and beta. The detergent octylglucoside induces the stepwise dissociation of LH1 into B820 (an alphabeta dimer) and B777 (monomeric polypeptides), both of which still retain their bound bacteriochlorophyll molecules. We have investigated the absorption properties of B820 as a function of temperature, whereby a spectral population called 'B851' has been characterised. We show evidence that it is a dimer of the B820 complex. This may represent an intermediate oligomeric form in the process of the LH1 ring formation, as its existence was predicted from global analysis of the absorption spectra of the LH1/B820 equilibrium [Pandit et al. (2001) Biochemistry 40, 12913-12924]. Stabilisation of this dissociated form of LH1 may help in understanding both the electronic properties and the association process of these integral membrane proteins.     

Time-resolved dissociation of the light-harvesting 1 complex of Rhodospirillum rubrum,studied by infrared laser temperature jump

For the first time, data are presented on the time-resolved disassembly reaction of a highly organized membrane protein complex in vitro. The photosynthetic core light-harvesting complex of the bacterial strain Rhodospirillum rubrum G9 consists of 12-16 dimeric subunits that in vivo are associated with the photosynthetic reaction center in a ringlike manner. Isolated in a detergent solution, its appearance either as a ringlike complex (called B873 and absorbing at 873 nm), subunit dimer (called B820 and absorbing at 820 nm), or monomeric form (called B777 and absorbing at 777 nm) is strongly temperature-dependent. In thermodynamic equilibria between B820 and B873, intermediate-sized complexes have also been observed that have absorption maxima around 850 nm. It is unknown whether these structures appear as intermediates in the kinetic B820-B873 (dis)assembly reaction. In this paper disassembly of the light-harvesting complex into its dimeric subunits was followed spectroscopically on a time scale up to 200 ms, upon applying an infrared laser-induced temperature jump. The full dissociation process appears to take place on a time scale of tens to hundreds of milliseconds, the rates becoming faster at higher starting temperatures. Applying the same technique, the dissociation reaction of dimeric subunits into monomers also could be established. This dissociation process occurred on a much faster time scale and took place within the 500 micros response time of our detection system.     

Exciton transfer between LH1 antenna complex and photosynthetic reaction center dimer

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.     

Interactions stabilizing the structure of the core light-harvesting complex (LH1) of photosynthetic bacteria and its subunit (B820)

Reconstitution experiments with a chemically synthesized core light-harvesting (LH1) beta-polypeptide analogue having 3-methylhistidine instead of histidine in the position that normally donates the coordinating ligand to bacteriochlorophyll (Bchl) have provided the experimental data needed to assign to B820 one of the two possible alphabeta.2Bchl pairs that are observed in the crystal structure of LH2 from Phaeospirillum (formerly Rhodospirillum) molischianum, the one with rings III and V of Bchl overlapping. Consistent with the assigned structure, experimental evidence is provided to show that significant stabilizing interactions for both the subunit complex (B820) and LH1 occur between the N-terminal regions of the alpha- and beta-polypeptides. On the basis of the results with the chemically synthesized polypeptides used in this study, along with earlier results with protease-modified polypeptides, mutants, and chemically synthesized polypeptides, the importance of a stretch of 9-13 amino acids at the N-terminal end of the alpha- and beta-polypeptides is underscored. A progressive loss of interaction with the LH1 beta-polypeptide was found as the first three N-terminal amino acids of the LH1 alpha-polypeptide were removed. The absence of the N-terminal formylmethionine (fMet), or conversion of the sulfur in this fMet to the sulfoxide, resulted in a decrease in LH1 formation. In addition to the removal of fMet, removal of the next two amino acids also resulted in a decrease in K(assoc) for B820 formation and nearly eliminated the ability to form LH1. It is suggested that the first three amino acids (fMetTrpArg) of the LH1 alpha-polypeptide of Rhodospirillum rubrum form a cluster that is most likely involved in close interaction with the side chain of His -18 (see Figure 1 for numbering of amino acids) of the beta-polypeptide. The results provide evidence that the folding motif of the alpha- and beta-polypeptides in the N-terminal region observed in crystal structures of LH2 is also present in LH1 and contributes significantly to stabilizing the complex.     

The effect of growth conditions on the light-harvesting apparatus in Rhodopseudomonas acidophila

The detailed effect on the light-harvesting apparatus of three different wild-type strains of Rhodopseudomonas acidophila in response to changes in both light-intensity and temperature have been investigated. In all three strains at high light-intensities (160 mol s m² and above) the only LH2 antenna complex synthesised is the B800–850 complex. In strains 7050 and 7750 as the light-intensity is lowered the B800–850 complex is gradually replaced by another type of LH2 the B800–820 complex. However, at no light-intensities studied is this changeover complete when the cells are grown at 30°C. If however, the light-intensity is lowered at temperatures below 25°C with strain 7750 there is a complete replacement of the B800–850 complex by the B800–820 complex. At all light-intensities and temperatures tested, strain 10050 only synthesised the B800–850 complex. Strain 7050 also responded to changes in light-intensity by altering its carotenoid composition. At high light-intensity the major carotenoids were rhodopin and rhodopin-glucoside, while at low light-intensities the major ones were rhodopinal and rhodopinal-glucoside. This change in carotenoid content started to occur at rather higher light-intensities than the switchover from B800–850 to B800–820.     

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 17899

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodopin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphenylamine (DPA) concentrations not exceeding 53 μM, while at illumination intensity decreased to 500 lx they could grow at 71 μM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoene synthetase (resulting in a decrease in the total carotenoid content) and of phytoene desaturase, which results in formation of neurosporene hydroxy derivatives and ζ-carotene. In the cells grown at 500 lx, small amounts of spheroidene and OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was ~15% of the control (without inhibition) for the B800-850 and ~20% of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with carotenoids due to disintegration of some carotenoidless complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.     

Functional LH1 antenna complexes influence electron transfer in bacterial photosynthetic reaction centers

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.     

Mutants of Rhodobacter sphaeroides lacking one or more pigment-protein complexes and complementation with reaction-centre, LH1, and LH2 genes

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.     

Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

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.     

Spectroscopic studies of two spectral variants of light-harvesting complex 2 (LH2) from the photosynthetic purple sulfur bacterium Allochromatium vinosum

Two spectral forms of the peripheral light-harvesting complex (LH2) from the purple sulfur photosynthetic bacterium Allochromatium vinosum were purified and their photophysical properties characterized. The complexes contain bacteriochlorophyll a (BChl a) and multiple species of carotenoids. The composition of carotenoids depends on the light conditions applied during growth of the cultures. In addition, LH2 grown under high light has a noticeable split of the B800 absorption band. The influence of the change of carotenoid distribution as well as the spectral change of the excitonic absorption of the bacteriochlorophylls on the light-harvesting ability was studied using steady-state absorption, fluorescence and femtosecond time-resolved absorption at 77K. The results demonstrate that the change of the distribution of the carotenoids when cells were grown at low light adapts the absorptive properties of the complex to the light conditions and maintains maximum photon-capture performance. In addition, an explanation for the origin of the enigmatic split of the B800 absorption band is provided. This spectral splitting is also observed in LH2 complexes from other photosynthetic sulfur purple bacterial species. According to results obtained from transient absorption spectroscopy, the B800 band split originates from two spectral forms of the associated BChl a monomeric molecules bound within the same complex.     

Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

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.     

Stark effect spectroscopy of carotenoids in photosynthetic antenna and reaction center complexes

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.     

Determination of the B820 subunit size of a bacterial core light-harvesting complex by small-angle neutron scattering

The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl beta-D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11,400 +/- 500 and radius of gyration of 11.0 +/- 1.0 A, corresponding to a heterodimer consisting of one pair of alphabeta-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30,000 to 50,000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual alpha- and beta-polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The alpha-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the beta-polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between alpha- and beta-polypeptides in reconstitution with bacteriochlorophyll a molecules.