Role of the core region of the PufX protein in inhibition of reconstitution of the core light-harvesting complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus.

PufX, the protein encoded by the pufX gene of Rhodobacter capsulatus and Rhodobacter sphaeroides, has been further characterized. The mature forms of these proteins contain 9 and 12 fewer amino acids, respectively, at the C-terminal end of the protein than are encoded by their pufX genes. To identify the portion of PufX responsible for inhibition of LH1 formation in reconstitution experiments, different regions (N-terminus and several core regions containing different lengths of the C-terminus) of Rb. sphaeroides and Rb. capsulatus PufX were chemically synthesized. Neither the N- nor C-terminal polypeptides of Rb. sphaeroides were inhibitory to LH1 reconstitution. However, all core segments were active, causing 50% inhibition at a concentration ratio of between 3:1 and 6:1 relative to the LH1 alpha-polypeptides whose concentrations were 3-4 microM. CD measurements indicated that the core segment containing 39 amino acids of Rb. sphaeroides PufX exhibited 47% alpha-helix in trifluoroethanol while the core segment containing 43 amino acids of Rb. capsulatus PufX exhibited 59 and 55% alpha-helix in trifluoroethanol and in 0.80% octylglucoside in water, respectively. Approximately 50% alpha-helix was also indicated by a PHD (Burkhard-Rost) structure prediction. Binding of bacteriochlorophyll to these PufX core segments is implicated.

The solution structure of Rhodobacter sphaeroides LH1beta reveals two helical domains separated by a more flexible region: structural consequences for the LH1 complex.

Here, the solution structure of the Rhodobacter sphaeroides core light-harvesting complex beta polypeptide solubilised in chloroform:methanol is presented. The structure, determined by homonuclear NMR spectroscopy and distance geometry, comprises two alpha helical regions (residue -34 to -15 and -11 to +6, using the numbering system in which the conserved histidine residue is numbered zero) joined by a more flexible four amino acid residue linker. The C-terminal helix forms the membrane spanning region in the intact LH1 complex, whilst the N-terminal helix must lie in the lipid head groups or in the cytoplasm, and form the basis of interaction with the alpha polypeptide. The structure of a mutant beta polypeptide W(+9)F was also determined. This mutant, which is deficient in a hydrogen bond donor to the bacteriochlorophyll, showed an identical structure to the wild-type, implying that observed differences in interaction with other LH1 polypeptides must arise from cofactor binding. Using these structures we propose a modification to existing models of the intact LH1 complex by replacing the continuous helix of the beta polypeptide with two helices, one of which lies at an acute angle to the membrane plane. We suggest that a key difference between LH1 and LH2 is that the beta subunit is more bent in LH1. This modification puts the N terminus of LH1beta close to the reaction centre H subunit, and provides a rationale for the different ring sizes of LH1 and LH2 complexes.

A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions.

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.

In vitro reconstitution of the core and peripheral light-harvesting complexes of Rhodospirillum molischianum from separately isolated components.

In most purple bacteria, the core light-harvesting complex (LH1) differs from the peripheral light-harvesting complex (LH2) in spectral properties and amino acid sequences. In Rhodospirillum (Rs. )molischianum, however, the LH2 closely resembles the LH1 of many species in amino acid sequence identity and in some spectral properties (e.g., circular dichroism and resonance Raman). Despite these similarities to LH1, the LH2 of Rs. molischianum displays an absorption spectrum similar to the LH2 complexes of other bacteria. Moreover, its crystal structure is very similar to the LH2 of Rhodopseudomonas (Rps.) acidophila. To better understand the basis of the biochemical and spectral differences between LH1 and LH2, we isolated the alpha and beta polypeptides of the LH2 complexes from an LH2-only strain of Rhodobacter (Rb.) sphaeroides as well as the alpha and beta polypeptides from both the LH1 and LH2 complexes from Rs. molischianum. We then examined their behavior in reconstitution assays with bacteriochlorophyll (Bchl). The Rb. sphaeroides LH2 alpha and beta polypeptides were inactive in reconstitution assays, whether alone, paired with each other, or paired in hybrid assays with the complementary LH1 polypeptides of Rs. rubrum, Rb. sphaeroides, Rb. capsulatus, or Rps. viridis. The LH1 beta polypeptide of Rs. molischianum behaved similarly to the LH1 beta polypeptides of Rs. rubrum, Rb. sphaeroides, Rb. capsulatus, and Rps. viridis, forming a subunit-type complex with or without an alpha polypeptide, and forming an LH1 complex when combined with a native LH1 alpha polypeptide. Interestingly, the LH2 beta polypeptide of Rs. molischianum, in the absence of other polypeptides, also formed a subunit-type complex as well as a further red-shifted complex whose spectrum resembled the 850 nm absorbance band of LH2. In the presence of the LH1 alpha polypeptide of Rs. rubrum or Rs. molischianum, it formed an LH1-type complex, but in the presence of the LH2 alpha polypeptide of Rs. molischianum it formed an LH2 complex. This is the first reported reconstitution of an LH2 complex using only isolated LH2 polypeptides and Bchl. It is also the first example of an LH2 beta polypeptide that can form an LH1 subunit-type complex and an LH1-type complex when paired with an LH1 alpha polypeptide.

Isolation of the PufX protein from Rhodobacter capsulatus and Rhodobacter sphaeroides: evidence for its interaction with the alpha-polypeptide of the core light-harvesting complex.

Using mutant strains of Rhodobacter capsulatus and Rhodobacter sphaeroides in which the pufX gene had been deleted, it was possible to identify by HPLC membrane protein components present in pufX+ cells but absent in pufX- cells. In parallel preparations, membrane proteins soluble in chloroform/methanol containing ammonium acetate were first extracted from lyophilized membrane fractions of the pufX+ cells and separated from pigments and larger protein material by gel-filtration chromatography. Protein-containing fractions were examined by HPLC, and several peaks were collected from pufX+ material that were not present in pufX- material. From N-terminal amino acid sequencing, the PufX protein of Rb. capsulatus was identified, and from positive interaction with a PufX protein antibody, the Rb. sphaeroides PufX protein was identified. Although overall yields were very small, sufficient quantities of these proteins were isolated to evaluate their effect on the reconstitution of the core light-havesting antenna (LH1) and its subunit complex. From the behavior of the PufX protein and the alpha-polypeptide of LH1 on HPLC, qualitative evidence was obtained that the two proteins have a high affinity for each other. In reconstitution assays with bacteriochlorophyll (Bchl) and the LH1 alpha- and beta-polypeptides of Rb. capsulatus, the PufX protein of Rb. capsulatus was inhibitory to LH1 formation at low concentration. A similar inhibition was exhibited by Rb. sphaeroides PufX protein for reconstitution of LH1 with Bchl and the LH1 alpha- and beta-polypeptides of Rb. sphaeroides. In both cases, the ratios of concentrations of the PufX protein to the alpha-polypeptide causing 50% inhibition were approximately 0.5. Formation of the heterologous (alpha beta) subunit-type complex formed with Bchl and the alpha- and beta-polypeptides of LH1 of Rb. capsulatus was also inhibited by low concentrations of the Rb. capsulatus PufX protein (approximately 50% inhibition at PufX:alpha-polypeptide ratios = 0.5). However, neither PufX protein inhibited formation of a homologous (beta beta) subunit-type complex, which indicates that the PufX proteins do not interact with the beta-polypeptides.

Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 1. Minimal requirements for subunit formation.

Described are the chemical synthesis, isolation and characterization of each of three polypeptides whose amino acid sequences reproduce portions of the amino acid sequence of the beta-polypeptides of the core light-harvesting complex (LH1) of Rhodobacter sphaeroides or Rhodospirillum rubrum. The native beta-polypeptides of LH1 of these organisms contain 48 and 54 amino acids, respectively. The smallest synthetic polypeptide had an amino acid sequence identical to that of the last 16 amino acids of the beta-polypeptide of Rb. sphaeroides (sph beta 16) but failed to form either a subunit- or LH1-type complex under reconstitution conditions. Also, this polypeptide, lengthened on the N terminus by adding the sequence Lys-Ile-Ser-Lys to enhance solubility, failed to form a subunit- or LH1-type complex. In contrast, polypeptides containing either the 31 amino acids at the C terminus of the beta-polypeptide of Rb. sphaeroides (sph beta 31) or the equivalent 31 amino acids of the beta-polypeptide of Rs. rubrum (rr beta 31) were fully competent in forming a subunit-type complex and exhibited association constants for complex formation comparable to or exceeding those of the native beta-polypeptides. The absorption and CD spectra of these subunit-type complexes were nearly identical to those of subunit complexes formed with native beta-polypeptides. It may be concluded that all structural features required to make the subunit complex are present in the well-defined, chemically synthesized polypeptides. Neither polypeptide appeared to interact with the native alpha-polypeptides to form a LH1-type complex. However, sph beta 31 formed a LH1-type complex absorbing at 849 nm without an alpha-polypeptide. Although chemical syntheses of polypeptides of this size are common, the purification of membrane-spanning segments is much more challenging because the polypeptides lack solubility in water. The chemical syntheses reported here represent the first such syntheses of membrane-spanning polypeptides which display native activity upon reconstitution.

Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides. 2. Determination of structural features that stabilize complex formation and their implications for the structure of the subunit complex.

Chemically synthesized polypeptides have been utilized with a reconstitution assay to determine the role of specific amino acid side chains in stabilizing the core light-harvesting complex (LH1) of photosynthetic bacteria and its subunit complex. In the preceding paper [Meadows, K. A., Parkes-Loach, P. S., Kehoe, J. W., and Loach, P. A. (1998) Biochemistry 37, 3411-3417], it was demonstrated that 31-residue polypeptides (compared to 48 and 54 amino acids in the native polypeptides) having the same sequence as the core region of the beta-polypeptide of Rhodobacter sphaeroides (sph beta 31) or Rhodospirillum rubrum (rr beta 31) could form subunit-type complexes. However, neither polypeptide interacted with the native alpha-polypeptides to form a native LH1 complex. In this paper, it is demonstrated that larger segments of the native Rb. sphaeroides beta-polypeptide possess native behavior in LH1 formation. Polypeptides were synthesized that were six (sph beta 37) and ten amino acids (sph beta 41) longer than sph beta 31. Although sph beta 37 exhibited behavior nearly identical to that of sph beta 31, sph beta 41 behaved more like the native polypeptide. In the case of rr beta 31, a polypeptide with four additional amino acids toward the C terminus was synthesized (rr beta 35). Because LH1-forming behavior was not recovered with this longer polypeptide, one or more of the three remaining amino acids at the C-terminal end of the native beta-polypeptide seem to play an important role in LH1 stabilization in Rs. rubrum. Three analogues of the core region of the Rb. sphaeroides beta-polypeptide were synthesized, in each of which one highly conserved amino acid was changed. Evidence was obtained that the penultimate amino acid, a Trp residue, is especially important for subunit formation. When it was changed to Phe, the lambda Max of the subunit shifted from 823 to 811 nm and the association constant decreased about 500-fold. Changing each of two other amino acids had smaller effects on subunit formation. Changing Trp to Phe at the location six amino acid residues toward the C terminus from the His coordinated to Bchl resulted in an approximately 10-fold decrease in the association constant for subunit formation but did not affect the formation of a LH1-type complex compared to sph beta 31. Finally, changing Arg to Leu at the location seven amino acid residues toward the C terminus from the His coordinated to Bchl decreased the association constant for subunit formation by about 30-fold. In this case, no LH1-type complex could be formed. On the basis of these results, in comparison with the crystal structure of the LH2 beta-polypeptide of Rhodospirillum molischianum, two possible structures for the subunit complex are suggested.

Evaluation of structure-function relationships in the core light-harvesting complex of photosynthetic bacteria by reconstitution with mutant polypeptides.

Seven mutant LH1 polypeptides of Rhodobactor sphaeroides have been isolated, and their behaviors in in vitro reconstitution of LH1 and its subunit complex have been characterized. Two mutants were selected to address the increased stability of the subunit complex of Rb. sphaeroides compared with that of Rhodobacter capsulatus. We found that this difference can be largely ascribed to the existence of Tyr at position +4 in the beta-polypeptide (the numbering system used assigns position 0 to the His which provides the coordinating ligand to bacteriochlorophyll) of the former bacterium compared to Met in that position in the latter. The amount of energy involved in the increased interaction was 1.6 kcal/mol, which would be consistent with a hydrogen bond involving Tyr. Mutation of the His at position 0 to Asn allows an estimate of the binding energy for subunit formation contributed by coordination of the imidazole group of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/mol per BChl. Finally, an evaluation of the role of amino acids in the C-terminal region of the alpha-polypeptide was begun. Reconstitution of a mutant alpha-polypeptide in which Trp at position +11 was changed to Phe resulted in optimal formation of an LH1-type complex whose lambda(max) was blue-shifted to 853 nm, the same as observed in the intact bacterium harboring this mutation. These results provide further confirmation that the environment of BChl in reconstituted LH1 complexes is the same as in vivo and support the assignment of this residue to a role in hydrogen bonding with the C3(1) carbonyl group of BChl. Two other mutants of the alpha-polypeptide in which 5 and 14 amino acids in the C-terminus were deleted were also examined. These were of interest because the latter mutant, unlike the former, resulted in a low level of expression of LH1 in intact cells. However, with both of these mutant polypeptides, reconstitution appeared identical to that of the native system. In the case of the mutant shortened by 14 amino acids, a small blue-shift in lambda(max) to 861 nm was observed, again reproducing the blue-shift exhibited by the intact cells. Thus, these results suggest that the lowered levels of in vivo expression observed in these two mutants are due to reduced incorporation of the alpha-polypeptide into the membrane or its increased degradation, rather than to decreased stabilization of the LH1 complex.

Comparison of the structural requirements for bacteriochlorophyll binding in the core light-harvesting complexes of Rhodospirillum rubrum and Rhodospirillum sphaeroides using reconstitution methodology with bacteriochlorophyll analogs.

Bacteriochlorophyll (BChl) structural requirements for formation of the core light-harvesting complex (LH1) and its structural subunit complex were examined by reconstitution with BChl analogs and the alpha- and beta-polypeptides of Rhodospirillum rubrum and Rhodobacter sphaeroides. Comparable results were obtained with most of the BChl analogs and the polypeptides of each bacterium, indicating the conservation of BChl binding sites. These systems showed the following common requirements for formation of the subunit complex and LH1: (1) Mg or a metal of similar size and coordination chemistry (e.g., Zn, Cd, Ni), (2) a bacteriochlorin oxidation state of the macrocyclic ring, (3) a 13(2)-carbomethoxy group, and (4) an intact ring V. Some structural features were not as critically important. For example, the subunit complex and LH1 could be formed with both sets of polypeptides and BChl b, as well as with analogs containing either short (ethanol) or long (phytol) esterifying alcohols. Two derivatives were identified that behave differently with the two sets of polypeptides. The 3-acetyl group is required to form LH1 in both bacteria, although a subunit-type complex was readily formed with [3-vinyl]BChl a and the polypeptides of Rs. rubrum but formed only slightly under special conditions with polypeptides of Rb. sphaeroides. [13(2)-OH]BChl a(p) formed both subunit- and LH1-type complexes with the alpha- and beta-polypeptides of Rb. sphaeroides but not with those of Rs. rubrum. Thus, some subtle differences in the BChl binding sites exist in the LH1 complexes of these two bacteria.

Reconstitution of the bacterial core light-harvesting complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with isolated alpha- and beta-polypeptides, bacteriochlorophyll alpha, and carotenoid.

Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll alpha with isolated light-harvesting complex I (LHI) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum. Reconstitution techniques first developed in this laboratory using the LHI polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll alpha spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supplied either as organic-solvent extracts of chromatophores or as thin-layer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacteriochlorophyll a spectral properties characteristic of native LHI complexes of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environment in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal appropriate carotenoid to bacteriochlorophyll alpha energy-transfer efficiencies based on comparisons with the in vivo systems. In the case of R. rubrum reconstitutions, carotenoids afford protection from photodynamic degradation. Thus, carotenoids reconstituted into LHI exhibit spectral and functional characteristics associated with native pigments. Heterologous reconstitutions demonstrate the applicability of the developed assay in dissecting the molecular environment of carotenoids in light-harvesting complexes.

Enzymatic and chemical cleavage of the core light-harvesting polypeptides of photosynthetic bacteria: determination of the minimal polypeptide size and structure required for subunit and light-harvesting complex formation.

To ascertain the minimal structural requirements for formation of the subunit and core light-harvesting complex (LH1), the alpha- and beta-polypeptides of the LH1 from three purple photosynthetic bacteria were enzymatically or chemically truncated or modified. These polypeptides were then used in reconstitution experiments with bacteriochlorophyll a (BChla), and the formation of subunit and LH1 complexes was evaluated using absorbance and circular dichroism spectroscopies. Truncation or modification outside of the conserved core sequence region of the polypeptides had no effect on subunit or LH1 formation. However, the extent of formation and stability of the subunit and LH1 decreased as the polypeptide was shortened inside the core region within the N-terminal domain. This behavior was suggested to be due to the loss of potential ion-pairing and/or hydrogen-bonding interactions between the polypeptides. While the spectroscopic properties of the subunit complexes generated using truncated polypeptides were analogous to those obtained using native polypeptides, in some cases the resulting LH1 complex absorption was blue-shifted relative to the control. Thus, truncation within the N-terminal domain may have long-range effects on the immediate BChla binding environment, since the putative BChla binding site resides near the C-terminal end of the polypeptides. It was also demonstrated that the His located within the membrane-spanning domain on the N-terminal end of the beta-polypeptide is not participating in ligation of the BChla in the reconstituted subunit and therefore probably not in LH1.

Reconstitution of a functional photosynthetic receptor complex with isolated subunits of core light-harvesting complex and reaction centers.

The B820 subunit form of the core light-harvesting complex LHI, isolated from the photosynthetic bacterium Rhodospirillum rubrum, was combined in a reassociation assay with the reaction center (RC) isolated from the same or related bacteria. This reassociation produced a photoreceptor complex (PRC) which appeared, by absorption spectroscopy, circular dichroism measurements, and kinetic absorption spectroscopy measuring transient photochanges, as analogous to the PRC in the intact bacteria. Energy transfer between the LHI and reaction center progressed with almost 100% efficiency and indicated a cooperative pattern of transfer. Treatment of the RC with proteinase K resulted in peptide cleavages of all three polypeptides of the RC but did not alter the light-induced charge separation in the RC or prevent the reassociation of the LHI and modified RC. Energy transfer efficiency from LHI to RC still approached 100% but the cooperative behavior seen in reconstitutions with the intact RC was not observed. Initial experiments using interspecies reassociations (LHI from Rhodobacter sphaeroides and RC from Rs. rubrum) showed a low efficiency of energy transfer from LHI to RC. Possible association domains for the LHI-RC interaction based on considerations of the comparative amino acid sequences of the RC of each bacteria and the most feasible remaining residues in the proteinase K treated RC are considered.

Probing protein structural requirements for formation of the core light-harvesting complex of photosynthetic bacteria using hybrid reconstitution methodology.

The α- and ß-polypeptides of LH1 isolated from four different photosynthetic bacteria (Rhodospirillum rubrum, Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas viridis) were used for homologous and hybrid reconstitution experiments with bacteriochlorophyll a. Formation of B820-type subunit complexes and LH1-type complexes were evaluated. The ß-polypeptides of R. rubrum, Rb. sphaeroides and Rb. capsulatus behaved similarly and formed B820-type subunit complexes in the absence of an α-polypeptide. The α- and ß-polypeptides were both required to form a LH1-type complex with each of these three homologous systems. In hybrid experiments where the ß-polypeptides were tested for reconstitution with α-polypeptides other than their homologous partners, half of the twelve possible combinations resulted in formation of both B820- and LH1-type complexes. Three of the combinations that did not result in formation of a LH1-type complex involved the ß-polypeptide of R. rubrum. It is suggested that these latter results can be explained by charge repulsion between the Lys at position-17 (assigning the conserved His located nearest to the C-terminus as position 0) in the ß-polypeptide of R. rubrum and each of the heterologous α-polypeptides tested, all of which have an Arg at this location. Conclusions that can be derived from these experimental results include: (1) the experimental data support the idea that a central core region of approximately 40 amino acids exists in each of the polypeptides, which contains sufficient information to allow formation of both the B820- and LH1-type complexes and (2) a specific portion of the N-terminal hydrophilic region of each polypeptide was found in which ion pairs between oppositely charged groups on the α- and ß-polypeptides seem to stabilize complex formation.

Probing the structure of the core light-harvesting complex (LH1) of Rhodopseudomonas viridis by dissociation and reconstitution methodology.

A subunit complex was formed from the core light-harvesting complex (LH1) of bacteriochlorophyll(BChl)-b-containing Rhodopseudomonas viridis. The addition of octyl glucoside to a carotenoid-depleted Rps. viridis membrane preparation resulted in a subunit complex absorbing at 895 nm, which could be quantitatively dissociated to free BChl b and then reassociated to the subunit. When carotenoid was added back, the subunit could be reassociated to LH1 with a 25% yield. Additionally, the Rps. viridis α- and ß-polypeptides were isolated, purified, and then reconstituted with BChl b. They formed a subunit absorbing near 895 nm, similar to the subunit formed by titration of the carotenoid depleted membrane, but did not form an LH1-type complex at 1015 nm. The same results were obtained with the ß-polypeptide alone and BChl b. Isolated polypeptides were also tested for their interaction with BChl a. They formed subunit and LH1-type complexes similar to those formed using polypeptides isolated from BChl-a-containing bacteria but displayed 6-10 nm smaller red shifts in their long-wavelength absorption maxima. Thus, the larger red shift of BChl-b-containing Rps. viridis is not attributable solely to the protein structure. The ß-polypeptide of Rps. viridis differed from the other ß-polypeptides tested in that it could form an LH1-type complex with BChl a in the absence of the α- and γ-polypeptides. It apparently contains the necessary information required to assemble into an LH1-type complex. When the γ-polypeptide was tested in reconstitution with BChl a and BChl b with the α- and ß-polypeptides, it had no effect; its role remains undetermined.

Fluorescence polarization and low-temperature absorption spectroscopy of a subunit form of light-harvesting complex I from purple photosynthetic bacteria.

Measurements of polarized fluorescence and CD were made on light-harvesting complex 1 and a subunit form of this complex from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus. The subunit form of LH1, characterized by a near-infrared absorbance band at approximately 820 nm, was obtained by titration of carotenoid-depleted LH1 complexes with the detergent n-octyl beta-D-glucopyranoside as reported by Miller et al. (1987) [Miller J. F., Hinchigeri, S. B., Parkes-Loach, P. S., Callahan, P. M., Sprinkle, J. R., & Loach, P. A. (1987) Biochemistry 26, 5055-5062]. Fluorescence polarization and CD measurements at 77 K suggest that this subunit form must consist of an interacting bacteriochlorophyll a dimer in all three bacterial species. A small, local decrease in the polarization of the fluorescence is observed upon excitation at the blue side of the absorption band of the B820 subunit. This decrease is ascribed to the presence of a high-energy exciton component, perpendicular to the main low-energy exciton component. From the extent of the depolarization, we estimate the oscillator strength of the high-energy component to be at most 3% of the main absorption band. The optical properties of B820 are best explained by a Bchl a dimer that has a parallel or antiparallel configuration with an angle between the Qy transition dipoles not larger than 33 degrees. The importance of this structure is emphasized by the results showing that core antennas from three different purple bacteria have a similar structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a structural subunit from the core light-harvesting complex of Rhodobacter sphaeroides 2.4.1 and puc705-BA.

A method for isolating a structural subunit, B825, from the B875 core light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1 (wild-type) and a B800-B850(-) mutant, puc705-BA, is presented. This method, based on one developed to prepare a similar subunit, B820, from the core LHC of Rhodospirillum rubrum [Miller et al., Biochemistry 26, 5055-5062 (1987)], requires the dissociation of treated chromatophores with the detergent, octyl-glucoside. A subsequent gel filtration step separates B800-850 (if present), reaction centers, and free bacteriochlorophyll from the subunit complex. B825 was quantitatively reassociated into an 873 nm absorbing form which resembled the in vivo complex as judged by its absorption properties. The polypeptides in B825 and B800-850 were isolated by HPLC and identified by N-terminal amino acid sequencing. Two polypeptides, alpha and beta, were found in each complex in a 1:1 ratio. The spectral and biochemical properties of the subunits isolated from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus are compared.

Isolation and characterization of a subunit form of the B875 light-harvesting complex from Rhodobacter capsulatus.

A structural subunit (called B816) has been isolated from the B875 light-harvesting complex of Rhodobacter capsulatus using a detergent-mediated dissociation of chromatophores. Rb. capsulatus MW442 (B800-850- B875+ car+) chromatophores were extracted with benzene and titrated with octyl glucoside (OG) to shift the near-infrared absorption maximum from 873 to 816 nm. Gel filtration chromatography was then used to separate B816 from reaction centers. B816 could be quantitatively shifted back to a B875-like form (lambda max = 875 nm) by decreasing the OG concentration. A similar B816 species could be isolated in low yield from wildtype (B800-850+ B875+ car+) cells but not from SB203E (B800-850- B875+ car-). In the latter case, the B816 subunit seemed too unstable to be isolated under equivalent conditions. The alpha:beta polypeptide ratio, the CD spectrum, and the ability to reversibly dissociate B816 to free bacteriochlorophyll and alpha- and beta-polypeptides were found to be similar to those of the B820 subunit of Rs. rubrum previously reported by our laboratory.

Probing the bacteriochlorophyll binding site by reconstitution of the light-harvesting complex of Rhodospirillum rubrum with bacteriochlorophyll a analogues.

Structural features of bacteriochlorophyll (BChl) a that are required for binding to the light-harvesting proteins of Rhodospirillum rubrum were determined by testing for reconstitution of the B873 or B820 (structural subunit of B873) light-harvesting complexes with BChl a analogues. The results indicate that the binding site is very specific; of the analogues tested, only derivatives of BChl a with ethyl, phytyl, and geranylgeranyl esterifying alcohols and BChl b (phytyl) successfully reconstituted to form B820- and B873-type complexes. BChl analogues lacking magnesium, the C-3 acetyl group, or the C-13(2) carbomethoxy group did not reconstitute to form B820 or B873. Also unreactive were 13(2)-hydroxyBChl a and 3-acetylchlorophyll a. Competition experiments showed that several of these nonreconstituting analogues significantly slowed BChl a binding to form B820 and blocked BChl a-B873 formation, indicating that the analogues may competitively bind to the protein even though they do not form red-shifted complexes. With the R. rubrum polypeptides, BChl b formed complexes that were further red-shifted than those of BChl a; however, the energies of the red shifts, binding behavior, and circular dichroism (CD) spectra were similar. B873 complexes reconstituted with the geranylgeranyl BChl a derivative, which contains the native esterifying alcohol for R. rubrum, showed in-vivo-like CD features, but the phytyl and ethyl B873 complexes showed inverted CD features in the near infrared. The B820 complex with the ethyl derivative was about 30-fold less stable than the two longer esterifying alcohol derivatives, but all formed stable B873 complexes.

Spectroscopic characterization of the light-harvesting complex of Rhodospirillum rubrum and its structural subunit.

The spectroscopic properties of the light-harvesting complex of Rhodospirillum rubrum, B873, and a detergent-isolated subunit form, B820, are presented. Absorption and circular dichroism spectra suggest excitonically interacting bacteriochlorophyll alpha (BChl alpha) molecules give B820 its unique spectroscopic properties. Resonance Raman results indicate that BCHl alpha is 5-coordinate in both B820 and B873 but that the interactions with the BChl C2 acetyl in B820 and B873 are different. The reactivity of BChl alpha in B820 in light and oxygen, or NaBH4, suggests that it is exposed to detergent and the aqueous environment. Excited-state lifetimes of the completely dissociated 777-nm-absorbing form [1.98 ns in 4.5% octyl glucoside (OG)], the intermediate subunit B820 (0.72 ns in 0.8% OG), and the in vivo like reassociated B873 (0.39 ns in 0.3% OG) were measured by single-photon counting. The fluorescence decays were exponential when emission was detected at wavelengths longer than 864 nm. An in vivo like B873 complex, as judged by its spectroscopic properties, can be formed from B820 without the presence of a reaction center.