Solution structures of the core light-harvesting alpha and beta polypeptides from Rhodospirillum rubrum: implications for the pigment-protein and protein-protein interactions

We have determined the solution structures of the core light-harvesting (LH1) alpha and beta-polypeptides from wild-type purple photosynthetic bacterium Rhodospirillum rubrum using multidimensional NMR spectroscopy. The two polypeptides form stable alpha helices in organic solution. The structure of alpha-polypeptide consists of a long helix of 32 amino acid residues over the central transmembrane domain and a short helical segment at the N terminus that is followed by a three-residue loop. Pigment-coordinating histidine residue (His29) in the alpha-polypeptide is located near the middle of the central helix. The structure of beta-polypeptide shows a single helix of 32 amino acid residues in the membrane-spanning region with the pigment-coordinating histidine residue (His38) at a position close to the C-terminal end of the helix. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R.rubrum LH1 alpha and beta-polypeptides are different from those previously reported for the LH1 beta-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 alpha and beta-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 alpha and beta-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2-Gln6) of alpha-polypeptide and a GxxxG motif in the beta-polypeptide.     

Purification and Characterization of the Polypeptides of Core Light-Harvesting Complexes from Purple Sulfur Bacteria

Although the polypeptides of core light-harvesting complexes (LH1) from many purple nonsulfur bacteria have been well characterized, little information is available on the polypeptides of LH1 from purple sulfur photosynthetic organisms. We present here the results of isolation and characterization of LH1 polypeptides from two purple sulfur bacteria, Thermochromatium (Tch.) tepidum and Allochromatium (Ach.) vinosum. Native LH1 complexes were extracted and purified in a reaction center (RC)-associated form with the Qy absorption at 914 nm and 889 nm for Tch. tepidum and Ach. vinosum, respectively. Three components were confirmed from reverse-phase HPLC for the LH1 apopolypeptides of Tch. tepidum. The beta-polypeptide was found to be methylated at N-terminus, and two alpha-polypeptides were identified with one of them being modified by a formyl group at the N-terminal methionine residue. Two alpha- and two beta-polypeptides were confirmed for the LH1 complex of Ach. vinosum, and their primary structures were precisely determined. Homologous and hybrid reconstitution abilities were examined using bacteriochlorophyll a and separated alpha- and beta-polypeptides. The beta-polypeptide from Tch. tepidum was capable of forming uniform structural subunit not only with the alpha-polypeptide of Tch. tepidum but also with the alpha-polypeptide from a nonsulfur bacterium Rhodospirillum rubrum. The alpha-polypeptide alone or beta-polypeptide alone appeared only to result in incomplete subunits in the reconstitution experiments.     

Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated alpha- and beta-polypeptides and bacteriochlorophyll a

The light-harvesting complex of Rhodospirillum rubrum was reversibly dissociated into its component parts: bacteriochlorophyll and two 6-kilodalton polypeptides. The dissociation of the complex by n-octyl beta-D-glucopyranoside was accompanied by a shift of the absorbance maximum from 873 to 820 nm (a stable intermediate form) and finally to 777 nm. In the latter state, bacteriochlorophyll was shown to be free from the protein. Complexes absorbing at 820 and 873 nm could be re-formed from the fully dissociated state with over 80% yield by dilution of the detergent. Absorbance and circular dichroism properties of the re-formed B820 complex were essentially identical with those of B820 formed from chromatophores. Phospholipids and higher concentrations of complex were required to obtain the in vivo circular dichroism spectrum for reassociated B873. Reconstitution of the light-harvesting complexes from separately isolated alpha- and beta-polypeptides and bacteriochlorophyll was also demonstrated. Absorbance and circular dichroism spectra of these complexes were identical with those of complexes formed by the reassociation of the dissociated complex. Bacteriochlorophyll and the beta-polypeptide alone formed a complex that had an absorbance at 820 nm, but an 873-nm complex could not be formed without addition of the alpha-polypeptide. The alpha-polypeptide alone with bacteriochlorophyll did not form any red-shifted complex. In preliminary structure-function studies, some analogues of bacteriochlorophyll were also tested for reconstitution.     

Methionine oxidation and its effect on the stability of a reconstituted subunit of the light-harvesting complex from Rhodospirillum rubrum.

An additional component in the purified core light-harvesting complex (LH1) from wild-type purple photosynthetic bacterium Rhodospirillum rubrum has been identified as an oxidized species of alpha-polypeptide by MALDI-TOF mass spectrometry. This component appears as a slightly earlier-eluting peak in the RP-HPLC chromatogram compared with the authentic alpha-polypeptide. The oxidation site has been determined to be the N-terminal methionine residue by high-resolution NMR spectroscopy, where the methionine is oxidized to methionine sulfoxide in a diastereoisomeric form. Interconversion between the oxidized and authentic alpha-polypeptides has been confirmed by selective oxidation and reduction. The oxidative modification of methionine is shown to have discernible effects on the ability to form B820 subunit with beta-polypeptide and bacteriochlorophyll a, and on the stability of the reconstituted B820 subunit. Both the ability and the stability for the samples using the oxidized alpha-polypeptide are moderately reduced, indicating that the oxidation-induced conformational change in the N-terminal domain of alpha-polypeptide may affect the pigment-binding environment through a long-range interaction. The MALDI-TOF mass results also reveal that the N-terminus of alpha-polypeptide is formylated and no phosphorylation has occurred in this polypeptide.     

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.     

Interaction of bacteriochlorophyll with the LH1 and PufX polypeptides of photosynthetic bacteria: use of chemically synthesized analogs and covalently attached fluorescent probes

The protein components of the reaction center (RC) and core light-harvesting (LH 1) complexes of photosynthetic bacteria have evolved to specifically, but non-covalently, bind bacteriochlorophyll (Bchl). The contribution to binding of specific structural elements in the protein and Bchl may be determined for the LH 1 complex because its subunit can be studied by reconstitution under equilibrium conditions. Important to the determination and utilization of such information is the characterization of the interacting molecular species. To aid in this characterization, a fluorescent probe molecule has been covalently attached to each of the LH 1 polypeptides. The fluorescent probes were selected for optimal absorption and emission properties in order to facilitate their unique excitation and to enable the detection of energy transfer to Bchl. Oregon Green 488 carboxylic acid and 7-diethylaminocoumarin-3-carboxylic acid seemed to fulfill these requirements. Each of these probes were utilized to derivatize the LH1 β-polypeptide of Rhodobacter sphaeroides. It was demonstrated that the β-polypeptides did not interact with each other in the absence of Bchl. When Bchl was present, the probe-labeled β-polypeptides interacted with Bchl to form subunit-type complexes much as those formed with the native polypeptides. Energy transfer from the probe to Bchl occurred with a high efficiency. The α-polypeptide from LH 1 of Rb. sphaeroides and that from Rhodospirillum rubrum were also derivatized in the same manner. Since these polypeptides do not oligomerize in the absence of a β-polypeptide, reversible binding of a single Bchl to a single polypeptide could be measured. Dissociation constants for complex formation were estimated. The relevance of these data to earlier studies of equilibria involving subunit complexes is discussed. Also involved in the photoreceptor complex of Rb. sphaeroides and Rhodobacter capsulatus is another protein referred to as PufX. Two large segments of this protein were chemically synthesized, one reproducing the amino acid sequence of the core segment predicted for Rb. sphaeroides PufX and the other reproducing the amino acid sequence predicted for the core segment of Rb. capsulatus PufX. Each polypeptide was covalently labeled with a fluorescent probe and tested for energy transfer to Bchl. Each was found to bind Bchl with an affinity similar to the affinity of the LH 1 polypeptides for Bchl. It is suggested that PufX binds Bchl and interacts with a Bchlċα-polypeptide component of LH 1 to truncate, or interupt, the LH 1 ring adjacent to the location of the Q_B binding site of the RC. This revised version was published online in June 2006 with corrections to the Cover Date.     

The light-harvesting core-complex and the B820-subunit from Rhodopseudomonas marina. Part I. Purification and characterisation.

The BChla-containing B880-complex (core-complex) of Rhodopseudomonas marina (Rhodospirillaceae) was isolated with a new purification method. The isolation of the B880-complex was performed by solubilisation of the photosynthetic membranes with the detergent LDAO and subsequent fractionated ammonium-sulfate precipitation with about 50% recovery. The B880-complex retained its original spectral properties as revealed with absorption, fluorescence and circular dichroism spectroscopy. Furthermore, we dissociated the B880-complex with the detergent n-octyl-beta-glucoside (OG) and purified the developed subcomplex by the method of Miller et al. [1], which showed an absorption maximum at 820 nm (B820). The alpha- to beta-polypeptide ratio and the alpha- or beta-polypeptide to BChla ratio, respectively, were estimated to be 1:1 in both complexes. The molecular weights of the B880 and the B820-complexes, determined by gel filtration chromatography, were 181 and 32 kDa, respectively. Thus, it appears that the B880-complex of Rp. marina consists of 24 polypeptides and the B820-complex of four polypeptides. Six B820-complexes or possible subunits could form the B880-complex. On the basis of these data we propose a model for the structure of BChla containing core-complexes.     

Multiple copies of the coding regions for the light-harvesting B800-850 alpha- and beta-polypeptides are present in the Rhodopseudomonas palustris genome.

A reverse-phase HPLC System for isolation of the water insoluble alpha- and beta-polypeptides of the light-harvesting complex II (LH II) of Rhodopseudomonas (Rps.) palustris without employment of any detergent was developed. The material obtained was of high purity and suitable for direct microsequence analysis. Chromatographic analysis could resolve at least two major beta-polypeptides, beta a and beta b, two major alpha-polypeptides, alpha a and alpha b, and two additional minor polypeptides. N-terminal amino acid sequencing shows that the resolved peaks correspond to different polypeptide species and that the minor species have an N-terminal sequence identical to that of the alpha b polypeptide. An oligonucleotide derived from the amino terminal sequence of the alpha a polypeptide was utilized to screen a genomic library from Rps.palustris. Several independent clones have been characterized by Southern blot and nucleotide sequence analysis. We show that Rps.palustris contains at least four different clusters of beta and alpha genes. Two clones contain sequences potentially coding for beta a-alpha a and beta b-alpha b polypeptides; and two additional clones potentially coding for beta and alpha peptides which we named beta c-alpha c and beta d-alpha d, which did not correspond to the major purified polypeptides. In addition to the protein chemistry data, the conservation at the amino acid level and the presence of canonical ribosomal binding sites upstream of each of the identified genes strongly suggest that all four coding regions are expressed.     

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.     

Calcium ions are involved in the unusual red shift of the light-harvesting 1 Qy transition of the core complex in thermophilic purple sulfur bacterium Thermochromatium tepidum

Thermophilic purple sulfur bacterium, Thermochromatium tepidum, can grow at temperatures up to 58 degrees C and exhibits an unusual Qy absorption at 915 nm for the core light-harvesting complex (LH1), an approximately 35-nm red shift from those of its mesophilic counterparts. We demonstrate in this study, using a highly purified LH1-reaction center complex, that the LH1 Qy transition is strongly dependent on metal cations and Ca2+ is involved in the unusual red shift. Removal of the Ca2+ resulted in formation of a species with the LH1 Qy absorption at 880 nm, and addition of the Ca2+ to the 880-nm species recovered the native 915-nm form. Interchange between the two forms is fully reversible. Based on spectroscopic and isothermal titration calorimetry analyses, the Ca2+ binding to the LH1 complex was estimated to occur in a stoichiometric ratio of Ca2+/alphabeta-subunit = 1:1 and the binding constant was in 10(5) m(-1) order of magnitude, which is comparable with those for EF-hand Ca2+-binding proteins. Despite the high affinity, conformational changes in the LH1 complex upon Ca2+ binding were small and occurred slowly, with a typical time constant of approximately 6 min. Replacement of the Ca2+ with other metal cations caused blue shifts of the Qy bands depending on the property of the cations, indicating that the binding site is highly selective. Based on the amino acid sequences of the LH1 complex, possible Ca2+-binding sites are proposed that consist of several acidic amino acid residues near the membrane interfaces of the C-terminal region of the alpha-polypeptide and the N-terminal region of the beta-polypeptide.     

Solution structure of the Rhodobacter sphaeroides PufX membrane protein: implications for the quinone exchange and protein-protein interactions

PufX membrane protein is found in Rhodobacter species of purple photosynthetic bacteria and has been known to play an essential role in ubiquinone/ubiquinol exchange between the reaction center and cytochrome bc1 complex and also contribute to the dimerization of the reaction center-light-harvesting core complex. We have determined the solution structure of the Rhodobacter sphaeroides PufX using multidimensional NMR spectroscopy. The PufX, functionally expressed in Escherichia coli, forms a stable alpha helix consisting of 21 residues over the central transmembrane domain. The overall structure of the PufX is very similar to those of the LH1 alpha- and beta-polypeptides from Rhodospirillum rubrum and LH2 polypeptides. A short segment (Lys28-Gly35) rich in Gly and Ala residues revealed a relatively fast exchange between the backbone amide protons and deuteriums in the hydroxyl groups of the solvent, indicating that the backbone of this segment is more easily accessible to the surrounding solvent molecules compared to those of its neighboring portions. The Gly- and Ala-rich segment is located in the middle of the central helix and forms an extensive groove-like conformation on the surface with the neighboring residues, where the residues with large side chains are aligned on one side of the helix, and small residues are aligned on the other face. Such a structural motif may serve as a functional site responsible for ubiquinol transport from the core complex to the membrane phase and for sequence-specific helix-helix interactions with the neighboring polypeptides.     

The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms

Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.     

Projection structure of the photosynthetic reaction centre-antenna complex of Rhodospirillum rubrum at 8.5 A resolution

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.     

Hydrophobic pockets at the membrane interface: an original mechanism for membrane protein interactions

The effect of partial digestion by trypsin and GluC protease on the association of the membrane polypeptides of LH1 from Rhodospirillum (Rsp.) rubrum was studied. Trypsin and GluC protease treatments of LH1 result in the cleavage of the first three amino acids from the alpha polypeptide and of the first 18 amino acids from the beta polypeptide, respectively, without any noticeable reorganization of their secondary structure, as measured by attenuated total reflectance Fourier transform IR spectroscopy. However, the enthalpy variation accompanying dimer formation was dramatically reduced by the protease attacks by as much as 80%. Our results show that the alphabeta heterodimer is mainly stabilized by hydrophobic interactions which involve the amino-terminal extensions of the participating polypeptides. Using the close homology between the polypeptides of Rsp. rubrum LH1 and that of Rsp. molischianum LH2, whose structure is known, a structural model for these "hydrophobic pockets" lying close to the membrane interface is proposed.     

Amino acid sequence of the nucleotide binding region of chloroplast coupling factor 1

The labeling of chloroplast coupling factor 1 by 3'-O-(4-benzoyl)benzoyl-ATP (BzATP) was studied. When the enzyme was incubated with approximately 10 microM BzATP and 6 mM MgCl2 at pH 7.9 for approximately 20 min and passed through two Sephadex G-50 centrifuge columns, three BzATP molecules were bound per coupling factor molecule. Photolysis of radioactive enzyme-bound BzATP followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the BzATP bound primarily to the beta-polypeptide. If unbound BzATP was not removed by centrifuge columns prior to photolysis, significant labeling of the alpha-polypeptide also occurred. After photolysis, the BzATP-labeled enzyme was treated with trypsin, and two radioactive peptides were isolated by high-performance liquid chromatography on a C18 column. The two peptides were sequenced and found to correspond to amino acids 360-378 and 393-397 of the beta-polypeptide. For the sequence 360-378, two specific amino acids were found to be radioactive (Tyr-362 and Asp-369). This region of the polypeptide is highly conserved in several different species and probably corresponds to part of the nucleotide binding region of the catalytic site. In the case of amino acids 393-397, a very low level of radioactivity was found for all amino acids. The significance of this peptide for the binding of nucleotides to coupling factor 1 could not be established.     

Cyclic adenosine monophosphate and phorbol ester, like gonadotropin-releasing hormone, stimulate the biosynthesis of luteinizing hormone polypeptide chains in a nonadditive manner

We have previously reported that GnRH stimulates the synthesis of both the alpha- and beta-polypeptide chains of LH. In the present study, in order to investigate the mechanisms involved in the GnRH regulation of LH subunit synthesis, we have explored the effects of cAMP and a phorbol ester [12-O-tetradecanoyl phorbol 13-acetate (TPA)] using anterior pituitary cells in primary culture incubated in the presence of [35S]methionine. The radioactivity incorporated into alpha and LH beta immunologically related polypeptides was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled material immunoextracted from cells and media with specific antisera. The cAMP analog 8-Br-cAMP (at concentrations 0.25-2 mM), the cholera toxin (6-60 nM), and forskolin (10-100 microM) induced, like GnRH, an increase in the [35S]methionine incorporation into alpha- and LH beta-subunits. On the other hand, TPA (50-100 nM) also enhanced the synthesis of LH subunits. After a 5-h incubation in the presence of GnRH, 8-Br-cAMP, and TPA in different combinations, no cumulative effect was observed. These results demonstrate that intracellular cAMP and TPA are potent activators of both alpha- and LH beta-polypeptide chain synthesis, suggesting that cAMP as well as diacylglycerols may act as intracellular mediators of the GnRH effect on LH subunit synthesis.     

Role of the C-terminal extrinsic region of the alpha polypeptide of the light-harvesting 2 complex of Rhodobacter sphaeroides: a domain swap study

The LH1 and LH2 complexes of Rhodobacter sphaeroides form ring structures of 16 and 9 protomers, respectively, comprising alpha and beta polypeptides, bacteriochlorophylls (Bchl), and carotenoids. Using the LH2 complex as a starting point, two chimeric LH complexes were constructed incorporating the alphaC-terminal domain of either the Rb. sphaeroides LH1 complex or the Rhodospirillum molischianum LH2 complex. The LH1 domain swap produced a new red-shifted component that comprised approximately 30% of the total absorbance. In the LH1alpha C-terminal mutant this new red-shifted species acts as the terminal emitter, with the new emission maximum located 10 nm further to the red than for the WT. Raman spectroscopy indicates that a fraction of the B850 Bchls is involved in relatively weak H-bonds, possibly involving the alphaTrp(+11) residue within the new alphaC-terminus, consistent with a more LH1-like character for one of the Bchls. The CD data indicate that the domain swaps have perturbed the native arrangement of the B850 Bchls, including the site energy difference between the alpha- and beta-bound Bchls. Thus, the normal energetic structure of the ring system has been disrupted, with one component blue shifted due to the presumed loss of an H-bond donor and the other red shifted by the influence of the new alphaC-terminal domain. The dichotomous response of the mutants to the carotenoids incorporated, spheroidenone or neurosporene, strongly suggests that the C-terminal region of the alpha polypeptide is involved in binding a carotenoid. The projection map of the LH1alpha C-terminal mutant complex was determined in negative stain at 25 A resolution, and it shows a diameter of 53 A, compared to 50 A for the WT. Hence these new spectral properties have not been accompanied by an alteration in ring size.     

On the influence of pigment-protein interactions on the energy transfer processes in photosynthetic membrane structures. 2. LH2 complex of Rhodobacter sphaeroides

Low-temperature heterogeneous absorption and circular dichroism spectra of the Rb. sphaeroides LH2 complexes are calculated within the framework of the mini-exciton theory and diagonal static random disorder for the pure electronic transitions of the monomeric Bchl molecules. The coupling of Bchl molecules with the surrounding amino acid residues has been shown to change both the exciton distribution between the pigment molecules in each of the exciton states. The value of the delocalization index depends on the excitation wavelength and varies between 2-6 Bchl molecules. The optical transitions occurring at 780-790 and 820 nm have been found to be strongly mixed so that all Bchl molecules of the LH2 complex predetermine absorption in these spectral regions. On the other hand, absorption at 800 and 850 nm is mainly determined by the cycles of 9 and 18 Bchl molecules, respectively. Thus, the light energy absorbed by the B800 molecules at 800 nm is transferred to the B850 molecules by the interlevel exciton relaxation processes due to the population of the heavily mixed 820-nm exciton levels. The width of the heterogeneous absorption band for the cyclic monomeric aggregate has been shown to decrease as compared with the monomeric absorption band by square root(Ndel) time, where Ndel is the mean number of pigments over which the exciton is delocalized within the excited absorption band.     

Isolation and characterization of a minor legumin and its constituent polypeptides from Pisum sativum (pea).

The purification and characterization of a minor legumin species from Pisum sativum is described. Electrophoretic data indicate that it corresponds to a legumin subunit pair previously designated L1. The beta-polypeptides of the minor legumin have a phenylalanine N-terminus. This is the first time that an amino acid other than glycine has been reported as the N-terminus of the basic polypeptides from legumin-like proteins from any plant species. Sequence analyses of the isolated alpha- and beta-polypeptides of the minor legumin show that it does not correspond to any of the three legumin gene families that have previously been defined on the basis of DNA hybridizations and genetic analyses.     

Rhodospirillum rubrum possesses a variant of the bchP gene, encoding geranylgeranyl-bacteriopheophytin reductase

The bchP gene product of Rhodobacter sphaeroides is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in Rhodospirillum rubrum encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that Rhodospirillum rubrum is a naturally occurring bchP mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to Rhodospirillum rubrum, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of Rhodospirillum rubrum for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to bchP, other members of a photosynthesis gene cluster were identified in Rhodospirillum rubrum, including a bchG gene, demonstrated to encode a functional Bchl synthetase by complementation of a Rhodobacter sphaeroides mutant.