Unique Peripheral Antennas in the Photosystems of the Streptophyte Alga Mesostigma viride.

Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI-PSII megacomplex, PSII-LHCII, two PSI-LHCI-LHCIIs, PSI-LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI-LHCI-LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII-LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI-LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.

Effect of Detergents and Osmolytes on Thermal Stability of Native and Mutant Rhodobacter sphaeroides Reaction Centers.

Photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides is one of the most well-studied transmembrane pigment-protein complexes. It is a relatively stable protein with established conditions for its isolation from membranes, purification, and storage. However, it has been shown that some amino acid substitutions can affect stability of the RC, which results in a decrease of the RCs yield during its isolation and purification, disturbs spectral properties of the RCs during storage, and can lead to sample heterogeneity. To optimize conditions for studying mutant RCs, the effect of various detergents and osmolytes on thermal stability of the complex was examined. It was shown that trehalose and, to a lesser extent, sucrose, maltose, and hydroxyectoin at 1 M concentration slow down thermal denaturation of RCs. Sodium cholate was found to have significant stabilizing effect on the structure of native and genetically modified RCs. The use of sodium cholate as a detergent has several advantages and can be recommended for the storage and investigation of the unstable mutant membrane complexes of purple bacteria in long-term experiments.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Expression and purification of affinity-tagged variants of the photochemical reaction center from Heliobacterium modesticaldum.

The heliobacterial photochemical reaction center (HbRC) from the chlorophototrophic Firmicutes bacterium Heliobacterium modesticaldum is the only homodimeric type I RC whose structure is known. Using genetic techniques recently established in our lab, we have developed a rapid heterologous expression system for the HbRC core polypeptide PshA. Our system relies on rescue of the non-chlorophototrophic ∆pshA::cbp2p-aph3 strain of Hbt. modesticaldum by expression of a heterologous pshA gene from a replicating shuttle vector. In addition, we constructed two tagged variants of PshA, one with an N-terminal octahistidine tag and one with an internal hexahistidine tag, which facilitate rapid purification of pure, active HbRC cores in milligram quantities. We constructed a suite of shuttle vectors bearing untagged or tagged versions of pshA driven by various promoters. Surprisingly, we found that the eno and gapDH_2 promoters from Clostridium thermocellum drive better expression of pshA than fragments of DNA derived from the region upstream of the pshA locus on the Hbt. modesticaldum genome. This "pshA rescue" strategy also provided a useful window into how Hbt. modesticaldum regulates pigment synthesis and growth rate when chlorophototrophic output decreases.

Use of new strains of Rhodobacter sphaeroides and a modified simple culture medium to increase yield and facilitate purification of the reaction centre.

A new gene expression system was developed in Rhodobacter sphaeroides, replacing a pRK415-based system used previously. The broad host-range IPTG-inducible plasmid pIND4 was used to create the plasmid pIND4-RC1 for expression of the puhA and pufQBALMX genes, encoding the reaction centre (RC) and light-harvesting complex 1 (LH1) proteins. The strain R. sphaeroides ΔRCLH was used to make a knockout of the rshI restriction endonuclease gene, enabling electroporation of DNA into the bacterium; a subsequent knockout of ppsR was made, creating the strain R. sphaeroides RCx lacking this oxygen-sensing repressor of the photosynthesis gene cluster. Using pIND4-RC1, LH1 levels were increased by a factor of about 8 over pRS1 per cell in cultures grown semi-aerobically. In addition, the ppsR knockout allowed for photosynthetic pigment-protein complex synthesis in the presence of high concentrations of molecular oxygen; here, LH1 levels per cell increased by 20 % when grown under high aeration conditions. A new medium (called RLB) is the E. coli medium LB supplemented with MgCl2 and CaCl2, which was found to increase growth rates and final cell culture densities, with an increase of 30 % of LH1 per cell detected in R. sphaeroides RCx(pIND4-RC1) grown in RLB versus LB medium. Furthermore, cell density was about three times greater in RLB compared to semi-aerobic conditions. The combination of all the modifications resulted in an increase of LH1 and RC per mL of culture volume by approximately 35-fold, and a decrease in the length of culture incubation time from about 5 days to ~36 h.

Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species.

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

Bimodal intramolecular excitation energy transfer in a multichromophore photosynthetic model system: hybrid fusion proteins comprising natural phycobilin- and artificial chlorophyll-binding domains.

The phycobilisomes of cyanobacteria and red-algae are highly efficient peripheral light-harvesting complexes that capture and transfer light energy in a cascade of excitation energy transfer steps through multiple phycobilin chromophores to the chlorophylls of core photosystems. In this work, we focus on the last step of this process by constructing simple functional analogs of natural phycobilisome-photosystem complexes that are based on bichromophoric protein complexes comprising a phycobilin- and a chlorophyll- or porphyrin-binding domain. The former is based on ApcE(1-240), the N-terminal chromophore-binding domain of the phycobilisome's L(CM) core-membrane linker, and the latter on HP7, a de novo designed four-helix bundle protein that was originally planned as a high-affinity heme-binding protein, analogous to b-type cytochromes. We fused a modified HP7 protein sequence to ApcEΔ, a water-soluble fragment of ApcE(1-240) obtained by excising a putative hydrophobic loop sequence of residues 77-153. HP7 was fused either to the N- or the C-terminus of ApcEΔ or inserted between residues 76 and 78, thereby replacing the native hydrophobic loop domain. We describe the assembly, spectral characteristics, and intramolecular excitation energy transfer of two unique systems: in the first, the short-wavelength absorbing zinc-mesoporphyrin is bound to the HP7 domain and serves as an excitation-energy donor to the long-wavelength absorbing phycocyanobilin bound to the ApcE domain; in the second, the short-wavelength absorbing phycoerythrobilin is bound to the ApcE domain and serves as an excitation energy donor to the long-wavelength absorbing zinc-bacteriochlorophyllide bound to the HP7 domain. All the systems that were constructed and tested exhibited significant intramolecular fluorescence resonance energy transfer with yields ranging from 21% to 50%. This confirms that our modular, covalent approach for studying EET between the cyclic and open chain tetrapyrroles is reasonable, and may be extended to larger structures mimicking light-harvesting in cyanobacteria. The design, construction, and characterization process demonstrated many of the advances in constructing such model systems, particularly in our ability to control the fold and aggregation state of protein-based systems. At the same time, it underlines the potential of exploiting the versatility and flexibility of protein-based systems in assembling multiple pigments into effective light-harvesting arrays and tuning the spectral properties of multichromophore systems.

Expression, purification, crystallization and preliminary X-ray structure analysis of wild-type and L(M196)H-mutant Rhodobacter sphaeroides reaction centres.

The electron and proton transport mediated by protein-bound cofactors in photosynthesis have been investigated by various methods in order to determine the energetics, the dynamics and the pathway of this process. In purple bacteria, primary photosynthetic charge separation and the build-up of a proton gradient across the periplasmic membrane are catalyzed by the photosynthetic reaction centre (RC). Here, the purification, crystallization and preliminary X-ray analysis of wild-type and L(M196)H-mutant RCs of Rhodobacter sphaeroides are presented, enabling study of the influence of the protein environment of the primary electron donor on the spectral properties and photochemical activity of the RC.

Purification of the photosynthetic reaction center from Heliobacterium modesticaldum.

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC-MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756-6764, 2006; Romberger et al., Photosynth Res 104:293-303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c (553) and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g', two 8(1)-OH-Chl a (F), and one 4,4'-diaponeurosporene. It lacks the PshB polypeptide binding the F(A) and F(B) [4Fe-4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P(800) (+)F(X)(-) state is 10-15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K (M) of 10 µM and a k (cat) of 9.5 s(-1) under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.

Oligomeric characterization of the photosynthetic apparatus of Rhodobacter sphaeroides R26.1 by nondenaturing electrophoresis methods.

Blue and colorless native gel electrophoresis in combination with LC-ESI-MS/MS are powerful tools in the analysis of protein networks in biological membranes. We used these techniques in the present study to generate a comprehensive overview on a proteome-wide scale of intracytoplasmic membrane (ICM) associated proteins in order to investigate the native supramolecular organization of Rhodobacter sphaeroides R26.1 photosynthetic apparatus. The results obtained were compared with past proteomic data, as well as with models for the topology of photosynthetic membranes as derived from previously published atomic force microscopy studies. We identified 52 proteins organized in 10 different multiprotein complexes. We were able to demonstrate the existence of different oligomeric states for the integral membrane pigment-protein complexes dedicated to bacterial photosynthesis. Specifically, we found dimers and trimers, as well as supercomplexes of light-harvesting (LH) 2 at very high molecular weights (around 10,000 kDa). We recovered the monomeric form of the photochemical reaction center (RC), as well as the monomer and dimer of the reaction center-light harvesting 1-PufX (RC-LH1-PufX) complex. Curiously, no type of LH1 complex was detected. Lastly, ATP synthase and cytochrome bc(1) complexes were only recovered in their monomeric states. Purified ICM vesicles were shown to be rich in newly discovered gene products, including three proteins with unknown functions (RSP_2125, RSP_3238, RSP_6207), a possible alkane hydroxylase and a spheroidene monooxygenase. Other multiprotein complexes were found to be localized in the ICM, including succinate dehydrogenase in trimeric form and sarcosine oxidase in two different aggregation states. These findings contribute to the growing body of evidence that the bacterial ICM is a specialized bioenergetic membrane hosting, not only photosynthesis, but many other critical activities.

Unifying principles in homodimeric type I photosynthetic reaction centers: properties of PscB and the FA, FB and FX iron-sulfur clusters in green sulfur bacteria.

The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from FA- and FB- and were unable to reduce NADP+. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the FA and FB clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. Mössbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe-4S]2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe-2S]2+,1+ clusters and rubredoxin-like Fe3+,2+ centers, and that the [4Fe-4S]2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g=5.4 and 4.4 assigned to a S=3/2 ground spin state [4Fe-4S]1+ cluster and at g=1.77 assigned to a S=1/2 ground spin state [4Fe-4S]1+ cluster, both from FX-. These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the FA and FB iron-sulfur clusters are present on a salt-dissociable subunit, and FX is present as an interpolypeptide [4Fe-4S]2+,1+ cluster with a significant population in a S=3/2 ground spin state.

Two-dimensional crystals of photosystem II: biochemical characterization, cryoelectron microscopy and localization of the D1 and cytochrome b559 polypeptides.

Photosystem II (PS II) is a photosynthetic reaction center found in higher plants which has the unique ability to evolve oxygen from water. Several groups have formed two-dimensional PS II crystals or have isolated PS II complexes and studied them by electron microscopy and image analysis. The majority of these specimens have not been well characterized biochemically and have yielded relatively low resolution two-dimensional projection maps with a variety of unit cell sizes. We report the characterization of the polypeptide and lipid content of tubular crystals of PS II. The crystals contain the reaction center core polypeptides D1, D2, cytochrome b559, as well as the chlorophyll-binding polypeptides (CP) CP47, CP43, CP29, CP26, CP24, and CP22. The lipid composition was similar to the lipids found in the stacked portion of thylakoids. We also report a 2.0-nm resolution projection map determined by electron microscopy and image analysis of frozen, hydrated PS II crystals. This projection map includes information on the portion of the complex buried in the lipid bilayer. The unit cell is a dimer with unit vectors of 17.0 and 11.4 nm separated by an angle of 106.6 degrees. In addition, Fab fragments against D1 and cytochrome b559 were used to localize those two polypeptides, and thus the reaction center, within the PS II complex. The results indicate that D1 and cytochrome b559 are found within one of the heaviest densities of the monomeric unit.

Internal electrostatic control of the primary charge separation and recombination in reaction centers from Rhodobacter sphaeroides revealed by femtosecond transient absorption.

We report the observation of two conformational states of closed RCs from Rhodobacter sphaeroides characterized by different P(+)H(A)(-) --> PH(A) charge recombination lifetimes, one of which is of subnanosecond value (700 +/- 200 ps). These states are also characterized by different primary charge separation lifetimes. It is proposed that the distinct conformations are related to two protonation states either of reduced secondary electron acceptor, Q(A)(-), or of a titratable amino acid residue localized near Q(A). The reaction centers in the protonated state are characterized by faster charge separation and slower charge recombination when compared to those in the unprotonated state. Both effects are explained in terms of the model assuming modulation of the free energy level of the state P(+)H(A)(-) by the charges on or near Q(A) and decay of the P(+)H(A)(-) state via the thermally activated P(+)B(A)(-) state.

Purification and proteomic characterization of plastids from Brassica napus developing embryos.

Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy.

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P(Zn)), the accessory Zn-BChl a (B(Zn)), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P(Zn)(*), measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B(Zn)(*) to P(Zn), and the latter, the electron transfer from P(Zn) to H. The angle between the transition dipoles of B(Zn) and P(Zn) was estimated to be 36 degrees based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P(Zn).

Isolation and characterization of oxygen-evolving thylakoid membranes and photosystem II particles from a marine diatom Chaetoceros gracilis.

Thylakoid membranes retaining high oxygen-evolving activity (about 250 micromol O(2)/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 micromol O(2)/mg Chl/h in the absence and presence of CaCl(2), respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.

Expression, purification and characterization of a high potential iron-sulfur protein from Acidithiobacillus ferrooxidans.

The high potential iron-sulfur protein (HiPIP) is involved in the iron respiratory electron transport chain of Acidithiobacillus ferrooxidans but its exact role is unclear. The gene of HiPIP from A. ferrooxidans ATCC 23270 was cloned and expressed in Escherichia coli, and the protein then purified by one-step affinity chromatography to homogeneity. The molecular mass of the HiPIP monomer was 7250.43 Da by MALDI-TOF MS, indicating the presence of the [Fe(4)S(4)] cluster. The optical and EPR spectra results of the recombinant protein confirmed that the iron-sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys25, Cys28, Cys37 and Cys50 were involved in ligating to the iron-sulfur cluster.

Substitution of isoleucine L177 by histidine in Rhodobacter sphaeroides reaction center results in the covalent binding of PA bacteriochlorophyll to the L subunit.

In this work, we report the unique case of bacteriochlorophyll (BChl) - protein covalent attachment in a photosynthetic membrane complex caused by a single mutation. The isoleucine L177 was substituted by histidine in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Pigment analysis revealed that one BChl molecule was missing in the acetone-methanol extract of the I(L177)H RCs. SDS-PAGE demonstrated that this BChl molecule could not be extracted with organic solvents apparently because of its stable covalent attachment to the mutant RC L-subunit. Our data indicate that the attached bacteriochlorophyll is one of the special pair BChls, P(A). The chemical nature of this covalent interaction remains to be identified.

Molecular assembly of artificial photosynthetic antenna core complex on an amino-terminated ITO electrode.

Bacterial photosynthetic membrane proteins, light-harvesting antenna complex (LH1), reaction center (RC), and their combined 'core' complex (LH1-RC) are functional elements in the primary photosynthetic events, i.e., capturing and transferring light energy and subsequent charge separation. These photosynthetic units (PSUs) isolated from Rhodospirillum rubrum (Rs. rubrum) were assembled onto an ITO electrode modified with 3-aminopropyltriethoxysilane (APS-ITO). The near IR absorption spectra of PSUs on the assembled electrodes were identical to those of solutions, indicating that the LH1 and LH1-RC core complexes were native on the electrode. Photocurrent response of PSUs on the electrode was examined upon illumination of the LH1 complex at 880 nm. The LH1-RC and a mixed assembly of LH1 and RC exhibited photocurrent response, but not LH1 only, consistent with the function of these PSUs, capturing light energy and transferring electron. This result provides useful methodology for building an artificial fabrication of PSUs on the electrode.

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.