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Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, are a resource for photosynthetic, photonic and fluorescence labeling elements. They cover an exceptionally broad spectral range, but the complex superstructure and assembly have been an obstacle. By replacing in Synechocystis sp. PCC 6803 the biliverdin reductases, we studied the role of chromophores in the assembly of the phycobilisome core. Introduction of the green-absorbing phycoerythrobilin instead of the red-absorbing phycocyanobilin inhibited aggregation. A novel, trimeric allophycocyanin (Dic-APC) was obtained. In the small (110 kDa) unit, the two chromophores, phycoerythrobilin and phytochromobilin, cover a wide spectral range (550 to 660 nm). Due to efficient energy transfer, it provides an efficient artificial light-harvesting element. Dic-APC was generated in vitro by using the contained core-linker, L

Robert John Porra (7.8.1931–16.5.2019) is probably best known for his substantial practical contributions to plant physiology and photosynthesis by addressing the problems of both the accurate spectroscopic estimation and the extractability of chlorophylls in many organisms. Physiological data and global productivity estimates, in particular of marine primary productivity, are often quoted on a chlorophyll basis. He also made his impact by work on all stages of tetrapyrrole biosynthesis: he proved the C5 pathway to chlorophylls, detected an alternative route to protoporphyrin in anaerobes and the different origin of the oxygen atoms in anaerobes and aerobes. A brief review of his work is supplemented by personal memories of the authors.

Constantin A. (Tino) Rebeiz, a pioneer in the field of chlorophyll biosynthesis, and a longtime member of the University of Illinois community of plant biologists, passed away on July 25, 2019. He came to the USA at a time that was difficult for members of minority groups to be in academia. However, his passion for the complexity of the biochemical origin of chlorophylls drove a career in basic sciences which extended into applied areas of environmentally friendly pesticides and treatment for skin cancer. He was a philanthropist; in retirement, he founded the Rebeiz Foundation for Basic Research which recognized excellence and lifetime achievements of selected top scientists in the general area of photosynthesis research. His life history, scientific breakthroughs, and community service hold important lessons for the field.

Chlorophylls provide the basis for photosynthesis and thereby most life on Earth. Besides their involvement in primary charge separation in the reaction center, they serve as light-harvesting and light-sensing pigments, they also have additional functions, e.g., in inter-system electron transfer. Chlorophylls also have a wealth of applications in basic science, medicine, as colorants and, possibly, in optoelectronics. Considering that there has been more than 200 years of chlorophyll research, one would think that all has been said on these pigments. However, the opposite is true: ongoing research evidenced in this Special Issue brings together current work on chlorophylls and on their carotenoid counterparts. These introductory notes give a very brief and in part personal account of the history of chlorophyll research and applications, before concluding with a snapshot of this year’s publications.

Phycobilisomes are large light-harvesting complexes attached to the stromal side of thylakoids in cyanobacteria and red algae. They can be remodeled or degraded in response to changing light and nutritional status. Both the core and the peripheral rods of phycobilisomes contain biliproteins. During biliprotein biosynthesis, open-chain tetrapyrrole chromophores are attached covalently to the apoproteins by dedicated lyases. Another set of non-bleaching (Nb) proteins has been implicated in phycobilisome degradation, among them NblA and NblB. We report in vitro experiments with lyases, biliproteins and NblA/B which imply that the situation is more complex than currently discussed: lyases can also detach the chromophores and NblA and NblB can modulate lyase-catalyzed binding and detachment of chromophores in a complex fashion. We show: (i) NblA and NblB can interfere with chromophorylation as well as chromophore detachment of phycobiliprotein, they are generally inhibitors but in some cases enhance the reaction; (ii) NblA and NblB promote dissociation of whole phycobilisomes, cores and, in particular, allophycocyanin trimers; (iii) while NblA and NblB do not interact with each other, both interact with lyases, apo- and holo-biliproteins; (iv) they promote synergistically the lyase-catalyzed chromophorylation of the β-subunit of the major rod component, CPC; and (v) they modulate lyase-catalyzed and lyase-independent chromophore transfers among biliproteins, with the core protein, ApcF, the rod protein, CpcA, and sensory biliproteins (phytochromes, cyanobacteriochromes) acting as potential traps. The results indicate that NblA/B can cooperate with lyases in remodeling the phycobilisomes to balance the metabolic requirements of acclimating their light-harvesting capacity without straining the overall metabolic economy of the cell.

Robert John Porra (7.8.1931-16.5.2019) is probably best known for his substantial practical contributions to plant physiology and photosynthesis by addressing the problems of both the accurate spectroscopic estimation and the extractability of chlorophylls in many organisms. Physiological data and global productivity estimates, in particular of marine primary productivity, are often quoted on a chlorophyll basis. He also made his impact by work on all stages of tetrapyrrole biosynthesis: he proved the C

Cyanobacterial phycobilisomes funnel the harvested light energy to the reaction centers via two terminal emitters, allophycocyanin B and the core-membrane linker. ApcD is the α-subunit of allophycocyanin B responsible for its red-shifted absorbance (λmax 665 nm). Far-red photo-acclimated cyanobacteria contain certain allophycocyanins that show even further red-shifted absorbances (λmax > 700 nm). We studied the chromophorylation of the three far-red induced ApcD subunits ApcD2, ApcD3 and ApcD4 from Chroococcidiopsis thermalis sp. PCC7203 during the expression in E. coli. The complex behavior emphasizes that a variety of factors contribute to the spectral red-shift. Only ApcD2 bound phycocyanobilin covalently at the canonical position C81, while ApcD3 and ApcD4 gave only traces of stable products. The product of ApcD2 was, however, heterogeneous. The major fraction had a broad absorption around 560 nm and double-peaked fluorescence at 615 and 670 nm. A minor fraction was similar to the product of conventional ApcD, with maximal absorbance around 610 nm and fluorescence around 640 nm. The heterogeneity was lost in C65 and C132 variants; in these variants only the conventional product was formed. With ApcD4, a red-shifted product carrying non-covalently bound phycocyanobilin could be detected in the supernatant after cell lysis. While this chromophore was lost during purification, it could be stabilized by co-assembly with a far-red light-induced β-subunit, ApcB3.

Cyanobacterial light-harvesting complexes, phycobilisomes, can undergo extensive remodeling under varying light conditions. Acclimation to far-red light involves not only generation of red-shifted chlorophylls in the photosystems, but also induction of additional copies of core biliproteins that have been related to red-shifted components of the phycobilisome (Gan et al., Life 5, 4, 2015). We are studying the molecular basis for these acclimations in Chroococcidiopsis thermalis sp. PCC7203. Five far-red induced allophycocyanin subunits (ApcA2, ApcA3, ApcB2, ApcB3 and ApcF2) were expressed in Escherichia coli, together with S-type chromophore-protein lyases and in situ generated chromophore, phycocyanobilin. Only one subunit, ApcF2, shows an unusual red-shift (λAmax~675nm, λFmax~698nm): it binds the chromophore non-covalently, thereby preserving its full conjugation length. This mechanism operates also in two Cys-variants of the induced subunits of bulky APC. All other wild-type subunits bind phycocyanobilin covalently to the conventional Cys-81 under catalysis of the lyase, CpcS1. Although three of them also show binding to additional cysteines, all absorb and fluoresce similar to conventional APC subunits (λAmax~610nm, λFmax~640nm). Another origin of red-shifted complexes was identified, however, when different wild-type α- and β-subunits of the far-red induced bulky APC were combined in a combinatorial fashion. Strongly red-shifted complexes (λFmax≤722nm) were formed when the α-subunit, PCB-ApcA2, and the β-subunit, PCB-ApcB2, were generated together in E. coli. This extreme aggregation-induced red-shift of ~90nm of covalently bound chromophores is reminiscent, but much larger, than the ~30nm observed with conventional APC.

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.

The genome of the cyanobacterium Nostoc sp. PCC 7120 encodes a large number of putative bacteriophytochrome and cyanobacteriochrome photoreceptors that, due to their long-wavelength absorption and fluorescence emission, might serve as fluorescent tags in intracellular investigations. We show that the PAS-GAF domain of the bacteriophytochrome, AphB, binds biliverdin covalently and exhibits, besides its reversible photochemistry, a moderate fluorescence in the near infrared (NIR) spectral region. It was selected for further increasing the brightness while retaining the NIR fluorescence. In the first step, amino acids assumed to improve fluorescence were selectively mutated. The resulting variants were then subjected to several rounds of random mutagenesis and screened for enhanced fluorescence in the NIR. The brightness of optimized PAS-GAF variants increased more than threefold compared to that of wt AphB(1-321), with only insignificant spectral shifts (Amax around 695 nm, and Fmax around 720 nm). In general, the brightness increases with decreasing wavelengths, which allows for a selection of the fluorophore depending on the optical properties of the tissue. A spectral heterogeneity was observed when residue His260, located in close proximity to the chromophore, was mutated to Tyr, emphasizing the strong effects of the environment on the electronic properties of the bound biliverdin chromophore.

Biliproteins have extended the spectral range of fluorescent proteins into the near-infrared region (NIR, 700–770 nm) of maximal transmission of most tissues and are also favorable for multiplex labeling. Their application, however, presents considerable challenges to increase their stability under physiological conditions and, in particular, to increase their brightness while maintaining the emission in near-infrared regions: their fluorescence yield generally decreases with increasing wavelengths, and their effective brightness depends strongly on the environmental conditions. We report a fluorescent biliprotein triad, termed BDFP1.1:3.1:1.1, that combines a large red-shift (722 nm) with high brightness in mammalian cells and high stability under changing environmental conditions. It is fused from derivatives of the phycobilisome core subunits, ApcE2 and ApcF2. These two subunits are induced by far-red light (FR, 650–700 nm) in FR acclimated cyanobacteria. Two BDFP1.1 domains engineered from ApcF2 covalently bind biliverdin that is accessible in most cells. The soluble BDFP3 domain, engineered from ApcE2, binds phytochromobilin non-covalently, generating BDFP3.1. This phytochromobilin chromophore was added externally; it is readily generated by an improved synthesis in E. coli and subsequent extraction. Excitation energy absorbed in the FR by covalently bound biliverdins in the two BDFP1.1 domains is transferred via fluorescence resonance energy transfer to the non-covalently bound phytochromobilin in the BDFP3.1 domain fluorescing in the NIR around 720 nm. Labeling of a variety of proteins by fusion to the biliprotein triad is demonstrated in prokaryotic and mammalian cells, including human cell lines.

A recent publication (Esteban in New Phytol 217:341–342, 2018) describes how the use and citation of the assay of chlorophylls a and b extracted in aqueous 80% acetone by Arnon (Plant Physiol 2:1–15, 1949) is increasing, even in journals with high impact factors. This is a very disconcerting situation: the assay is outdated because it relies on the seriously under-estimated extinction coefficients of Mackinney (J Biol Chem 140:315–322, 1941), and the assay of chlorophylls is one of the most important, and much reported, procedures in studies of photosynthesis and related plant biological fields. Using the assay has led to the accumulation of masses of inaccurate data and confusion during the resolution of some plant biological problems. A summary not only of an accurate assay of chlorophylls in aqueous 80% acetone but also of a long-known method to correct the data obtained by Arnon’s procedure (cf. Porra et al. in Biochim Biophys Acta 975:384–394, 1989) is briefly described below together with references to reliable assays in this and other solvents by other authors.

Far-red and near-infrared emitting chromophores extend applications of fluorescent proteins to regions of maximal transmission of most tissues, but present considerable engineering challenges. Far-red adapting cyanobacteria generate a novel set of biliproteins. One of them, ApcF2, from a thermophilic cyanobacterium was subjected to structure-guided, site-directed random and specific mutagenesis, and was screened for bright far-red emission. We report the generation of chromoproteins, termed BDFPs, that are small, bind auto-catalytically the ubiquitous biliverdin as chromophore, express well, and retain their fluorescence in mammalian cells and in the nematode, C. elegans. They are, moreover, photostable and tolerate high temperature, low pH and chemical denaturation. Homo-bichromophoric tandems of these proteins improve labeling, while hetero-bichromophoric systems with large Stokes shifts are suitable for applications like FRET, multi-channel or super-resolution microscopy. The BDFPs compare favorably to other biliproteins and provide a novel, extremely versatile labeling tool-box.

Photosynthesis relies on energy transfer from light-harvesting complexes to reaction centers. Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, attach to the membrane via the multidomain core-membrane linker, L(CM). The chromophore domain of L(CM) forms a bottleneck for funneling the harvested energy either productively to reaction centers or, in case of light overload, to quenchers like orange carotenoid protein (OCP) that prevent photodamage. The crystal structure of the solubly modified chromophore domain from Nostoc sp. PCC7120 was resolved at 2.2 Å. Although its protein fold is similar to the protein folds of phycobiliproteins, the phycocyanobilin (PCB) chromophore adopts ZZZssa geometry, which is unknown among phycobiliproteins but characteristic for sensory photoreceptors (phytochromes and cyanobacteriochromes). However, chromophore photoisomerization is inhibited in L(CM) by tight packing. The ZZZssa geometry of the chromophore and π-π stacking with a neighboring Trp account for the functionally relevant extreme spectral red shift of L(CM). Exciton coupling is excluded by the large distance between two PCBs in a homodimer and by preservation of the spectral features in monomers. The structure also indicates a distinct flexibility that could be involved in quenching. The conclusions from the crystal structure are supported by femtosecond transient absorption spectra in solution.

Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.

The brightly fluorescent phycobiliproteins are light-harvesting pigments in cyanobacteria and certain algae, covering almost the entire visible spectrum. Another type of biliproteins, the phytochrome family, comprises regulatory photoswitches in plants, fungi and many bacteria; their spectra cover an even wider range extending into the near-infrared and near-ultraviolet. In both types, the chromophores can be tuned by modulating chromophore-protein interactions, including the transition between fluorescence and photoswitching. These properties render biliproteins useful for bioimaging. Applications require not only the introduction of genes coding for apoproteins, but also genes coding for biosynthesis of the chromophores. Strategies for tuning and heterologous biosynthesis will be discussed.

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

Cyanobacteriochromes are a structurally and spectrally highly diverse class of phytochrome-related photosensory biliproteins. They contain one or more GAF domains that bind phycocyanobilin (PCB) autocatalytically; some of these proteins are also capable of further modifying PCB to phycoviolobilin or rubins. We tested the chromophorylation with the non-photochromic phycoerythrobilin (PEB) of 16 cyanobacteriochrome GAFs from Nostoc sp. PCC 7120, of Slr1393 from Synechocystis sp. PCC 6803, and of Tlr0911 from Thermosynechococcus elongatus BP-1. Nine GAFs could be autocatalytically chromophorylated in vivo/in E. coli with PEB, resulting in highly fluorescent biliproteins with brightness comparable to that of fluorescent proteins like GFP. In several GAFs, PEB was concomitantly converted to phycourobilin (PUB) during binding. This not only shifted the spectra, but also increased the Stokes shift. The chromophorylated GAFs could be oligomerized further by attaching a GCN4 leucine zipper domain, thereby enhancing the absorbance and fluorescence of the complexes. The presence of both PEB and PUB makes these oligomeric GAF-"bundles" interesting models for energy transfer akin to the antenna complexes found in cyanobacterial phycobilisomes. The thermal and photochemical stability and their strong brightness make these constructs promising orange fluorescent biomarkers.

Phycobiliproteins are brightly-fluorescent light-harvesting pigments for photosynthesis in cyanobacteria and red algae. They are also of interest as fluorescent biomarkers, but their heterologous generation in vivo has previously required multiple transformations. We report here a modular approach that requires only two DNA segments. The first codes for the apo-protein. The second codes for fusions capable of chromophore biosynthesis and its covalent attachment to the apo-protein; it contains the genes of heme oxygenase, a bilin reductase, and a chromophore lyase. Phycobiliproteins containing phycoerythrobilin (λ(fluor) ~ 560 nm), phycourobilin (λ(fluor) ~ 500 nm), phycocyanobilin (λ(fluor) ~ 630 nm) or phycoviolobilin (λ(fluor) ~ 580 nm) were obtained in high yield in E. coli. This approach facilitates chromophorylation studies of phycobiliproteins, as well as their use for fluorescence labeling based on their high fluorescence.

Proceedings of the National Academy of Sciences of the United States of America 114(50), 13170 - 13175 (2017). doi:10.1073/pnas.1715495114, The light-harvesting phycobilisome in cyanobacteria and red algae requires the lyase-catalyzed chromophorylation of phycobiliproteins. There are three functionally distinct lyase families known. The heterodimeric E/F type is specific for attaching bilins covalently to α-subunits of phycocyanins and phycoerythrins. Unlike other lyases, the lyase also has chromophore-detaching activity. A subclass of the E/F-type lyases is, furthermore, capable of chemically modifying the chromophore. Although these enzymes were characterized >25 y ago, their structures remained unknown. We determined the crystal structure of the heterodimer of CpcE/F from Nostoc sp. PCC7120 at 1.89-Å resolution. Both subunits are twisted, crescent-shaped α-solenoid structures. CpcE has 15 and CpcF 10 helices. The inner (concave) layer of CpcE (helices h2, 4, 6, 8, 10, 12, and 14) and the outer (convex) layer of CpcF (h16, 18, 20, 22, and 24) form a cavity into which the phycocyanobilin chromophore can be modeled. This location of the chromophore is supported by mutations at the interface between the subunits and within the cavity. The structure of a structurally related, isomerizing lyase, PecE/F, that converts phycocyanobilin into phycoviolobilin, was modeled using the CpcE/F structure as template. A H$_{87}$C$_{88}$ motif critical for the isomerase activity of PecE/F is located at the loop between h20 and h21, supporting the proposal that the nucleophilic addition of Cys-88 to C10 of phycocyanobilin induces the isomerization of phycocyanobilin into phycoviolobilin. Also, the structure of NblB, involved in phycobilisome degradation could be modeled using CpcE as template. Combined with CpcF, NblB shows a low chromophore-detaching activity., Published by National Acad. of Sciences, Washington, DC

Phytochromobilin (P Phi B), the chromophore of plant phytochromes, is difficult to isolate because phytochromes occur at very low concentrations in plants. It is, therefore, frequently replaced in plant phytochrome studies by phycocyanobilin, which is abundant in cyanobacteria. P Phi B is also an attractive chromophore for far-red emitting chromoproteins. In this work, we design and optimize a simple method to efficiently isolate useful quantities of P Phi B: The chromophore is generated in Escherichiacoli and transiently bound to a tailored chromophore-binding domain of ApcE2, the apo-protein of a core-membrane linker, from which it can subsequently be released. The ease and effectiveness of this method hinges not only on the enhanced biosynthesis of P Phi B in the presence of the ApcE2 construct from Synechococcus sp. PCC7335, but also on the noncovalent binding of the pigment to its apo-protein. The isolated P Phi B was successfully incorporated into phytochrome-related assemblies, and furthermore, the noncovalently bound P Phi B could be transferred directly from the ApcE2 construct to the apo-proteins of phytochromes, cyanobacteriochromes and phycobiliproteins, without loss of relevant biological activity.

Cyanobacteriochromes are photochromic sensory photoreceptors in cyanobacteria that are related to phytochromes but cover a much broader spectral range. Using a homology search, a group of putative blue-absorbing photoreceptors was identified in Nostoc sp. PCC 7120 that, in addition to the canonical chromophore-binding cysteine of cyanobacteriochromes, have a conserved extra cysteine in a DXCF motif. To assess their photochemical activities, putative chromophore-binding GAF domains were expressed in Escherichia coli together with the genes for phycocyanobilin biosynthesis. All except one covalently bound a chromophore and showed photoreversible photochromic responses, with absorption at approximately 420 nm for the 15Z states formed in the dark, and a variety of red-shifted absorption peaks in the 490-600 nm range for the 15E states formed after light activation. Under denaturing conditions, the covalently bound chromophores were identified as phycocyanobilin, phycoviolobilin or mixtures of both. The canonical cysteines and those of the DXCF motifs were mutated, singly or together. The canonical cysteine is responsible for stable covalent attachment of the bilin to the apo-protein at C3(1) . The second linkage from the cysteine in the DXCF motif, probably to C10 of the chromophore, yields blue-absorbing rubin-type 15Z chromophores, but is lost in most cases upon photoconversion to the 15E isomers of the chromophores, and also when denatured with acidic urea.

We study the fluorescence properties of peridinin-chlorophyll-protein light-harvesting complex reconstituted with both chlorophyll a and chlorophyll b coupled to spherical gold nanoparticles. Since there is a bidirectional energy transfer between the chlorophylls, the impact of plasmon excitations upon the energy transfer dynamics can be studied in such a hybrid nanostructure. The results show that the emission of both chlorophyll a and chlorophyll b in a reconstituted PCP is strongly enhanced when the light-harvesting complexes are separated from the metallic nanoparticles by a 12-nm-thick SiO2 layer, the average enhancement reaches a factor of 8. The analysis of fluorescence decay curves indicates that the energy transfer between chlorophyll a and chlorophyll b is also influenced by plasmon excitations in gold nanoparticles.

Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli , the fused phycobiliproteins absorbed light in the range of 610–660 nm, and fluoresced at ~ 670 nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540–575 nm, and fluorescing at ~ 585 nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.

Cyanobacteriochromes are phytochrome homologues in cyanobacteria that act as sensory photoreceptors. We compare two cyanobacteriochromes, RGS (coded by slr1393) from Synechocystis sp. PCC 6803 and AphC (coded by all2699) from Nostoc sp. PCC 7120. Both contain three GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA protein) domains (GAF1, GAF2 and GAF3). The respective full-length, truncated and cysteine point-mutated genes were expressed in Escherichia coli together with genes for chromophore biosynthesis. The resulting chromoproteins were analyzed by UV-visible absorption, fluorescence and circular dichroism spectroscopy as well as by mass spectrometry. RGS shows a red-green photochromism (λ(max) = 650 and 535 nm) that is assigned to the reversible 15Z/E isomerization of a single phycocyanobilin-chromophore (PCB) binding to Cys528 of GAF3. Of the three GAF domains, only GAF3 binds a chromophore and the binding is autocatalytic. RGS autophosphorylates in vitro; this reaction is photoregulated: the 535 nm state containing E-PCB was more active than the 650 nm state containing Z-PCB. AphC from Nostoc could be chromophorylated at two GAF domains, namely GAF1 and GAF3. PCB-GAF1 is photochromic, with the proposed 15E state (λ(max) = 685 nm) reverting slowly thermally to the thermostable 15Z state (λ(max) = 635 nm). PCB-GAF3 showed a novel red-orange photochromism; the unstable state (putative 15E, λ(max) = 595 nm) reverts very rapidly (τ ~ 20 s) back to the thermostable Z state (λ(max) = 645 nm). The photochemistry of doubly chromophorylated AphC is accordingly complex, as is the autophosphorylation: E-GAF1/E-GAF3 shows the highest rate of autophosphorylation activity, while E-GAF1/Z-GAF3 has intermediate activity, and Z-GAF1/Z-GAF3 is the least active state.

The phycobilin:cysteine 84-phycobiliprotein lyase, CpcS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin (PEB) attachment at nearly all cysteine 82 binding sites (consensus numbering) of phycoerythrin, phycoerythrocyanin, phycocyanin, and allophycocyanin (Zhao, K. H., Su, P., Tu, J. M., Wang, X., Liu, H., Ploscher, M., Eichacker, L., Yang, B., Zhou, M., and Scheer, H. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14300–14305). We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo- and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (

International audience; Most cyanobacteria harvest light with large antenna complexes called phycobilisomes. The diversity of their constituting phycobiliproteins contributes to optimize the photosynthetic capacity of these microorganisms. Phycobiliprotein biosynthesis, which involves several post-translational modifications including covalent attachment of the linear tetrapyrrole chromophores (phycobilins) to apoproteins, begins to be well understood. However, the biosynthetic pathway to the blue-green-absorbing phycourobilin (lambda(max) approximately 495 nm) remained unknown, although it is the major phycobilin of cyanobacteria living in oceanic areas where blue light penetrates deeply into the water column. We describe a unique trichromatic phycocyanin, R-PC V, extracted from phycobilisomes of Synechococcus sp. strain WH8102. It is evolutionarily remarkable as the only chromoprotein known so far that absorbs the whole wavelength range between 450 and 650 nm. R-PC V carries a phycourobilin chromophore on its alpha-subunit, and this can be considered an extreme case of adaptation to blue-green light. We also discovered the enzyme, RpcG, responsible for its biosynthesis. This monomeric enzyme catalyzes binding of the green-absorbing phycoerythrobilin at cysteine 84 with concomitant isomerization to phycourobilin. This reaction is analogous to formation of the orange-absorbing phycoviolobilin from the red-absorbing phycocyanobilin that is catalyzed by the lyase-isomerase PecE/F in some freshwater cyanobacteria. The fusion protein, RpcG, and the heterodimeric PecE/F are mutually interchangeable in a heterologous expression system in Escherichia coli. The novel R-PC V likely optimizes rod-core energy transfer in phycobilisomes and thereby adaptation of a major phytoplankton group to the blue-green light prevailing in oceanic waters.

Stephan Wormke, Sebastian Mackowski, Hugo Scheer, Christoph Brauchle Wesentlicher Grund fur den evolutionaren Erfolg der Photosynthese ist die funktionale Trennung der Primarreaktionen in Lichtsammelkomplexe und Reaktionszentren. Mit Silbernanopartikeln gekoppelte Komplexe steigern die Effizienz der Lichtsammlung um mehr als eine Grosenordnung.

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL2 and a heterotetrameric protein complex (ChlNB)2. The ChlL2 dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)2 complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)2. For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A–C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.

— Resonance-enhanced coherent anti-Stokes Raman spectra (CARS) are reported for monomers and for trimers with and without linker proteins of allophycocyanin isolated from Mastigocladus laminosus. The CARS spectrum of the monomer is independent of the presence of linker proteins and is very similar to that of phycocyanin monomers indicating that the equivalent chromophores exhibit like structures in both biliproteins. Large differences are, however, observed between the spectra of phycocyanin trimers and those of allophycocyanin trimers with or without linker proteins (Lc8,9). The observed differences between monomer and trimer spectra are consistent with a change of the α-chromophore-protein arrangement upon aggregation without linker. If linker proteins are present in the trimer, then additional geometry changes of the β-chromophores are induced; these could relate to a transition from the 15Z-anti to 15Z-syn conformation.

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (alphabeta)3 resulted first in monomerization (alphabeta), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore-protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.

Ensemble and single-molecule spectroscopy demonstrates that both emission and absorption of peridinin-chlorophyll-protein photosynthetic antennae can be largely enhanced through plasmonic interactions. We find up to 18-fold increase of the chlorophyll fluorescence for complexes placed near a silver metal layer. This enhancement, which leaves no measurable effects on the protein structure, is observed when exciting either chlorophyll or carotenoid and is attributed predominantly to an increase of the excitation rate in the antenna. The enhancement mechanism comes from plasmon-induced amplification of electromagnetic fields inside the complex. This result is an important step toward applying plasmonic nanostructures for controlling the optical response of complex biomolecules and improving the design and functioning of artificial light-harvesting systems.

Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these "half-mers", homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.

We combine ensemble and single-molecule spectroscopy to gain insight into the energy transfer between chlorophylls (Chls) in peridinin-chlorophyll-protein (PCP) complexes reconstituted with Chl a, Chl b, as well as both Chl a and Chl b. The main focus is the heterochlorophyllous system (Chl a/b-N-PCP), and reference information essential to interpret experimental observations is obtained from homochlorophyllous complexes. Energy transfer between Chls in Chl a/b-N-PCP takes place from Chl b to Chl a and also from Chl a to Chl b with comparable Förster energy transfer rates of 0.0324 and 0.0215ps−1, respectively. Monte Carlo simulations yield the ratio of 39:61 for the excitation distribution between Chl a and Chl b, which is larger than the equilibrium distribution of 34:66. An average Chl a/Chl b fluorescence intensity ratio of 66:34 is measured, however, for single Chl a/b-N-PCP complexes excited into the peridinin (Per) absorption. This difference is attributed to almost three times more efficient energy transfer from Per to Chl a than to Chl b. The results indicate also that due to bilateral energy transfer, the Chl system equilibrates only partially during the excited state lifetimes.

Peridinin-chlorophyll-proteins (PCP) were reconstituted with binary 1:1 chlorophyll (Chl) mixtures of Chl a, Chl b, [3-acetyl]-Chl a (acChl a), and studied by bulk and single-molecule fluorescence spectroscopy. The latter provides a way to distinguish in a given sample hetero-chlorophyllous complexes that contain two different Chls from homo-chlorophyllous ones containing the same Chl in both binding sites. The results are compared with those of homo-chlorophyllous PCP reconstituted with pure Chl a, Chl b, or acChl a. Relative intensities of the Chl fluorescence in hetero-chlorophyllous complexes were obtained and modeled using the Förster description of energy transfer combined with known variations of peridinin (Per)-Chl excitation transfer rates for the different Chl pigments. In the case of hetero-chlorophyllous complexes containing acChl a, the energy transfer is unidirectional in the energetically preferable direction, while it is bi-directional in the sample reconstituted with Chl a and Chl b.

The phycobilin: Cysteine-84-phycobiliprotein lyase, CpeS1, catalyzes phycocyanobilin (PCB) and phycoerythrobilin attachment to nearly all cysteine-84 (consensus sequence) binding sites of phycoerythrin, phycoerythrocyanin, phycocyanin and allophycocyanin (Zhao et al. (2007) Proc Natl Acad Sci 104:14300-14305). We now show that CpeS1 can bind PCB, as assayed by Ni(2+) chelating affinity chromatography. Binding is rapid, and the chromophore is bound in an extended conformation similar to that in phycobiliproteins but only poorly fluorescent. Upon addition of apo-biliproteins, the chromophore is transferred to the latter much slower ( approximately 1 h), indicating that chromophorylated CpeS1 is an intermediate in the enzymatic reaction. In addition, imidazole is bound to PCB, as shown by mass spectroscopy of tryptic digests of the intermediate CpeS1-PCB complex.

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the β-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the α-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

Biliproteins are post-translationally modified by chromophore addition. In phycoerythrocyanin, the heterodimeric lyase PecE/F covalently attaches phycocyanobilin (PCB) to cysteine-alpha84 of the apoprotein PecA, with concomitant isomerization to phycoviolobilin. We found that: (a) PecA adds autocatalytically PCB, yielding a low absorbance, low fluorescence PCB.PecA adduct, termed P645 according to its absorption maximum; (b) In the presence of PecE, a high absorbance, high fluorescence PCB.PecA adduct is formed, termed P641; (c) PecE is capable of transforming P645 to P641; (d) When in stop-flow experiments, PecA and PecE were preincubated before chromophore addition, a red-shifted intermediate (P650, tau=32 ms) was observed followed by a second, which was blue-shifted (P605, tau=0.5 s), and finally a third (P638, tau=14 s) that yielded the adduct (P641, tau=20 min); (e) The reaction was slower, and P605 was missing, if PecA and PecE were not preincubated; (f) Gel filtration gave no evidence of a stable complex between PecA and PecE; however, complex formation is induced by adding PCB; and (g) A red-shifted intermediate was also formed, but more slowly, with phycoerythrobilin, and denaturation showed that this is not yet covalently bound. We conclude, therefore, that PecA and PecE form a weak complex that is stabilized by PCB, that the first reaction step involves a conformational change and/or protonation of PCB, and that PecE has a chaperone-like function on the chromoprotein.

Single molecule spectroscopy experiments are reported for native peridinin–chlorophyll a–protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Forster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm− 1. In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.

Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD(+) complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD(+) as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.

The genome of the cyanobacterium Nostoc sp. PCC7120 carries three genes (all4978, all7016, and alr7522) encoding putative heme-binding GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) proteins that were annotated as transcriptional regulators. They are composed of an N-terminal cofactor domain and a C-terminal helix-turn-helix motif. All4978 showed the highest affinity for protoheme binding. The heme binding capability of All7016 was moderate, and Alr7522 did not bind heme at all. The "as isolated" form of All4978, identified by Soret band (λmax = 427 nm), was assigned by electronic absorption, EPR, and resonance Raman spectroscopy as a hexa-coordinated low spin Fe(III) heme with a distal cysteine ligand (absorption of δ-band around 360 nm). The protoheme cofactor is noncovalently incorporated. Reduction of the heme could be accomplished by chemically using sodium dithionite and electrospectrochemically; this latter method yielded remarkably low midpoint potentials of -445 and -453 mV (following Soret and α-band absorption changes, respectively). The reduced form of the heme (Fe(II) state) binds both NO and CO. Cysteine coordination of the as isolated Fe(III) protein is unambiguous, but interestingly, the reduced heme instead displays spectral features indicative of histidine coordination. Cys-His ligand switches have been reported as putative signaling mechanisms in other heme-binding proteins; however, these novel cyanobacterial proteins are the first where such a ligand-switch mechanism has been observed in a GAF domain. DNA binding of the helix-turn-helix domain was investigated using a DNA sequence motif from its own promoter region. Formation of a protein-DNA complex preferentially formed in ferric state of the protein.

Complexes were prepared of horse heart myoglobin with derivatives of (bacterio)chlorophylls and the linear tetrapyrrole, phycocyanobilin. Structural factors importantfor bindingare(i)the presenceofacentral metalwithopenligationsite,whicheveninducesbindingofphycocyanobilin, and (ii) the absence of the hydrophobic esterifying alcohol, phytol. Binding is further modulated by the stereochemistry at the isocyclic ring. The binding pocket can act as a reaction chamber: with enolizable substrates, apo-myoglobin acts as a 13 2 -epimerase converting, e.g., Zn-pheophorbide a' (13 2 S) to a (13 2 R). Light-induced reduction and oxidation of the bound pigments are accelerated as compared to solution. Some flexibility of the myoglobin is required for these reactions to occur; a nucleophile is required near the chromophores for photoreduction (Krasnovskii reaction), and oxygen for photooxidation. Oxidation of the bacteriochlorin in the complex and in aqueous solution continues in the dark. © 2006 Elsevier B.V. All rights reserved.

The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-β84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-β84 of the apo-β-subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mm NaCl and 500 mm potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a Km(PCB) = 2.7 and 2.3μm, and a kcat = 1.7 × 10-5 and 1.1 × 10-5 s-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS.

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