[2Fe-2S] Proteins in Chlorosomes: CsmI and CsmJ Participate in Light-Dependent Control of Energy Transfer in Chlorosomes of <i>Chlorobaculum tepidum</i>

Chlorosomes of <i>Chlorobaculum tepidum</i> are formed from stacks of <i>syn–anti</i> coordinated bacteriochlorophyll <i>c</i> dimers, which form a suprastructure comprised of coaxial nanotubes and are surrounded by a glycolipid monolayer envelope containing 10 proteins. Three of these proteins, CsmI, CsmJ, and CsmX, have sequences very similar in their N-terminal domains to those of [2Fe-2S] ferredoxins of the adrenodoxin/putidaredoxin subfamily. The roles of these proteins in chlorosomes were studied in single-, double-, and triple-mutant strains. In each mutant, only the protein(s) corresponding to the mutated gene(s) was missing, and the amounts of other chlorosome proteins did not vary significantly. Electrophoretic analyses and immunoblotting showed that CsmX was much less abundant than CsmI or CsmJ. The growth rates and the pigment and isoprenoid quinone contents of isolated chlorosomes of the mutants were similar to wild-type values. Quenching and recovery of energy transfer in isolated chlorosomes and intact cells were studied by measuring fluorescence emission after exposure to or removal of oxygen. Oxygen-induced activation of the quencher in isolated chlorosomes or in intact cells was largely independent of CsmI and CsmJ. This may be because oxygen can diffuse across the chlorosome envelope easily and directly reacts with the quencher. However, CsmI and CsmJ were required to restore energy transfer fully after isolated chlorosomes were exposed to oxygen. Studies with intact cells suggested that cells contain both light-dependent and light-independent pathways for reducing the quenching species in chlorosomes and that CsmI and CsmJ are components of a light-dependent pathway

Triplet Excited State Energies and Phosphorescence Spectra of (Bacterio)Chlorophylls

(Bacterio)­Chlorophyll ((B)­Chl) molecules play a major role in photosynthetic light-harvesting proteins, and the knowledge of their triplet state energies is essential to understand the mechanisms of photodamage and photoprotection, as the triplet excitation energy of (B)­Chl molecules can readily generate highly reactive singlet oxygen. The triplet state energies of 10 natural chlorophyll (Chl <i>a</i>, <i>b</i>, <i>c</i>₂, <i>d</i>) and bacteriochlorophyll (BChl <i>a</i>, <i>b</i>, <i>c</i>, <i>d</i>, <i>e</i>, <i>g</i>) molecules and one bacteriopheophytin (BPheo <i>g</i>) have been directly determined via their phosphorescence spectra. Phosphorescence of four molecules (Chl <i>c</i>₂, BChl <i>e</i> and <i>g</i>, BPheo <i>g</i>) was characterized for the first time. Additionally, the relative phosphorescence to fluorescence quantum yield for each molecule was determined. The measurements were performed at 77K using solvents providing a six-coordinate environment of the Mg²⁺ ion, which allows direct comparison of these (B)­Chls. Density functional calculations of the triplet state energies show good correlation with the experimentally determined energies. The correlation determined computationally was used to predict the triplet energies of three additional (B)­Chl molecules: Chl <i>c</i>₁, Chl <i>f</i>, and BChl <i>f</i>

Supporting Spectral and Mass Characterization from De novo synthetic biliprotein design, assembly and excitation energy transfer

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-α-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorbance, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette cysteines (Cys) <i>in vitro</i>. A blue-shifted tripyrrole (TPB) formed from maquette-bound PCB displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in <i>Escherichia coli</i> to improve the efficiency of bilin binding and alter bilin conformation. Bilins bind <i>in vitro</i> and <i>in vivo</i> to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices

Supplementary Data Fusion MS from Multi-step excitation energy transfer engineered in genetic fusions of natural and synthetic light-harvesting proteins

Synthetic proteins designed and constructed from first-principles with minimal reference to the sequence of any natural protein have proven robust and extraordinarily adaptable for engineering a range of functions. Here for the first time we describe expression and genetic fusion of a natural photosynthetic light-harvesting subunit with a synthetic protein designed for light energy capture and multi-step transfer. We demonstrate excitation energy transfer from the bilin of the CpcA subunit (phycocyanin α subunit) of the cyanobacterial photosynthetic light-harvesting phycobilisome to synthetic four-helix-bundle proteins accommodating sites that specifically bind a variety of selected photoactive tetrapyrroles positioned to enhance energy transfer by relay. The examination of combinations of different bilin, chlorin and bacteriochlorin cofactors has led to identification of the preconditions for directing energy from the bilin light-harvesting antenna into synthetic protein-cofactor constructs that can be customized for light-activated chemistry in the cell

Polymer–Chlorosome Nanocomposites Consisting of Non-Native Combinations of Self-Assembling Bacteriochlorophylls

Chlorosomes are one of the characteristic light-harvesting antennas from green sulfur bacteria. These complexes represent a unique paradigm: self-assembly of bacteriochlorophyll pigments within a lipid monolayer without the influence of protein. Because of their large size and reduced complexity, they have been targeted as models for the development of bioinspired light-harvesting arrays. We report the production of biohybrid light-harvesting nanocomposites mimicking chlorosomes, composed of amphiphilic diblock copolymer membrane bodies that incorporate thousands of natural self-assembling bacteriochlorophyll molecules derived from green sulfur bacteria. The driving force behind the assembly of these polymer–chlorosome nanocomposites is the transfer of the mixed raw materials from the organic to the aqueous phase. We incorporated up to five different self-assembling pigment types into single nanocomposites that mimic chlorosome morphology. We establish that the copolymer-BChl self-assembly process works smoothly even when non-native combinations of BChl homologues are included. Spectroscopic characterization revealed that the different types of self-assembling pigments participate in ultrafast energy transfer, expanding beyond single chromophore constraints of the natural chlorosome system. This study further demonstrates the utility of flexible short-chain, diblock copolymers for building scalable, tunable light-harvesting arrays for technological use and allows for an in vitro analysis of the flexibility of natural self-assembling chromophores in unique and controlled combinations

Structural Variability in Wild-Type and <i>bchQ bchR</i> Mutant Chlorosomes of the Green Sulfur Bacterium <i>Chlorobaculum tepidum</i>

The self-aggregated state of bacteriochlorophyll (BChl) <i>c</i> molecules in chlorosomes belonging to a <i>bchQ bchR</i> mutant of the green sulfur bacteria <i>Chlorobaculum tepidum</i>, which mostly produces a single 17²-farnesyl-(<i>R</i>)-[8-ethyl,12-methyl]­BChl <i>c</i> homologue, was characterized by solid-state nuclear magnetic resonance (NMR) spectroscopy and high-resolution electron microscopy. A nearly complete ¹H and ¹³C chemical shift assignment was obtained from well-resolved homonuclear ¹³C–¹³C and heteronuclear ¹H–¹³C NMR data sets collected from ¹³C-enriched chlorosome preparations. Pronounced doubling (1:1) of specific ¹³C and ¹H resonances revealed the presence of two distinct and nonequivalent BChl <i>c</i> components, attributed to all <i>syn-</i> and all <i>anti</i>-coordinated parallel stacks, depending on the rotation of the macrocycle with respect to the 3¹-methyl group. Steric hindrance from the 20-methyl functionality induces structural differences between the <i>syn</i> and <i>anti</i> forms. A weak but significant and reproducible reflection at 1/0.69 nm^–1 in the direction perpendicular to the curvature of cylindrical segments observed with electron microscopy also suggests parallel stacking of BChl <i>c</i> molecules, though the observed lamellar spacing of 2.4 nm suggests weaker packing than for wild-type chlorosomes. We propose that relaxation of the pseudosymmetry observed for the wild type and a related BChl <i>d</i> mutant leads to extended domains of alternating <i>syn</i> and <i>anti</i> stacks in the <i>bchQ bchR</i> chlorosomes. Domains can be joined to form cylinders by helical <i>syn–anti</i> transition trajectories. The phase separation in domains on the cylindrical surface represents a basic mechanism for establishing suprastructural heterogeneity in an otherwise uniform supramolecular scaffolding framework that is well-ordered at the molecular level

Cyanobacteriochrome Photoreceptors Lacking the Canonical Cys Residue

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that sense near-ultraviolet to far-red light. Like the distantly related phytochromes, all CBCRs reported to date have a conserved Cys residue (the “canonical Cys” or “first Cys”) that forms a thioether linkage to C3¹ of the linear tetrapyrrole (bilin) chromophore. Detection of ultraviolet, violet, and blue light is performed by at least three subfamilies of two-Cys CBCRs that require both the first Cys and a second Cys that forms a second covalent linkage to C10 of the bilin. In the well-characterized DXCF subfamily, the second Cys is part of a conserved Asp-Xaa-Cys-Phe motif. We here report novel CBCRs lacking the first Cys but retaining the DXCF Cys as part of a conserved Asp-Xaa-Cys-Ile-Pro (DXCIP) motif. Phylogenetic analysis demonstrates that DXCIP CBCRs are a sister to a lineage of DXCF CBCR domains from phototaxis sensors. Three such DXCIP CBCR domains (cce_4193g1, Cyan8802_2776g1, and JSC1_24240) were characterized after recombinant expression in <i>Escherichia coli</i> engineered to produce phycocyanobilin. All three covalently bound bilin and showed unidirectional photoconversion in response to green light. Spectra of acid-denatured proteins in the dark-adapted state do not correspond to those of known bilins. One DXCIP CBCR, cce_4193g1, exhibited very rapid dark reversion consistent with a function as a power sensor. However, Cyan8802_2776g1 exhibited slower dark reversion and would not have such a function. The full-length cce_4193 protein also possesses a DXCF CBCR GAF domain (cce_4193g2) with a covalently bound phycoviolobilin chromophore and a blue/green photocycle. Our studies indicate that CBCRs need not contain the canonical Cys residue to function as photochromic light sensors and that phototaxis proteins containing DXCIP CBCRs may potentially perceive both light quality and light intensity

Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus elongatus

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (<i>Te</i>CpcS-III). <i>Te</i>CpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. <i>Te</i>CpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. <i>Te</i>CpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of <i>Te</i>CpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and <i>Te</i>CpcS was produced