Identification and characterization of the low molecular mass ferredoxins involved in central metabolism in Heliomicrobium modesticaldum.

The homodimeric Type I reaction center (RC) from Heliomicrobium modesticaldum lacks the PsaC subunit found in Photosystem I and instead uses the interpolypeptide [4Fe-4S] cluster FX as the terminal electron acceptor. Our goal was to identify which of the small mobile dicluster ferredoxins encoded by the H. modesticaldum genome are capable of accepting electrons from the heliobacterial RC (HbRC) and pyruvate:ferredoxin oxidoreductase (PFOR), a key metabolic enzyme. Analysis of the genome revealed seven candidates: HM1_1462 (PshB1), HM1_1461 (PshB2), HM1_2505 (Fdx3), HM1_0869 (FdxB), HM1_1043, HM1_0357, and HM1_2767. Heterologous expression in Escherichia coli and studies using time-resolved optical spectroscopy revealed that only PshB1, PshB2, and Fdx3 are capable of accepting electrons from the HbRC and PFOR. Modeling studies using AlphaFold show that only PshB1, PshB2, and Fdx3 should be capable of docking on PFOR at a positively charged patch that overlays a surface-proximal [4Fe-4S] cluster. Proteomic analysis of wild-type and gene deletion strains ΔpshB1, ΔpshB2, ΔpshB1pshB2, and Δfdx3 grown under nitrogen-replete conditions revealed that Fdx3 is undetectable in the wild-type, ΔpshB1, and Δfdx3 strains, but it is present in the ΔpshB2 and ΔpshB1pshB2 strains, implying that Fdx3 may substitute for PshB2. When grown under nitrogen-deplete conditions, Fdx3 is present in the wild-type and all deletion strains except for Δfdx3. None of the knockout strains demonstrated significant impairment during chemotrophic dark growth on pyruvate, photoheterotrophic light growth on pyruvate, or phototrophic growth on acetate+CO2, indicating a high degree of redundancy among these three electron transfer proteins. Loss of both PshB1 and PshB2, but not FdxB, resulted in poor growth under N2-fixing conditions.

Phylloquinone is the principal Mehler reaction site within photosystem I in high light.

Photosynthesis is a vital process, responsible for fixing carbon dioxide, and producing most of the organic matter on the planet. However, photosynthesis has some inherent limitations in utilizing solar energy, and a part of the energy absorbed is lost in the reduction of O2 to produce the superoxide radical (O2•-) via the Mehler reaction, which occurs principally within photosystem I (PSI). For decades, O2 reduction within PSI was assumed to take place solely in the distal iron-sulfur clusters rather than within the two asymmetrical cofactor branches. Here, we demonstrate that under high irradiance, O2 photoreduction by PSI primarily takes place at the phylloquinone of one of the branches (the A-branch). This conclusion derives from the light dependency of the O2 photoreduction rate constant in fully mature wild-type PSI from Chlamydomonas reinhardtii, complexes lacking iron-sulfur clusters, and a mutant PSI, in which phyllosemiquinone at the A-branch has a significantly longer lifetime. We suggest that the Mehler reaction at the phylloquinone site serves as a release valve under conditions where both the iron-sulfur clusters of PSI and the mobile ferredoxin pool are highly reduced.

The PshX subunit of the photochemical reaction center from Heliobacterium modesticaldum acts as a low-energy antenna.

The anoxygenic phototrophic bacterium Heliobacterium modesticaldum contains a photochemical reaction center protein complex (called the HbRC) consisting of a homodimer of the PshA polypeptide and two copies of a newly discovered polypeptide called PshX, which is a single transmembrane helix that binds two bacteriochlorophyll g molecules. To assess the function of PshX, we produced a ∆pshX strain of Hbt. modesticaldum by leveraging the endogenous Hbt. modesticaldum Type I-A CRISPR-Cas system to aid in mutant selection. We optimized this system by separating the homologous recombination and CRISPR-based selection steps into two plasmid transformations, allowing for markerless gene replacement. Fluorescence and low-temperature absorbance of the purified HbRC from the wild-type and ∆pshX strains showed that the bacteriochlorophylls bound by PshX have the lowest site energies in the entire HbRC. This indicates that PshX acts as a low-energy antenna subunit, participating in entropy-assisted uphill energy transfer toward the P800 special bacteriochlorophyll g pair. We further discuss the role that PshX may play in stability of the HbRC, its conservation in other heliobacterial species, and the evolutionary pressure to produce and maintain single-TMH subunits in similar locations in other reaction centers.

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells.

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. We used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was over 100 times slower in the ∆petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P800 (the primary donor of the photochemical reaction center) were much higher in the ∆petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The ∆petCBDA mutant was unable to grow phototrophically on acetate plus CO2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

Reconstitution of the heliobacterial photochemical reaction center and cytochrome c553 into a proteoliposome system.

The heliobacterial reaction center (HbRC) is the simplest known photochemical reaction center, in terms of its polypeptide composition. In the heliobacterial cells, its electron donor is a cytochrome (cyt) c553 attached to the membrane via a covalent linkage with a diacylglycerol. We have reconstituted purified HbRC into liposomes mimicking the phospholipid composition of heliobacterial membranes. We also incorporated a lipid with a headgroup containing Ni(II):nitrilotriacetate (NTA) to provide a binding site for the soluble version of the heliobacterial cyt c553 in which the N-terminal membrane attachment site is replaced by a hexahistidine tag. The HbRC was inserted into the liposomes with the donor side preferentially exposed to the exterior; this bias increased to nearly 100% with higher concentrations (≥ 10 mol%) of the Ni(II)-NTA lipid in the membrane, and is most likely due to the net negative charge of the surface of the membrane. The HbRC in proteoliposomes without the Ni(II)-NTA lipid exhibited normal charge separation and subsequent charge recombination of the P800+FX- state in 15 ms; however, the oxidized primary donor (P800+) was not significantly reduced by added H6-cyt c553. In contrast, with proteoliposomes containing the Ni(II)-NTA lipid, addition of H6-cyt c553 resulted in a new kinetic component resulting from fast reduction (2-5 ms) of P800+ by H6-cyt c553. The contribution of this kinetic component varied with the concentration of added H6-cyt c553 and could represent 80% or more of the total P800+ decay. Thus, the HbRC and its interaction with its native electron donor have been reconstituted into an artificial membrane system.

Using the Endogenous CRISPR-Cas System of Heliobacterium modesticaldum To Delete the Photochemical Reaction Center Core Subunit Gene.

In Heliobacterium modesticaldum, as in many Firmicutes, deleting genes by homologous recombination using standard techniques has been extremely difficult. The cells tend to integrate the introduced plasmid into the chromosome by a single recombination event rather than perform the double recombination required to replace the targeted locus. Transformation with a vector containing only a homologous recombination template for replacement of the photochemical reaction center gene pshA produced colonies with multiple genotypes, rather than a clean gene replacement. To address this issue, we required an additional means of selection to force a clean gene replacement. In this study, we report the genetic structure of the type I-A and I-E CRISPR-Cas systems from H. modesticaldum, as well as methods to leverage the type I-A system for genome editing. In silico analysis of the CRISPR spacers revealed a potential consensus protospacer adjacent motif (PAM) required for Cas3 recognition, which was then tested using an in vivo interference assay. Introduction of a homologous recombination plasmid that carried a miniature CRISPR array targeting sequences in pshA (downstream of a naturally occurring PAM sequence) produced nonphototrophic transformants with clean replacements of the pshA gene with ∼80% efficiency. Mutants were confirmed by PCR, sequencing, optical spectroscopy, and growth characteristics. This methodology should be applicable to any genetic locus in the H. modesticaldum genome.IMPORTANCE The heliobacteria are the only phototrophic members of the largely Gram-positive phylum Firmicutes, which contains medically and industrially important members, such as Clostridium difficile and Clostridium acetobutylicum Heliobacteria are of interest in the study of photosynthesis because their photosynthetic system is unique and the simplest known. Since their discovery in the early 1980s, work on the heliobacteria has been hindered by the lack of a genetic transformation system. The problem of introducing foreign DNA into these bacteria has been recently rectified by our group; however, issues still remained for efficient genome editing. The significance of this work is that we have characterized the endogenous type I CRISPR-Cas system in the heliobacteria and leveraged it to assist in genome editing. Using the CRISPR-Cas system allowed us to isolate transformants with precise replacement of the pshA gene encoding the main subunit of the photochemical reaction center.

A Molecular Biology Tool Kit for the Phototrophic Firmicute Heliobacterium modesticaldum.

The heliobacteria are members of the bacterial order Clostridiales and form the only group of phototrophs in the phylum Firmicutes Several physiological and metabolic characteristics make them an interesting subject of investigation, including their minimalist photosynthetic system, nitrogen fixation abilities, and ability to reduce toxic metals. While the species Heliobacterium modesticaldum is an excellent candidate as a model system for the family Heliobacteriaceae, since an annotated genome and transcriptomes are available, studies in this organism have been hampered by the lack of genetic tools. We adapted techniques for genetic manipulation of related clostridial species for use with H. modesticaldum Five heliobacterial DNA methyltransferase genes were expressed in an Escherichia coli strain engineered as a conjugative plasmid donor for broad-host-range plasmids. Premethylation of the shuttle vectors before conjugation into H. modesticaldum is absolutely required for production of transconjugant colonies. The introduced shuttle vectors are maintained stably and can be recovered using a modified minipreparation procedure developed to inhibit endogenous DNase activity. Furthermore, we describe the formulation of various growth media, including a defined medium for metabolic studies and isolation of auxotrophic mutants.IMPORTANCE Heliobacteria are anoxygenic phototrophic bacteria with the simplest known photosynthetic apparatus. They are unique in using bacteriochlorophyll g as their main pigment and lacking a peripheral antenna system. Until now, research on this organism has been hampered by the lack of a genetic transformation system. Without such a system, gene knockouts, site-directed mutations, and gene expression studies cannot be performed to help us further understand or manipulate the organism. Here we report the genetic transformation of a heliobacterium, which should enable future genetic studies in this unique phototrophic organism.

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.

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches.

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2A/ec3A or ec2B/ec3B) and a phylloquinone (PhQA or PhQB). The axial ligand to the central Mg2+ of the ec2A and ec2B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to FX electron transfer show that in both species, the PsaA-N604L mutation (near ec2B) results in a ~50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2A) results in a ~70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P700+PhQ- is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.

Evolution of photosynthetic reaction centers: insights from the structure of the heliobacterial reaction center.

The proliferation of phototrophy within early-branching prokaryotes represented a significant step forward in metabolic evolution. All available evidence supports the hypothesis that the photosynthetic reaction center (RC)-the pigment-protein complex in which electromagnetic energy (i.e., photons of visible or near-infrared light) is converted to chemical energy usable by an organism-arose once in Earth's history. This event took place over 3 billion years ago and the basic architecture of the RC has diversified into the distinct versions that now exist. Using our recent 2.2-Å X-ray crystal structure of the homodimeric photosynthetic RC from heliobacteria, we have performed a robust comparison of all known RC types with available structural data. These comparisons have allowed us to generate hypotheses about structural and functional aspects of the common ancestors of extant RCs and to expand upon existing evolutionary schemes. Since the heliobacterial RC is homodimeric and loosely binds (and reduces) quinones, we support the view that it retains more ancestral features than its homologs from other groups. In the evolutionary scenario we propose, the ancestral RC predating the division between Type I and Type II RCs was homodimeric, loosely bound two mobile quinones, and performed an inefficient disproportionation reaction to reduce quinone to quinol. The changes leading to the diversification into Type I and Type II RCs were separate responses to the need to optimize this reaction: the Type I lineage added a [4Fe-4S] cluster to facilitate double reduction of a quinone, while the Type II lineage heterodimerized and specialized the two cofactor branches, fixing the quinone in the QA site. After the Type I/II split, an ancestor to photosystem I fixed its quinone sites and then heterodimerized to bind PsaC as a new subunit, as responses to rising O2 after the appearance of the oxygen-evolving complex in an ancestor of photosystem II. These pivotal events thus gave rise to the diversity that we observe today.

Light-driven quinone reduction in heliobacterial membranes.

Photosynthetic reaction centers (RCs) evolved > 3 billion years ago and have diverged into Type II RCs reducing quinones and Type I RCs reducing soluble acceptors via iron-sulfur clusters. Photosystem I (PSI), the exemplar Type I RC, uses modified menaquinones as intermediate electron transfer cofactors, but it has been controversial if the Type I RC of heliobacteria (HbRC) uses its two bound menaquinones in the same way. The sequence of the quinone-binding site in PSI is not conserved in the HbRC, and the recently solved crystal structure of the HbRC does not reveal a quinone in the analogous site. We found that illumination of heliobacterial membranes resulted in reduction of menaquinone to menaquinol, suggesting that the HbRC can perform a function thought restricted to Type II RCs. Experiments on membranes and live cells are consistent with the hypothesis that the HbRC preferentially reduces soluble electron acceptors (e.g., ferredoxins) in low light, but switches to reducing lipophilic quinones in high light, when the soluble acceptor pool becomes full. Thus, the HbRC may represent a functional evolutionary intermediate between PSI and the Type II RCs.

Uphill energy transfer in photosystem I from Chlamydomonas reinhardtii. Time-resolved fluorescence measurements at 77 K.

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI-LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI-LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI-LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI-LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~ 12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~ 675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Thermodynamics of the Electron Acceptors in Heliobacterium modesticaldum: An Exemplar of an Early Homodimeric Type I Photosynthetic Reaction Center.

The homodimeric type I reaction center in heliobacteria is arguably the simplest known pigment-protein complex capable of conducting (bacterio)chlorophyll-based conversion of light into chemical energy. Despite its structural simplicity, the thermodynamics of the electron transfer cofactors on the acceptor side have not been fully investigated. In this work, we measured the midpoint potential of the terminal [4Fe-4S](2+/1+) cluster (FX) in reaction centers from Heliobacterium modesticaldum. The FX cluster was titrated chemically and monitored by (i) the decrease in the level of stable P800 photobleaching by optical spectroscopy, (ii) the loss of the light-induced g ≈ 2 radical from P800(+•) following a single-turnover flash, (iii) the increase in the low-field resonance at 140 mT attributed to the S = (3)/2 ground spin state of FX(-), and (iv) the loss of the spin-correlated P800(+) FX(-) radical pair following a single-turnover flash. These four techniques led to similar estimations of the midpoint potential for FX of -502 ± 3 mV (n = 0.99), -496 ± 2 mV (n = 0.99), -517 ± 10 mV (n = 0.65), and -501 ± 4 mV (n = 0.84), respectively, with a consensus value of -504 ± 10 mV (converging to n = 1). Under conditions in which FX is reduced, the long-lived (∼15 ms) P800(+) FX(-) state is replaced by a rapidly recombining (∼15 ns) P800(+)A0(-) state, as shown by ultrafast optical experiments. There was no evidence of the presence of a P800(+) A1(-) spin-correlated radical pair by electron paramagnetic resonance (EPR) under these conditions. The midpoint potentials of the two [4Fe-4S](2+/1+) clusters in the low-molecular mass ferredoxins were found to be -480 ± 11 mV/-524 ± 13 mV for PshBI, -453 ± 6 mV/-527 ± 6 mV for PshBII, and -452 ± 5 mV/-533 ± 8 mV for HM1_2505 as determined by EPR spectroscopy. FX is therefore suitably poised to reduce one [4Fe-4S](2+/1+) cluster in these mobile electron carriers. Using the measured midpoint potential of FX and a quasi-equilibrium model of charge recombination, the midpoint potential of A0 was estimated to be -854 mV at room temperature. The midpoint potentials of A0 and FX are therefore 150-200 mV less reducing than their respective counterparts in Photosystem I of cyanobacteria and plants. This places the redox potential of the FX cluster in heliobacteria approximately equipotential to the highest-potential iron-sulfur cluster (FA) in Photosystem I, consistent with its assignment as the terminal electron acceptor.

Species-dependent alteration of electron transfer in the early stages of charge stabilization in Photosystem I.

Electron transfer (ET) in Photosystem I (PS I) is bidirectional, occurring in two pseudosymmetric branches of cofactors. The relative use of two branches in the green alga Chlamydomonas reinhardtii and the cyanobacterium Synechocystis sp. PCC 6803 has been studied by changing the Met axial ligands of the chlorophyll a acceptor molecules, A0A and A0B, to His. The nature of the effect on the ET is found to be species dependent. In C. reinhardtii, transient absorption and transient EPR data show that in the M688HPsaA variant, forward ET from A0A to the quinone, A1A, is blocked in 100% of the PS I complexes. In contrast, in Synechocystis sp. PCC 6803, forward ET from A0A to A1A is blocked in only 50% of the PS I complexes, but in those PS I complexes in which electrons reach A1A, further transfer to the iron-sulfur cluster FX is blocked. Similar species differences are found for the corresponding B-branch variants. One possible explanation of this behavior is that it is the result of two conformers in which an H-bond between the His side chain and the O1 carbonyl group of A1 is either present or absent. The spectroscopic data suggest that the two conformers are present in nearly equal amounts in the Synechocystis sp. PCC 6803 variants, while only the conformer without the H-bond is present in the same variants of C. reinhardtii.

Controlling electron transfer between the two cofactor chains of photosystem I by the redox state of one of their components.

Two functional electron transfer (ET) chains, related by a pseudo-C2 symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA(-) oxidation. This is the largest change of PhQA(-) oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700(+). This situation allows a second photochemical charge separation event to be initiated before PhQA(-) has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA(-) does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed.

Excitation dynamics in Photosystem I from Chlamydomonas reinhardtii. Comparative studies of isolated complexes and whole cells.

Identical time-resolved fluorescence measurements with ~3.5-ps resolution were performed for three types of PSI preparations from the green alga, Chlamydomonas reinhardtii: isolated PSI cores, isolated PSI-LHCI complexes and PSI-LHCI complexes in whole living cells. Fluorescence decay in these types of PSI preparations has been previously investigated but never under the same experimental conditions. As a result we present consistent picture of excitation dynamics in algal PSI. Temporal evolution of fluorescence spectra can be generally described by three decay components with similar lifetimes in all samples (6-8ps, 25-30ps, 166-314ps). In the PSI cores, the fluorescence decay is dominated by the two fastest components (~90%), which can be assigned to excitation energy trapping in the reaction center by reversible primary charge separation. Excitation dynamics in the PSI-LHCI preparations is more complex because of the energy transfer between the LHCI antenna system and the core. The average trapping time of excitations created in the well coupled LHCI antenna system is about 12-15ps longer than excitations formed in the PSI core antenna. Excitation dynamics in PSI-LHCI complexes in whole living cells is very similar to that observed in isolated complexes. Our data support the view that chlorophylls responsible for the long-wavelength emission are located mostly in LHCI. We also compared in detail our results with the literature data obtained for plant PSI.

Purification and photobiochemical profile of photosystem 1 from a high-salt tolerant, oleaginous Chlorella (Trebouxiophycaea, Chlorophyta).

The eukaryotic green alga Chlamydomonas reinhardtii has been studied extensively within the biofuel industry as a model organism, as researchers look towards algae to provide chemical feedstocks (i.e., lipids) for the production of liquid transportation fuels. C. reinhardtii, however, is unsuitable for high-level production of such precursors due to its relatively poor lipid accumulation and fresh-water demand. In this study we offer insight into the primary light harvesting and electron transfer reactions that occur during phototropic growth in a high-salt tolerant strain of Chlorella (a novel strain introduced here as NE1401), a single-celled eukaryotic algae also in the phylum Chlorophyta. Under nutrient starvation many eukaryotic algae increase dramatically the amount of lipids stored in lipid bodies within their cell interiors. Microscopy and lipid analyses indicate that Chlorella sp. NE1401 may become a superior candidate for algal biofuels production. We have purified highly active Photosystem 1 (PS1) complexes to study in vitro, so that we may understand further the photobiochemisty of this promising biofuel producer and how its characteristics compare and contrast with that of the better understood C. reinhardtii. Our findings suggest that the PS1 complex from Chlorella sp. NE1401 demonstrates similar characteristics to that of C. reinhardtii with respect to light-harvesting and electron transfer reactions. We also illustrate that the relative extent of the light state transition performed by Chlorella sp. NE1401 is smaller compared to C. reinhardtii, although they are triggered by the same dynamic light stresses.

Purification and biochemical characterization of the ATP synthase from Heliobacterium modesticaldum.

Heliobacterium modesticaldum is an anaerobic photosynthetic bacterium that grows optimally at pH 6-7 and 52°C and is the only phototrophic member of the Firmicutes phylum family (gram-positive bacteria with low GC content). The ATP synthase of H. modesticaldum was isolated and characterized at the biochemical and biophysical levels. The isolated holoenzyme exhibited the subunit patterns of F-type ATP synthases containing a 5-subunit hydrophilic F1 subcomplex and a 3-subunit hydrophobic F0 subcomplex. ATP hydrolysis by the isolated HF1F0 ATP synthase was successfully detected after pretreatment with different detergents by an in-gel ATPase activity assay, which showed that the highest activity was detected in the presence of mild detergents such as LDAO; moreover, high catalytic activity in the gel was already detected after the initial incubation period of 0.5h. In contrast, HF1F0 showed extremely low ATPase activity in harsher detergents such as TODC. The isolated fully functional enzyme will form the basis for future structural studies.

The requirement for carotenoids in the assembly and function of the photosynthetic complexes in Chlamydomonas reinhardtii.

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b6f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b6f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.