GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6 f complex accumulation.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6 f complex (Cytb6 f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6 f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6 f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6 f.

Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly.

A Chlamydomonas reinhardtii mutant lacking CGL71, a thylakoid membrane protein previously shown to be involved in photosystem I (PSI) accumulation, exhibited photosensitivity and highly reduced abundance of PSI under photoheterotrophic conditions. Remarkably, the PSI content of this mutant declined to nearly undetectable levels under dark, oxic conditions, demonstrating that reduced PSI accumulation in the mutant is not strictly the result of photodamage. Furthermore, PSI returns to nearly wild-type levels when the O2 concentration in the medium is lowered. Overall, our results suggest that the accumulation of PSI in the mutant correlates with the redox state of the stroma rather than photodamage and that CGL71 functions under atmospheric O2 conditions to allow stable assembly of PSI. These findings may reflect the history of the Earth's atmosphere as it transitioned from anoxic to highly oxic (1-2 billion years ago), a change that required organisms to evolve mechanisms to assist in the assembly and stability of proteins or complexes with O2-sensitive cofactors.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism.

Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions.

The Use of Contact Mode Atomic Force Microscopy in Aqueous Medium for Structural Analysis of Spinach Photosynthetic Complexes.

To investigate the dynamics of photosynthetic pigment-protein complexes in vascular plants at high resolution in an aqueous environment, membrane-protruding oxygen-evolving complexes (OECs) associated with photosystem II (PSII) on spinach (Spinacia oleracea) grana membranes were examined using contact mode atomic force microscopy. This study represents, to our knowledge, the first use of atomic force microscopy to distinguish the putative large extrinsic loop of Photosystem II CP47 reaction center protein (CP47) from the putative oxygen-evolving enhancer proteins 1, 2, and 3 (PsbO, PsbP, and PsbQ) and large extrinsic loop of Photosystem II CP43 reaction center protein (CP43) in the PSII-OEC extrinsic domains of grana membranes under conditions resulting in the disordered arrangement of PSII-OEC particles. Moreover, we observed uncharacterized membrane particles that, based on their physical characteristics and electrophoretic analysis of the polypeptides associated with the grana samples, are hypothesized to be a domain of photosystem I that protrudes from the stromal face of single thylakoid bilayers. Our results are interpreted in the context of the results of others that were obtained using cryo-electron microscopy (and single particle analysis), negative staining and freeze-fracture electron microscopy, as well as previous atomic force microscopy studies.

The GreenCut: re-evaluation of physiological role of previously studied proteins and potential novel protein functions.

Based on comparative genomics, a list of proteins present in the green algal, flowering and nonflowering plant lineages, but not detected in nonphotosynthetic organisms, was assembled (Merchant et al., Science 318:245-250, 2007; Karpowicz et al., J Biol Chem 286:21427-21439, 2011). This protein grouping, previously designated the GreenCut, was established using stringent comparative genomic criteria; they are those Chlamydomonas reinhardtii proteins with orthologs in Arabidopsis thaliana, Physcomitrella patens, Oryza sativa, Populus tricocarpa and at least one of the three Ostreococcus species with fully sequenced genomes, but not in bacteria, yeast, fungi or mammals. Many GreenCut proteins are also present in red algae and diatoms and a subset of 189 have been identified as encoded on nearly all cyanobacterial genomes. Of the current GreenCut proteins (597 in total), approximately half have been studied previously. The functions or activities of a number of these proteins have been deduced from phenotypic analyses of mutants (defective for genes encoding specific GreenCut proteins) of A. thaliana, and in many cases the assigned functions do not exist in C. reinhardtii. Therefore, precise physiological functions of several previously studied GreenCut proteins are still not clear. The GreenCut also contains a number of proteins with certain conserved domains. Three of the most highly conserved domains are the FK506 binding, cyclophilin and PAP fibrillin domains; most members of these gene families are not well characterized. In general, our analysis of the GreenCut indicates that many processes critical to green lineage organisms remain unstudied or poorly characterized. We have begun to examine the functions of some GreenCut proteins in detail. For example, our work on the CPLD38 protein has demonstrated that it has an essential role in photosynthetic function and the stability of the cytochrome b 6 f complex.

Novel thylakoid membrane GreenCut protein CPLD38 impacts accumulation of the cytochrome b6f complex and associated regulatory processes.

Based on previous comparative genomic analyses, a set of nearly 600 polypeptides was identified that is present in green algae and flowering and nonflowering plants but is not present (or is highly diverged) in nonphotosynthetic organisms. The gene encoding one of these "GreenCut" proteins, CPLD38, is in the same operon as ndhL in most cyanobacteria; the NdhL protein is part of a complex essential for cyanobacterial respiration. A cpld38 mutant of Chlamydomonas reinhardtii does not grow on minimal medium, is high light-sensitive under photoheterotrophic conditions, has lower accumulation of photosynthetic complexes, reduced photosynthetic electron flow to P700(+), and reduced photochemical efficiency of photosystem II (ΦPSII); these phenotypes are rescued by a wild-type copy of CPLD38. Single turnover flash experiments and biochemical analyses demonstrated that cytochrome b6f function was severely compromised, and the levels of transcripts and polypeptide subunits of the cytochrome b6f complex were also significantly lower in the cpld38 mutant. Furthermore, subunits of the cytochrome b6f complex in mutant cells turned over much more rapidly than in wild-type cells. Interestingly, PTOX2 and NDA2, two major proteins involved in chlororespiration, were more than 5-fold higher in mutants relative to wild-type cells, suggesting a shift in the cpld38 mutant from photosynthesis toward chlororespiratory metabolism, which is supported by experiments that quantify the reduction state of the plastoquinone pool. Together, these findings support the hypothesis that CPLD38 impacts the stability of the cytochrome b6f complex and possibly plays a role in balancing redox inputs to the quinone pool from photosynthesis and chlororespiration.

A flavin binding cryptochrome photoreceptor responds to both blue and red light in Chlamydomonas reinhardtii.

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light-activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities.

A bioassay for the detection of perchlorate in the ppb range.

A bioassay for the determination of ppb (µg·L(-1)) concentrations of perchlorate has been developed and is described herein. The assay uses the enzyme perchlorate reductase (PR) from the perchlorate-reducing organism Dechloromonas agitata in purified and partially purified forms to detect perchlorate. The redox active dye phenazine methosulfate (PMS) is shown to efficiently shuttle electrons to PR from NADH. Perchlorate can be determined indirectly by monitoring NADH oxidization by PR. To lower the detection limit, we have shown that perchlorate can be concentrated on a solid-phase extraction (SPE) column that is pretreated with the cation decyltrimethylammonium bromide (DTAB). Perchlorate is eluted from these columns with a solution of 2 M NaCl and 200 mM morpholine propane sulfonic acid (MOPS, pH 12.5). By washing these columns with 15 mL of 2.5 mM DTAB and 15% acetone, contaminating ions, such as chlorate and nitrate, are removed without affecting the bioassay. Because of the effect of complex matrices on the SPE columns, the method of standard additions is used to analyze tap water and groundwater samples. The efficacy of the developed bioassay was demonstrated by analyzing samples from 2-17000 ppb in deionized lab water, tap water, and contaminated groundwater.

Phylogenomic analysis of the Chlamydomonas genome unmasks proteins potentially involved in photosynthetic function and regulation.

Chlamydomonas reinhardtii, a unicellular green alga, has been exploited as a reference organism for identifying proteins and activities associated with the photosynthetic apparatus and the functioning of chloroplasts. Recently, the full genome sequence of Chlamydomonas was generated and a set of gene models, representing all genes on the genome, was developed. Using these gene models, and gene models developed for the genomes of other organisms, a phylogenomic, comparative analysis was performed to identify proteins encoded on the Chlamydomonas genome which were likely involved in chloroplast functions (or specifically associated with the green algal lineage); this set of proteins has been designated the GreenCut. Further analyses of those GreenCut proteins with uncharacterized functions and the generation of mutant strains aberrant for these proteins are beginning to unmask new layers of functionality/regulation that are integrated into the workings of the photosynthetic apparatus.

Biogenesis of iron-sulfur clusters in photosystem I: holo-NfuA from the cyanobacterium Synechococcus sp. PCC 7002 rapidly and efficiently transfers [4Fe-4S] clusters to apo-PsaC in vitro.

The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, Mössbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro.

Heliobacterial photosynthesis.

Heliobacteria contain Type I reaction centers (RCs) and a homodimeric core, but unlike green sulfur bacteria, they do not contain an extended antenna system. Given their simplicity, the heliobacterial RC (HbRC) should be ideal for the study of a prototypical homodimeric RC. However, there exist enormous gaps in our knowledge, particularly with regard to the nature of the secondary and tertiary electron acceptors. To paraphrase S. Neerken and J. Amesz (2001 Biochim Biophys Acta 1507:278-290): with the sole exception of primary charge separation, little progress has been made in recent years on the HbRC, either with respect to the polypeptide composition, or the nature of the electron acceptor chain, or the kinetics of forward and backward electron transfer. This situation, however, has changed. First, the low molecular mass polypeptide that contains the terminal FA and FB iron-sulfur clusters has been identified. The change in the lifetime of the flash-induced kinetics from 75 ms to 15 ms on its removal shows that the former arises from the P798+ [FA/FB]- recombination, and the latter from P798+ FX- recombination. Second, FX has been identified in HbRC cores by EPR and Mössbauer spectroscopy, and shown to be a [4Fe-4S]1+,2+ cluster with a ground spin state of S=3/2. Since all of the iron in HbRC cores is in the FX cluster, a ratio of approximately 22 Bchl g/P798 could be calculated from chemical assays of non-heme iron and Bchl g. Third, the N-terminal amino acid sequence of the FA/FB-containing polypeptide led to the identification and cloning of its gene. The expressed protein can be rebound to isolated HbRC cores, thereby regaining both the 75 ms kinetic phase resulting from P798+ [FA/FB]- recombination and the light-induced EPR resonances of FA- and FB-. The gene was named 'pshB' and the protein 'PshB' in keeping with the accepted nomenclature for Type I RCs. This article reviews the current state of knowledge on the structure and function of the HbRC.

Identification and characterization of PshB, the dicluster ferredoxin that harbors the terminal electron acceptors F(A) and F(B) in Heliobacterium modesticaldum.

The Type I homodimeric photosynthetic reaction center found in anaerobic gram-positive bacteria of the genus Heliobacteriaceae incorporates FA- and FB-like iron-sulfur clusters similar to those found in Photosystem I as terminal electron acceptors. We recently isolated the PshB protein that harbors the iron-sulfur clusters from the reaction centers of Heliobacterium modesticaldum. Here, we report the cloning of a candidate gene and the properties of its product. Genuine PshB was dissociated from the reaction center with 1 M NaCl and purified using an affinity strategy. After acquiring its N-terminal amino acid sequence, an fd2-like gene encoding a 5.5-kDa dicluster ferredoxin was identified as a candidate for PshB. The Fd2-like apoprotein was expressed in Escherichia coli with a His tag, and the Fe/S clusters were inserted using inorganic reagents. The optical absorbance and EPR spectra of the Fd2-like holoprotein were similar to those of genuine PshB. The Fd2-like holoprotein was coeluted with P798-FX cores on both G-75 gel filtration and Ni affinity columns. Consistent with binding, the EPR resonances at g = 2.067, 1.933, and 1.890 from [FA/FB]- were restored after illumination at 15 K, and the long-lived, room-temperature charge recombination kinetics between P798+ and [FA/FB]- reappeared on a laser flash. These characteristics indicate that the long-sought gene and polypeptide harboring the FA- and FB-like clusters in heliobacteria have been identified. The amino acid sequence of PshB indicates an entirely different mode of binding with the reaction center core than PsaC, its counterpart in Photosystem I.

Identification of FX in the heliobacterial reaction center as a [4Fe-4S] cluster with an S = 3/2 ground spin state.

Type I homodimeric reaction centers, particularly the class present in heliobacteria, are not well understood. Even though the primary amino acid sequence of PshA in Heliobacillus mobilis has been shown to contain an F(X) binding site, a functional Fe-S cluster has not been detected by EPR spectroscopy. Recently, we reported that PshB, which contains F(A)- and F(B)-like Fe-S clusters, could be removed from the Heliobacterium modesticaldum reaction center (HbRC), resulting in 15 ms lifetime charge recombination between P798(+) and an unidentified electron acceptor [Heinnickel, M., Shen, G., Agalarov, R., and Golbeck, J. H. (2005) Biochemistry 44, 9950-9960]. We report here that when a HbRC core is incubated with sodium dithionite in the presence of light, the 15 ms charge recombination is replaced with a kinetic transient in the sub-microsecond time domain, consistent with the reduction of this electron acceptor. Concomitantly, a broad and intense EPR signal arises around g = 5 along with a minor set of resonances around g = 2 similar to the spectrum of the [4Fe-4S](+) cluster in the Fe protein of Azotobacter vinelandii nitrogenase, which exists in two conformations having S = (3)/(2) and S = (1)/(2) ground spin states. The Mössbauer spectrum in the as-isolated HbRC core shows that all of the Fe is present in the form of a [4Fe-4S](2+) cluster. After reduction with sodium dithionite in the presence of light, approximately 65% of the Fe appears in the form of a [4Fe-4S](+) cluster; the remainder is in the [4Fe-4S](2+) state. Analysis of the non-heme iron content of HbRC cores indicates an antenna size of 21.6 +/- 1.1 BChl g molecules/P798. The evidence indicates that the HbRC contains a [4Fe-4S] cluster identified as F(X) that is coordinated between the PshA homodimer; in contrast to F(X) in other type I reaction centers, this [4Fe-4S] cluster exhibits an S = (3)/(2) ground spin state.

Resolution and reconstitution of a bound Fe-S protein from the photosynthetic reaction center of Heliobacterium modesticaldum.

The photosynthetic reaction center of Heliobacterium modesticaldum (HbRC) was isolated from membranes using n-dodecyl beta-D-maltopyranoside followed by sucrose density ultracentrifugation. The low-temperature EPR spectra of whole cells, isolated membranes, and HbRC complexes are similar, showing a single Fe-S cluster with g values of 2.067, 1.933, and 1.890 after illumination at 20 K, and a complex spectrum attributed to exchange interaction from two Fe-S clusters after illumination during freezing. The protein containing the Fe-S clusters was removed from the HbRC by washing it with 1.0 M NaCl and purified by ultrafiltration over a 30 kDa cutoff membrane. Analysis of the filtrate by SDS-PAGE showed a major band at approximately 8 kDa that was weakly stained with Coomassie Brilliant Blue and strongly stained with silver. The optical spectrum of the oxidized Fe-S protein shows a maximum at 410 nm, and the EPR spectrum of the reduced Fe-S protein shows a complex set of resonances similar to those found in 2[4Fe-4S] ferredoxins. The HbRC core was purified by DEAE ion-exchange chromatography and resolved by SDS-PAGE. The purified HbRC was composed of a band at ca. 40 kDa, which is identified as PshA, and several additional proteins. The isolated Fe-S protein rebinds spontaneously to purified HbRC cores, and the light-induced EPR signals of the Fe-S clusters are recovered. The flash-induced kinetics of the HbRC complex show two kinetic phases at room temperature, one with a lifetime of 75 ms and the other with a lifetime of 15 ms. The 75 ms component is lost when the Fe-S protein is removed from the HbRC complex, and it is regained when the Fe-S protein is rebound to HbRC cores. Thus, the 75 ms kinetic phase is derived from recombination of a terminal Fe-S cluster with P798(+), and the 15 ms kinetic phase is derived from recombination with an earlier acceptor, probably F(X). We suggest that the bound Fe-S protein present in the HbRC be designated PshB.