Mark Heinnickel

A Flavin-Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

An animal-like cryptochrome (aCRY) functions as a sensory blue light receptor in the green alga Chlamydomonas; in addition, this flavoprotein unexpectedly acts as a sensory red light receptor. For plant cryptochromes, the dark form is proposed to contain an oxidized flavin, whereas for aCRY, the broad spectral responses point to the neutral radical state in the dark. 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.

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-FX 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 [FA/FB]- 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.

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 b6f complex.

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

Significance Our results suggest that particular ferredoxins in photosynthetic organisms are tailored to serve as electron carriers that sustain day-time and night-time metabolism and that the chloroplast-localized ferredoxin-5 (FDX5) appears to function in the desaturation of fatty acids required for maintaining the correct ratio of the dominant lipids in the thylakoid membranes and the integration of chloroplast and mitochondrial metabolism, which is absolutely required for growth in the dark. The most important messages from this work are that redox components associated with critical activities in photosynthetic organisms must be tuned to the redox conditions of the cells and the overall carbon budget of photosynthetic cells requires an understanding of metabolic features that accompany the movement of cells between light and dark conditions. 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.

Novel Thylakoid Membrane GreenCut Protein CPLD38 Impacts Accumulation of the Cytochrome b6f Complex and Associated Regulatory Processes

Background: The GreenCut is a group of ∼600 green-lineage-specific proteins hypothetically involved in photosynthesis. Results: A Chlamydomonas reinhardtii mutant disrupted for GreenCut gene CPLD38 has a marked reduction in cytochrome b6f and an increase in chlororespiration. Conclusion: CPLD38 is essential for accumulating cytochrome b6f and balancing chlororespiration and photosynthesis. Significance: This analysis demonstrates the importance of a GreenCut protein in photosynthesis. 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.

Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly

Significance Our results demonstrate that Chlamydomonas reinhardtii CGL71, a tetratricopeptide repeat protein, is involved in protecting photosystem I from oxidative disruption during assembly; this process may reflect oxygen sensitivity of the iron sulfur clusters that are integral to the complex. During the early evolution of photosynthesis, the atmosphere of the Earth was anoxic, making protection of complexes and assembly processes from the highly reactive oxygen molecule unnecessary. However, as atmospheric oxygen accumulated, mechanisms and factors evolved to stabilize the complexes during assembly. This need for oxidative protection is not exclusive to the photosynthetic machinery but would apply to any complex with cofactors and features susceptible to oxidizing conditions. 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.

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

Characterization of spinach grana membranes by contact mode atomic force microscopy in aqueous medium distinguishes molecular features and the distribution of the lumen-exposed domains of PSII. 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.

Wiring photosystem I for electron transfer to a tethered redox dye

We have recently reported the assembly of biological/organic hybrid nanoconstructs that generate H2 in the light (Grimme et al., Dalton Trans., 2009, 10106, Lubner et al., Biochemistry, 2010, 49, 10264). In these constructs, electrons are transferred directly from a photochemical module, Photosystem I (PS I), to a catalytic module, either a Pt nanoparticle (NP) or an [FeFe]-hydrogenase (H2ase), through the use of a covalently attached molecular wire. In neither case are any spectroscopic changes visible that would allow electron transfer to be monitored between the photochemical and catalytic modules. In this study, the catalytic module was replaced with an organic cofactor consisting of 1-(3-thiopropyl)-1′-(methyl)-4,4′-bipyridinium chloride that allowed electron transfer to be measured to a spectroscopically observable marker. EPR and optical spectroscopy showed that the tethered redox cofactor was attached to PS I through the FB cluster of PsaC. Under steady-state illumination, the rate of reduction of the 4,4′-bipyridinium cofactor was comparable to the rate of H2 evolution observed for the PS I—molecular wire—Pt-NP and PS I—molecular wire—[FeFe]-H2ase nanoconstructs. These observations provide proof-of-concept for incorporating a redox cofactor in the molecular wire, thereby setting the stage for monitoring the rate and yield of electron transfer between PS I and the tethered [FeFe]-H2ase.

Kinetics of Pigment-Acceptor Interaction Induced by Continuous Illumination in Synechocystis spaeroides Photosystem I Preparations Cooled to 160 K in the Dark and Light

The kinetics of dark reduction of chlorophyll P700 oxidized by continuous light in preparations of photosystem I reaction centers from cyanobacterium Synechosystis spharoides cooled in the dark to 160 K is essentially nonexponential. The characteristic times of the components range from fractions of a second to minutes or more. During the cooling of reaction center preparations under illumination with actinic light, most of the chlorophyll P700 molecules are fixed in the oxidized state at 160 K. The kinetics of dark reduction of P700+ in the fraction of reaction centers that retain photochemical activity under these conditions is somewhat faster compared to the samples cooled in the dark. A theoretical analysis of substantial deceleration of P700+ dark recovery kinetics was done for preparations of photosystem I reaction centers oxidized by continuous light at 160 K in comparison to the experiments where reaction centers were oxidized by short single light flashes. This slowing down of the kinetics in samples excited by continuous illumination can be explained by microconformational relaxation processes related to proton shifts in the reaction center.