Search

Hydrogenasen sind H2-umsetzende Metalloenzyme und enthalten katalytische Kofaktoren, deren Eisenionen durch biologisch ungewöhnliche Kohlenmonoxid- (CO) und Cyanid- (CN−) Liganden koordiniert sind. Externes CO und CN−hemmt Hydrogenasen jedoch. Der molekulare Mechanismus der Bindung von CN− an [FeFe]-Hydrogenasen ist unbekannt. In dieser Studie präsentieren wir Kristallstrukturen der mit CN− behandelten [FeFe]-Hydrogenase CpI aus Clostridium pasteurianum. Auf Grund der hohen Auflösung von 1.39 Å können wir die intrinsischen CO- und CN−-Liganden voneinander unterscheiden. Wir zeigen, dass externes CN− die offene Bindestelle des Kofaktors besetzt, an die auch externes CO bindet. Im Gegensatz zu anderen Inhibitoren zeigen die CN−-behandelten Kristalle Konformationsänderungen konservierter Reste des Protonentransferpfads, die einen direkten Austausch von Protonen zwischen den Aminosäuren E279 und S319 ermöglichen. Diese Konformation wurde als notwendig für einen effizienten Protonentransfer vorgeschlagen, doch wurde sie bisher nicht strukturell nachgewiesen.

Hemoglobins (Hbs) utilize heme b as a cofactor and are found in all kingdoms of life. The current knowledge reveals an enormous variability of Hb primary sequences, resulting in topological, biochemical and physiological individuality. As Hbs appear to modulate their reactivities through specific combinations of structural features, predicting the characteristics of a given Hb is still hardly possible. The unicellular green alga Chlamydomonas reinhardtii contains 12 genes encoding diverse Hbs of the truncated lineage, several of which possess extended N- or C-termini of unknown function. Studies on some of the Chlamydomonas Hbs revealed yet unpredictable structural and biochemical variations, which, along with a different expression of their genes, suggest diverse physiological roles. Chlamydomonas thus represents a promising system to analyze the diversification of Hb structure, biochemistry and physiology. Here, we report the crystal structure, resolved to 1.75 Å, of the heme-binding domain of cyanomet THB11 (Cre16.g662750), one of the pentacoordinate algal Hbs, which offer a free Fe-coordination site in the reduced state. The overall fold of THB11 is conserved, but individual features such as a kink in helix E, a tilted heme plane and a clustering of methionine residues at a putative tunnel exit appear to be unique. Both N- and C-termini promote the formation of oligomer mixtures, and the absence of the C terminus results in reduced nitrite reduction rates. This work widens the structural and biochemical knowledge on the 2/2Hb family and suggests that the N- and C-terminal extensions of the Chlamydomonas 2/2Hbs modulate their reactivity by intermolecular interactions.

Metal cofactors considerably widen the catalytic space of naturally occurring enzymes whose specific and enantioselective catalytic activity constitutes a blueprint for economically relevant chemical syntheses. To optimize natural enzymes and uncover novel reactivity, we need a detailed understanding of cofactor–protein interactions, which can be challenging to obtain in the case of enzymes with sophisticated cofactors. As a case study, we summarize recent research on the [FeFe]-hydrogenases, which interconvert protons, electrons and dihydrogen at a unique iron-based active site. We can now chemically synthesize the complex cofactor and incorporate it into an apo-protein to afford functional enzymes. By varying both the cofactor and the polypeptide components, we have obtained detailed knowledge on what is required for a metal cluster to process H2. In parallel, the design of artificial proteins and catalytically active nucleic acids are advancing rapidly. In this Perspective, we introduce these fields and outline how chemists and biologists can use this knowledge to develop novel tailored semisynthetic catalysts. The study of [FeFe]-hydrogenases exemplifies how one can manipulate even sophisticated metal clusters to afford insights into structure–function relationships of biological catalysts. This Perspective describes developments in designing artificial proteins and catalytically active nucleic acids towards minimalistic and robust semi-biological catalysts for chemical synthesis.

Several species of unicellular microalgae such as the model species Chlamydomonas reinhardtii possess plastid-localized [FeFe]-hydrogenases which, via ferredoxin, can accept electrons from photosynthetic electron transport. Thereby, under specific conditions, these algae light-dependently produce molecular hydrogen (H 2 ), which offers a sustainable way to generate a “green” and efficient fuel. Until today, the most common way to induce sustained H 2 production is to deprive Chlamydomonas of macronutrients such as sulfur (S) which results in a downregulation of photosynthetic production of molecular oxygen (O 2 ) and of assimilatory processes. These acclimation responses allow the O 2 sensitive algal [FeFe]-hydrogenases to become active and serve as an alternative electron sink of photosynthesis. Despite much progress in the field and a general understanding of the underlying mechanisms, many basic and applied aspects of the photosynthetic H 2 metabolism of eukaryotic algae remain to be elucidated. One rarely investigated factor is that microalgae have also been reported to consume H 2 , especially as a response to high H 2 concentrations. Here, we analyzed the H 2 uptake activities of S-deprived Chlamydomonas cells incubated in different PBRs providing different gas phase volumes, either in continuous light or in the dark. We show that H 2 uptake occurs after prolonged incubation in the light as well as in sudden darkness. Dark-induced H 2 uptake can be delayed adding the phosphoribulose kinase inhibitor glycolaldehyde, suggesting a connection to carbohydrate metabolism. The results indicate that PBR setups as well as envisioned outdoor cultivation systems with natural light-dark cycles have to be carefully designed to prevent efficiency losses.

Proteins carrying an iron-porphyrin (heme) cofactor are essential for biological O2 management. The nature of Fe-O2 bonding in hemoproteins is debated for decades. We used energy-sampling and rapid-scan X-ray Kβ emission and K-edge absorption spectroscopy as well as quantum chemistry to determine molecular and electronic structures of unligated (deoxy), CO-inhibited (carboxy), and O2-bound (oxy) hemes in myoglobin (MB) and hemoglobin (HB) solutions and in porphyrin compounds at 20-260 K. Similar metrical and spectral features revealed analogous heme sites in MB and HB and the absence of low-spin (LS) to high-spin (HS) conversion. Amplitudes of Kβ main-line emission spectra were directly related to the formal unpaired Fe(d) spin count, indicating HS Fe(II) in deoxy and LS Fe(II) in carboxy. For oxy, two unpaired Fe(d) spins and, thus by definition, an intermediate-spin iron center, were revealed by our static and kinetic X-ray data, as supported by (time-dependent) density functional theory and complete-active-space self-consistent-field calculations. The emerging Fe-O2 bonding situation includes in essence a ferrous iron center, minor superoxide character of the noninnocent ligand, significant double-bond properties of the interaction, and three-center electron delocalization as in ozone. It resolves the apparently contradictory classical models of Pauling, Weiss, and McClure/Goddard into a unifying view of O2 bonding, tuned toward reversible oxygen transport.

Old Yellow Enzymes (OYEs) selectively reduce carbon‑carbon double bonds of a broad range of substrates with excellent stereoselectivity. Current challenges of their application in bio-based industrial processes are the cost-efficient regeneration of the nicotinamide co-substrate and expanding the range of industrially relevant substrates. Microalgae represent a rich and mostly untapped source for OYEs with novel catalytic properties and are promising whole-cell factories that regenerate nicotinamide by photosynthesis. By comparative phylogenetics, we identified 20 putative OYEs in eleven algal species. Three recombinant OYEs from the unicellular green alga Chlamydomonas reinhardtii (CrOYEs) reveal diverse biocatalytic properties and potential for enzyme optimization. High substrate conversion activities of living algal wild type and mutant cells linked the biocatalytic profiles to in vivo functionality and demonstrate the potential of microalgae as sources for distinctive OYE activities and for light-driven biotransformations.

Oxygenic phototrophs frequently encounter environmental conditions that result in intracellular energy crises. Growth of the unicellular green alga Chlamydomonas reinhardtii in hypoxia in the light depends on acclimatory responses of which the induction of photosynthetic cyclic electron flow is essential. The microalga cannot grow in the absence of molecular oxygen (O2 ) in the dark, although it possesses an elaborate fermentation metabolism. Not much is known about how the microalga senses and signals the lack of O2 or about its survival strategies during energy crises. Recently, nitric oxide (NO) has emerged to be required for the acclimation of C. reinhardtii to hypoxia. In this study, we show that the soluble guanylate cyclase (sGC) CYG12, a homologue of animal NO sensors, is also involved in this response. CYG12 is an active sGC, and post-transcriptional down-regulation of the CYG12 gene impairs hypoxic growth and gene expression in C. reinhardtii. However, it also results in a disturbed photosynthetic apparatus under standard growth conditions and the inability to grow heterotrophically. Transcriptome profiles indicate that the mis-expression of CYG12 results in a perturbation of responses that, in the wild-type, maintain the cellular energy budget. We suggest that CYG12 is required for the proper operation of the photosynthetic apparatus which, in turn, is essential for survival in hypoxia and darkness.

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H2) production and H2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity).

In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H2) as one of several fermentation products. H2 is generated mostly by the [Fe-Fe]-hydrogenase HYDA1, which uses plant type ferredoxin PETF/FDX1 (PETF) as an electron donor. Dark fermentation of the alga is mainly of the mixed acid type, because formate, ethanol, and acetate are generated by a pyruvate:formate lyase pathway similar to Escherichia coli. However, C. reinhardtii also possesses the pyruvate:ferredoxin oxidoreductase PFR1, which, like pyruvate:formate lyase and HYDA1, is localized in the chloroplast. PFR1 has long been suggested to be responsible for the low but significant H2 accumulation in the dark because the catalytic mechanism of pyruvate:ferredoxin oxidoreductase involves the reduction of ferredoxin. With the aim of proving the biochemical feasibility of the postulated reaction, we have heterologously expressed the PFR1 gene in E. coli. Purified recombinant PFR1 is able to transfer electrons from pyruvate to HYDA1, using the ferredoxins PETF and FDX2 as electron carriers. The high reactivity of PFR1 toward oxaloacetate indicates that in vivo, fermentation might also be coupled to an anaerobically active glyoxylate cycle. Our results suggest that C. reinhardtii employs a clostridial type H2 production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H2 evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA. Background: The pathway(s) of light-independent H2 production in green algae are yet unknown. Results: Pyruvate:ferredoxin oxidoreductase PFR1 and [Fe-Fe]-hydrogenase HYDA1 of Chlamydomonas can be coupled for pyruvate-dependent H2 production. Conclusion: H2 production by green algae in the dark is similar to bacterial PFOR-dependent fermentation. Significance: Understanding the fermentation metabolism of green algae allows insights into plastid bioenergetic pathways.

Chlamydomonas is a soil-dwelling freshwater alga that probably encounters hypoxic or anoxic conditions frequently in its natural habitat. Its repertoire of fermentative enzymes is remarkably complex and flexible. Despite this adaptability, at least under laboratory conditions, Chlamydomonas does not tolerate dark anoxia very well. It appears to respond to this condition by a state of quiescence, but still does not survive without light and molecular oxygen (O2) longer than a couple of days. The amounts of hundreds of transcripts change in anoxic algal cultures, but not much is known about the regulation of the anoxic response in Chlamydomonas. The phenotypes of mutants deficient for one or the other fermentative enzyme indicate a significant level of control by metabolic signals, and a subset of genes is coordinately regulated in copper and O2 deficiency by the copper response regulator1 (CRR1). However, the majority of genes that are differentially expressed in anaerobic Chlamydomonas cells are regulated CRR1 independently. While details on O2 sensing in Chlamydomonas remain elusive, principles and patterns begin to emerge, such as a possible influence of light and the involvement of nitric oxide (NO) as a signaling molecule. This chapter summarizes knowledge on the anoxic metabolism and its regulation in Chlamydomonas and hypothesizes on putative signaling pathways based on existing reports and observations.

Molecular hydrogen (H-2) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H-2 production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H-2 production comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H-2-producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.

The green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism characterized by a plastidic hydrogenase (HYD1) coupled to photosynthesis and a bacterial-type fermentation system in which pyruvate formate lyase (PFL1) is the central fermentative enzyme. To identify mutant strains with altered hydrogen metabolism, a C. reinhardtii nuclear transformant library was screened. Mutant strain 48F5 showed lower light-dependent hydrogen (H₂) evolution rates and reduced in vitro hydrogenase activity, and fermentative H₂ production in the dark was enhanced. The transformant has a single integration of the paromomycin resistance cassette within the PFL1 gene, and is unable to synthesize PFL1 protein. 48F5 secretes no formate, but produces more ethanol, D-lactate and CO₂ than the wild type. Moreover, HYD1 transcript and HYD1 protein levels were lower in the pfl1 mutant strain. Complementation of strain 48F5 with an intact copy of the PFL1 gene restored formate excretion and hydrogenase activity to the wild type level. This analysis shows that the PFL1 pathway has a significant impact on hydrogen metabolism in C. reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism and reacts to hypoxic or anaerobic conditions with the induced expression of many genes. One gene which is upregulated particularly strongly is the FDX5 gene, encoding one of at least six ferredoxin isoforms in C. reinhardtii . Fdx5 is a typical plant-type 2Fe2S protein that is located in the chloroplast. The FDX5 promoter region contains three GTAC motifs, which are known to be the binding sites for copper response regulator 1 (Crr1) and other SQUAMOSA promoter binding proteins (SBPs). This study shows that two of these GTAC sites are essential to confer oxygen and also copper responsiveness to a reporter gene. The SBP domain of Crr1 is able to bind to both of these GTAC sites in in vitro binding assays. Moreover, in a Crr1-deficient C. reinhardtii strain, FDX5 is not expressed. These results clearly indicate that Crr1 is involved in the transcriptional regulation of the FDX5 gene in the absence of oxygen or copper.

Several species of unicellular green algae, such as the model green microalga Chlamydomonas reinhardtii, can operate under either aerobic photosynthesis or anaerobic metabolism conditions. A particularly interesting metabolic condition is that of "anaerobic oxygenic photosynthesis", whereby photosynthetically generated oxygen is consumed by the cell's own respiration, causing anaerobiosis in the culture in the light, and induction of the cellular "hydrogen metabolism" process. The latter entails an alternative photosynthetic electron transport pathway, through the oxygen-sensitive FeFe-hydrogenase, leading to the light-dependent generation of molecular hydrogen in the chloroplast. The FeFe-hydrogenase is coupled to the reducing site of photosystem-I via ferredoxin and is employed as an electron-pressure valve, through which electrons are dissipated, thus permitting a sustained electron transport in the thylakoid membrane of photosynthesis. This hydrogen gas generating process in the cells offers testimony to the unique photosynthetic metabolism that can be found in many species of green microalgae. Moreover, it has attracted interest by the biotechnology and bioenergy sectors, as it promises utilization of green microalgae and the process of photosynthesis in renewable energy production. This article provides an overview of the principles of photobiological hydrogen production in microalgae and addresses in detail the process of induction and analysis of the hydrogen metabolism in the cells. Furthermore, methods are discussed by which the interaction of photosynthesis, respiration, cellular metabolism, and H(2) production in Chlamydomonas can be monitored and regulated.

The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen-sensitive [Fe-Fe] hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and, in addition, a formate- and ethanol-producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl; EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes but only rarely in eukaryotes. Both the hydrogen- and the formate/ethanol-producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulfur depletion in illuminated cultures. Before now, the presence of a Pfl protein in C. reinhardtii was predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate-producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl-deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii . We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulfur depletion. Genetic and biochemical analyses show that sulfur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and also potentially ethanol-producing enzymes, such as pyruvate decarboxylase (EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.

Main conclusion Annotated hemoglobin genes inChlamydomonas reinhardtiiform functional globins, despite unusual architectures. Spectral characteristics show subtle biochemical differences. Multiple globins might help the alga to cope with its versatile environment. The unicellular green alga C. reinhardtii is a photosynthetic, often soil-dwelling organism, subjected to a changeable environment in nature. The alga contains 12 genes encoding so-called truncated hemoglobins that feature a two-on-two helical fold instead of the three-on-three helix arrangement of the long-studied vertebrate globins or plant symbiotic and non-symbiotic hemoglobins. In plants, non-symbiotic hemoglobins often play a role in acclimation to stress, and we could show recently that one of the C. reinhardtii globin genes is vital for anoxic growth. Here, three further globin encoding transcripts (Cre16.g661000.t1.1, Cre16.g661300.t2.1 and Cre16.g662750.t1.2) were heterologously expressed along with the recently studied THB1. UV–Vis and X-ray absorption spectroscopy analyses show that the sequences indeed encode functional hemoglobins, despite their uncommon primary sequences, which include long C-termini without any predictable function, or a split heme-binding domain. The proteins show some variations regarding the coordination of the heme iron or the interaction with diatomic ligands, indicating different functionalities. The respective transcripts are not responsive to the nitrogen source, in contrast to results reported for THB1, but they accumulate in darkness. This work advances experimental data on the very large globin family in general, and, more specifically, on hemoglobins in photosynthetic organisms.

International audience; Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts.

Recent studies indicate that [Fe]-hydrogenases and H 2 metabolism are widely distributed among green algae. The enzymes are simple structured and catalyze H 2 evolution with similar rates than the more complex [Fe]-hydrogenases from bacteria. Different green algal species developed diverse strategies to survive under sulfur deprivation. Chlamydomonas reinhardtii evolves large quantities of hydrogen gas in the absence of sulfur. In a sealed culture of C. reinhardtii , the photosynthetic O 2 evolution rate drops below the rate of respiratory O 2 consumption due to a reversible inhibition of photosystem II, thus leading to an intracellular anaerobiosis. The algal cells survive under these anaerobic conditions by switching their metabolism to a kind of photo-fermentation. Although possessing a functional [Fe]-hydrogenase gene, the cells of Scenedesmus obliquus produce no significant amounts of H 2 under S-depleted conditions. Biochemical analyses indicate that S. obliquus decreases almost the complete metabolic activities while maintaining a low level of respiratory activity.

Plastids are the compartments in which oxygenic photosynthesis of higher plants and eukaryotic microalgae converts the energy of sunlight into chemical energy. The latter is used by green cells to generate the whole palette of organic molecules needed to build a cell. Mankind has made use of the green powerhouses from the beginning of its existence on, but in the last decades, products other than food or fire wood have gained importance. Facing the deprivation of fossil fuels and climate changes due to anthropologically caused greenhouse effects, we want to use photosynthetically converted light energy as an energy source to generate renewable energy carriers. Above all, the synthesis of biodiesel made from plant or algal lipids is a promising strategy. As the cultivation of microalgae does not compete with food production, research focuses on understanding and engineering lipid biosynthesis in these unicellular organisms. Additionally, in contrast to higher plants, some green algae are capable of using the process of photosynthesis for the generation of another biofuel: molecular hydrogen. In this chapter, the pathways resulting in the generation of hydrogen and lipids in the plastid are reviewed. Additionally, proven and anticipated targets of biotechnological optimization are highlighted.

Metalloproteins utilize metal cofactors to catalyze essential reactions in all organisms. They carry out thermodynamically challenging substrate conversions such as the oxidation of water or hydrocarbons, the reduction of nitrogen to ammonium, and generation of molecular hydrogen. Besides their fundamental role in nature, metalloenzymes have promising biotechnological applications that aim to generate high-value chemicals, drugs, nutrients, biofuels, or electricity. Recent reports that a chemically synthesized compound is able to reconstitute [2Fe]-hydrogenases, harboring an especially elaborate and highly efficient metal cofactor, promise to pave the way for gaining much deeper insight into the function of even complex metal enzymes. What is more, synthetic biology approaches such as the chemical synthesis of artificial hydrogenases seem to be in reach.

In aerobes, anoxia impairs mitochondrial energy generation as well as biosynthesis and degradation of essential cell components. In a recent analysis we have shown that the unicellular green alga Chlamydomonas reinhardtii responds to anaerobiosis in the dark by significant changes of the transcriptome, which, in summary, were directed at saving and economizing energy. Several of the transcriptional changes were related to photosynthesis and were accompanied by reduced amounts of chlorophylls and plastid lipids as well as lowered photosystem 2 quantum yields. A further noticeable pattern was a transcriptional upregulation of various genes encoding O 2 dependent enzymes of central biosynthetic pathways. However, cells do not divide in dark-anoxia, indicating that C. reinhardtii cannot compensate for the lack of O 2 and light. Upon return to aeration and light, cultures show severe photo-bleaching, which might be a stress reaction, but also part of an acclimation process or its disturbance.

Anaerobiosis is a stress condition for aerobic organisms and requires extensive acclimation responses. We used RNA-Seq for a whole-genome view of the acclimation of Chlamydomonas reinhardtii to anoxic conditions imposed simultaneously with transfer to the dark. Nearly 1.4 × 103 genes were affected by hypoxia. Comparing transcript profiles from early (hypoxic) with those from late (anoxic) time points indicated that cells activate oxidative energy generation pathways before employing fermentation. Probable substrates include amino acids and fatty acids (FAs). Lipid profiling of the C. reinhardtii cells revealed that they degraded FAs but also accumulated triacylglycerols (TAGs). In contrast with N-deprived cells, the TAGs in hypoxic cells were enriched in desaturated FAs, suggesting a distinct pathway for TAG accumulation. To distinguish transcriptional responses dependent on COPPER RESPONSE REGULATOR1 (CRR1), which is also involved in hypoxic gene regulation, we compared the transcriptomes of crr1 mutants and complemented strains. In crr1 mutants, ∼40 genes were aberrantly regulated, reaffirming the importance of CRR1 for the hypoxic response, but indicating also the contribution of additional signaling strategies to account for the remaining differentially regulated transcripts. Based on transcript patterns and previous results, we conclude that nitric oxide–dependent signaling cascades operate in anoxic C. reinhardtii cells.

Hemoglobins are recognized today as a diverse family of proteins present in all kingdoms of life and performing multiple reactions beyond O 2 chemistry. The physiological roles of most hemoglobins remain elusive. Here, we show that a 2-on-2 (“truncated”) hemoglobin, termed THB8, is required for hypoxic growth and the expression of anaerobic genes in Chlamydomonas reinhardtii . THB8 is 1 of 12 2-on-2 hemoglobins in this species. It belongs to a subclass within the 2-on-2 hemoglobin class I family whose members feature a remarkable variety of domain arrangements and lengths. Posttranscriptional silencing of the THB8 gene results in the mis-regulation of several genes and a growth defect under hypoxic conditions. The latter is intensified in the presence of an NO scavenger, which also impairs growth of wild-type cells. As recombinant THB8 furthermore reacts with NO, the results of this study indicate that THB8 is part of an NO-dependent signaling pathway.

The unicellular green alga Chlamydomonas reinhardtii is able to use photosynthetically provided electrons for the production of molecular hydrogen by an [FeFe]-hydrogenase HYD1 accepting electrons from ferredoxin PetF. Despite the severe sensitivity of HYD1 towards oxygen, a sustained and relatively high photosynthetic hydrogen evolution capacity is established in C. reinhardtii cultures when deprived of sulfur. One of the major electron sources for proton reduction under this condition is the oxidation of starch and subsequent non-photochemical transfer of electrons to the plastoquinone pool. Here we report on the induction of photosynthetic hydrogen production by Chlamydomonas upon nitrogen starvation, a nutritional condition known to trigger the accumulation of large deposits of starch and lipids in the green alga. Photochemistry of photosystem II initially remained on a higher level in nitrogen-starved cells, resulting in a 2-day delay of the onset of hydrogen production compared with sulfur-deprived cells. Furthermore, though nitrogen-depleted cells accumulated large amounts of starch, both hydrogen yields and the extent of starch degradation were significantly lower than upon sulfur deficiency. Starch breakdown rates in nitrogen or sulfur-starved cultures transferred to darkness were comparable in both nutritional conditions. Methyl viologen treatment of illuminated cells significantly enhanced the efficiency of photosystem II photochemistry in sulfur-depleted cells, but had a minor effect on nitrogen-starved algae. Both the degradation of the cytochrome b 6 f complex which occurs in C. reinhardtii upon nitrogen starvation and lower ferredoxin amounts might create a bottleneck impeding the conversion of carbohydrate reserves into hydrogen evolution.

The green alga Chlamydomonas reinhardtii has a complex anaerobic metabolism characterized by a plastidic hydrogenase (HYD1) coupled to photosynthesis and a bacterial-type fermentation system in which pyruvate formate lyase (PFL1) is the central fermentative enzyme. To identify mutant strains with altered hydrogen metabolism, a C. reinhardtii nuclear transformant library was screened. Mutant strain 48F5 showed lower light-dependent hydrogen (H₂) evolution rates and reduced in vitro hydrogenase activity, and fermentative H₂ production in the dark was enhanced. The transformant has a single integration of the paromomycin resistance cassette within the PFL1 gene, and is unable to synthesize PFL1 protein. 48F5 secretes no formate, but produces more ethanol, D-lactate and CO₂ than the wild type. Moreover, HYD1 transcript and HYD1 protein levels were lower in the pfl1 mutant strain. Complementation of strain 48F5 with an intact copy of the PFL1 gene restored formate excretion and hydrogenase activity to the wild type level. This analysis shows that the PFL1 pathway has a significant impact on hydrogen metabolism in C. reinhardtii.

Oxygenic photosynthesis uses light as energy source to generate an oxidant powerful enough to oxidize water into oxygen, electrons and protons. Upon linear electron transport, electrons extracted from water are used to reduce NADP+ to NADPH. The oxygen molecule has been integrated into the cellular metabolism, both as the most efficient electron acceptor during respiratory electron transport and as oxidant and/or “substrate” in a number of biosynthetic pathways. Though photosynthesis of higher plants, algae and cyanobacteria produces oxygen, there are conditions under which this type of photosynthesis operates under hypoxic or anaerobic conditions. In the unicellular green alga Chlamydomonas reinhardtii, this condition is induced by sulfur deficiency, and it results in the production of molecular hydrogen. Research on this biotechnologically relevant phenomenon has contributed largely to new insights into additional pathways of photosynthetic electron transport, which extend the former concept of linear electron flow by far. This review summarizes the recent knowledge about various electron sources and sinks of oxygenic photosynthesis besides water and NADP+ in the context of their contribution to hydrogen photoproduction by C. reinhardtii. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

The unicellular green alga Chlamydomonas reinhardtii has at least six plant-type ferredoxins (FDX). Besides the long-known photosynthetic ferredoxin PetF the isoforms Fdx2–Fdx6 have been identified. The FDX genes are differentially expressed under various environmental conditions such as the availability of oxygen, copper, iron and ammonium. Recently, the anaerobically induced Fdx5 as well as Fdx2, which is involved in nitrite reduction were characterized in more detail. Moreover, it was shown that PetF, the central and most abundant FDX of C. reinhardtii , is a suitable partner of the hydrogenase HydA1. Using mutant variants of both PetF and HydA1, amino acid residues essential for the interaction of both proteins could be identified. These findings will help to tailor PetF for achieving an optimized photobiotechnological hydrogen production in C. reinhardtii , which might also benefit from new insights into the mechanism of how oxygen attacks the active site metal cluster of HydA1. This review gives an update on recent advances in understanding the function of ferredoxins and the hydrogenase in C. reinhardtii .

Background Sealed Chlamydomonas reinhardtii cultures evolve significant amounts of hydrogen gas under conditions of sulfur depletion. However, the eukaryotic green alga goes through drastic metabolic changes during this nutritional stress resulting in cell growth inhibition and eventually cell death. This study aimed at isolating C. reinhardtii transformants which produce hydrogen under normal growth conditions to allow a continuous hydrogen metabolism without the stressful impact of nutrient deprivation. Results To achieve a steady photobiological hydrogen production, a screening protocol was designed to identify C. reinhardtii DNA insertional mutagenesis transformants with an attenuated photosynthesis to respiration capacity ratio (P/R ratio). The screening protocol entails a new and fast method for mutant strain selection altered in their oxygen production/consumption balance. Out of 9000 transformants, four strains with P/R ratios varying from virtually zero to three were isolated. Strain apr1 was found to have a slightly higher respiration rate and a significantly lower photosynthesis rate than the wild type. Sealed cultures of apr1 became anaerobic in normal growth medium (TAP) under moderate light conditions and induced [FeFe]-hydrogenase activity, yet without significant hydrogen gas evolution. However, Calvin-Benson cycle inactivation of anaerobically adapted apr1 cells in the light led to a 2-3-fold higher in vivo hydrogen production than previously reported for the sulfur-deprived C. reinhardtii wild type. Conclusion Attenuated P/R capacity ratio in microalgal mutants constitutes a platform for achieving steady state photobiological hydrogen production. Using this platform, algal hydrogen metabolism can be analyzed without applying nutritional stress. Furthermore, these strains promise to be useful for biotechnological hydrogen generation, since high in vivo hydrogen production rates are achievable under normal growth conditions, when the photosynthesis to respiration capacity ratio is lowered in parallel to down regulated assimilative pathways.

The unicellular green alga Chlamydomonas reinhardtii possesses a [FeFe]-hydrogenase HydA1 (EC 1.12.7.2), which is coupled to the photosynthetic electron transport chain. Large amounts of H2 are produced in a light-dependent reaction for several days when C. reinhardtii cells are deprived of sulfur. Under these conditions, the cells drastically change their physiology from aerobic photosynthetic growth to an anaerobic resting state. The understanding of the underlying physiological processes is not only important for getting further insights into the adaptability of photosynthesis, but will help to optimize the biotechnological application of algae as H2 producers. Two of the still most disputed questions regarding H2 generation by C. reinhardtii concern the electron source for H2 evolution and the competition of the hydrogenase with alternative electron sinks. We analyzed the H2 metabolism of S-depleted C. reinhardtii cultures utilizing a special mass spectrometer setup and investigated the influence of photosystem II (PSII)- or ribulosebisphosphate-carboxylase/oxygenase (Rubisco)-deficiency. We show that electrons for H2-production are provided both by PSII activity and by a non-photochemical plastoquinone reduction pathway, which is dependent on previous PSII activity. In a Rubisco-deficient strain, which produces H2 also in the presence of sulfur, H2 generation seems to be the only significant electron sink for PSII activity and rescues this strain at least partially from a light-sensitive phenotype. The latter indicates that the down-regulation of assimilatory pathways in S-deprived C. reinhardtii cells is one of the important prerequisites for a sustained H2 evolution.

The photosynthetic green alga Chlamydomonas reinhardtii is capable of performing a complex fermentative metabolism which is related to the mixed acid fermentation of bacteria such as Escherichia coli. The fermentative pattern includes the products formate, ethanol, acetate, glycerol, lactate, carbon dioxide and molecular hydrogen (H2). H2 production is catalysed by an active [Fe]-hydrogenase (HydA) which is coupled with the photosynthetic electron-transport chain. The most important enzyme of the classic fermentation pathway is pyruvate formate-lyase, which is common in bacteria but seldom found in eukaryotes. An interaction between fermentation, photosynthesis and H2 evolution allows the algae to overcome long periods of anaerobiosis. In the absence of sulphur, the cells establish a photofermentative metabolism and accumulate large amounts of H2.

In Chlamydomonas reinhardtii cells, H 2 photoproduction can be induced in conditions of sulfur deprivation in the presence of acetate. The decrease in photosystem II (PSII) activity induced by sulfur deprivation leads to anoxia, respiration becoming higher than photosynthesis, thereby allowing H 2 production. Two different electron transfer pathways, one PSII dependent and the other PSII independent, have been proposed to account for H 2 photoproduction. In this study, we investigated the contribution of both pathways as well as the acetate requirement for H 2 production in conditions of sulfur deficiency. By using 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a PSII inhibitor, which was added at different times after the beginning of sulfur deprivation, we show that PSII-independent H 2 photoproduction depends on previously accumulated starch resulting from previous photosynthetic activity. Starch accumulation was observed in response to sulfur deprivation in mixotrophic conditions (presence of acetate) but also in photoautotrophic conditions. However, no H 2 production was measured in photoautotrophy if PSII was not inhibited by DCMU, due to the fact that anoxia was not reached. When DCMU was added at optimal starch accumulation, significant H 2 production was measured. H 2 production was enhanced in autotrophic conditions by removing O 2 using N 2 bubbling, thereby showing that substantial H 2 production can be achieved in the absence of acetate by using the PSII-independent pathway. Based on these data, we discuss the possibilities of designing autotrophic protocols for algal H 2 photoproduction.

Publisher Summary Future energy sources need to be unlimited, renewable, and environmentally neutral, and hydrogen (H 2 )-photoproduction by green algae from water and sunlight perfectly meets these requirements. The theoretical potential of [Fe]-hydrogenases for H 2 -evolution is indisputably high; however, the physiological activity of the up-to-now characterized algal strains has not reached the necessary efficiency for a biotechnological application. This chapter presents two different strategies, which, in combination, could lead to this aim. The identification and molecular biological characterization of the up-to-now only marginal examined alga species C. moewusii are described in the chapter. Particularities such as additional or displaced cystein residues or deviations in important regions of protein architecture indicate that the hydA proteins of C. moewusii belong to a new hydrogenase subtype. Therefore, C. moewusii seems to be an interesting new candidate for further physiological examinations. The chapter also presents a new technique for the generation and identification of mutant strains with an altered physiological H 2 production activity.

Green algae are the only known eukaryotes with both oxygenic photosynthesis and a hydrogen metabolism. Recent physiological and genetic discoveries indicate a close connection between these metabolic pathways. The anaerobically inducible hydA genes of algae encode a special type of highly active [Fe]-hydrogenase. Electrons from reducing equivalents generated during fermentation enter the photosynthetic electron transport chain via the plastoquinone pool. They are transferred to the hydrogenase by photosystem I and ferredoxin. Thus, the [Fe]-hydrogenase is an electron 'valve' that enables the algae to survive under anaerobic conditions. During sulfur deprivation, illuminated algal cultures evolve large quantities of hydrogen gas, and this promises to be an alternative future energy source.

We have found the transcript of one of at least six ferredoxin encoding genes of the green alga Chlamydomonas reinhardtii, FDX5, strongly accumulating in anaerobiosis, indicating a vital role of the encoded protein in the anaerobic metabolism of the cells. According to absorption and electron paramagnetic resonance spectroscopy, Fdx5 is a plant-type [2Fe2S]-ferredoxin with a redox potential similar to that of the ferredoxin PetF. However, although Fdx5 seems to be located in the chloroplast, it is not able to photoreduce nicotinamide adenine dinucleotide phosphate (NADP+) via ferredoxin-NADP-reductase, nor to be an electron donor to the plastidic [FeFe]-hydrogenase HydA1. Thus, Fdx5 seems to have a special role in a yet to be identified anaerobic pathway.