The Alga Uronema belkae Has Two Structural Types of [FeFe]-Hydrogenases with Different Biochemical Properties.

Several species of microalgae can convert light energy into molecular hydrogen (H2) by employing enzymes of early phylogenetic origin, [FeFe]-hydrogenases, coupled to the photosynthetic electron transport chain. Bacterial [FeFe]-hydrogenases consist of a conserved domain that harbors the active site cofactor, the H-domain, and an additional domain that binds electron-conducting FeS clusters, the F-domain. In contrast, most algal hydrogenases characterized so far have a structurally reduced, so-termed M1-type architecture, which consists only of the H-domain that interacts directly with photosynthetic ferredoxin PetF as an electron donor. To date, only a few algal species are known to contain bacterial-type [FeFe]-hydrogenases, and no M1-type enzymes have been identified in these species. Here, we show that the chlorophycean alga Uronema belkae possesses both bacterial-type and algal-type [FeFe]-hydrogenases. Both hydrogenase genes are transcribed, and the cells produce H2 under hypoxic conditions. The biochemical analyses show that the two enzymes show features typical for each of the two [FeFe]-hydrogenase types. Most notable in the physiological context is that the bacterial-type hydrogenase does not interact with PetF proteins, suggesting that the two enzymes are integrated differently into the alga's metabolism.

Insights into the Molecular Mechanism of Formaldehyde Inhibition of [FeFe]-Hydrogenases.

[FeFe]-hydrogenases are efficient H2 converting biocatalysts that are inhibited by formaldehyde (HCHO). The molecular mechanism of this inhibition has so far not been experimentally solved. Here, we obtained high-resolution crystal structures of the HCHO-treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum, showing HCHO reacts with the secondary amine base of the catalytic cofactor and the cysteine C299 of the proton transfer pathway which both are very important for catalytic turnover. Kinetic assays via protein film electrochemistry show the CpI variant C299D is significantly less inhibited by HCHO, corroborating the structural results. By combining our data from protein crystallography, site-directed mutagenesis and protein film electrochemistry, a reaction mechanism involving the cofactor's amine base, the thiol group of C299 and HCHO can be deduced. In addition to the specific case of [FeFe]-hydrogenases, our study provides additional insights into the reactions between HCHO and protein molecules.

Chlamydomonas reinhardtii mutants deficient for Old Yellow Enzyme 3 exhibit increased photooxidative stress.

Old Yellow Enzymes (OYEs) are flavin-containing ene-reductases that have been intensely studied with regard to their biotechnological potential for sustainable chemical syntheses. OYE-encoding genes are found throughout the domains of life, but their physiological role is mostly unknown, one reason for this being the promiscuity of most ene-reductases studied to date. The unicellular green alga Chlamydomonas reinhardtii possesses four genes coding for OYEs, three of which we have analyzed biochemically before. Ene-reductase CrOYE3 stood out in that it showed an unusually narrow substrate scope and converted N-methylmaleimide (NMI) with high rates. This was recapitulated in a C. reinhardtii croye3 mutant that, in contrast to the wild type, hardly degraded externally added NMI. Here we show that CrOYE3-mediated NMI conversion depends on electrons generated photosynthetically by photosystem II (PSII) and that the croye3 mutant exhibits slightly decreased photochemical quenching in high light. Non-photochemical quenching is strongly impaired in this mutant, and it shows enhanced oxidative stress. The phenotypes of the mutant suggest that C. reinhardtii CrOYE3 is involved in the protection against photooxidative stress, possibly by converting reactive carbonyl species derived from lipid peroxides or maleimides from tetrapyrrole degradation.

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway.

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN- ) ligands. Extrinsic CO and CN- , however, inhibit hydrogenases. The mechanism by which CN- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Šallowed us to distinguish intrinsic CN- and CO ligands and to show that extrinsic CN- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.

Distinctive structural properties of THB11, a pentacoordinate Chlamydomonas reinhardtii truncated hemoglobin with N- and C-terminal extensions.

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.

The soluble guanylate cyclase CYG12 is required for the acclimation to hypoxia and trophic regimes in Chlamydomonas reinhardtii.

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.

Effective intermediate-spin iron in O2-transporting heme proteins.

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 Kß 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 Kß 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.

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

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).

Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.

Molecular hydrogen (H2 ) 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 H2 production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H2 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 H2 -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.

Characterization of unusual truncated hemoglobins of Chlamydomonas reinhardtii suggests specialized functions.

MAIN CONCLUSION: Annotated hemoglobin genes in Chlamydomonas reinhardtii form 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.

Metalloprotein mimics - old tools in a new light.

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.

Photo-bleaching of Chlamydomonas reinhardtii after dark-anoxic incubation.

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.

Copper response regulator1-dependent and -independent responses of the Chlamydomonas reinhardtii transcriptome to dark anoxia.

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 × 10(3) 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.

Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic.

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.

Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide.

Hemoglobins are recognized today as a diverse family of proteins present in all kingdoms of life and performing multiple reactions beyond O2 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.

Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii.

In anaerobiosis, the green alga Chlamydomonas reinhardtii evolves molecular hydrogen (H(2)) as one of several fermentation products. H(2) 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 H(2) 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 H(2) production pathway in the dark, especially because C. reinhardtii PFR1 was also able to allow H(2) evolution in reaction mixtures containing Clostridium acetobutylicum 2[4Fe-4S]-ferredoxin and [Fe-Fe]-hydrogenase HYDA.

Differential expression of the Chlamydomonas [FeFe]-hydrogenase-encoding HYDA1 gene is regulated by the copper response regulator1.

The unicellular green alga Chlamydomonas reinhardtii adapts to anaerobic or hypoxic conditions by developing a complex fermentative metabolism including the production of molecular hydrogen by [FeFe]-hydrogenase isoform1 (HYDA1). HYDA1 transcript and hydrogenase protein accumulate in the absence of oxygen or copper (Cu). Factors regulating this differential gene expression have been unknown so far. In this study, we report on the isolation of a Chlamydomonas mutant strain impaired in HYDA1 gene expression by screening an insertional mutagenesis library for HYDA1 promoter activity using the arylsulfatase-encoding ARYLSULFATASE2 gene as a selection marker. The mutant strain has a deletion of the COPPER RESPONSE REGULATOR1 (CRR1) gene encoding for CRR1, indicating that this SQUAMOSA-PROMOTER BINDING PROTEIN (SBP) domain transcription factor is involved in the regulation of HYDA1 transcription. Treating the C. reinhardtii wild type with mercuric ions, which were shown to inhibit the binding of the SBP domain to DNA, prevented or deactivated HYDA1 gene expression. Reporter gene analyses of the HYDA1 promoter revealed that two GTAC motifs, which are known to be the cores of CRR1 binding sites, are necessary for full promoter activity in hypoxic conditions or upon Cu starvation. However, mutations of the GTAC sites had a much stronger impact on reporter gene expression in Cu-deficient cells. Electrophoretic mobility shift assays showed that the CRR1 SBP domain binds to one of the GTAC cores in vitro. These combined results prove that CRR1 is involved in HYDA1 promoter activation.

Nitrogen deprivation results in photosynthetic hydrogen production in Chlamydomonas reinhardtii.

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 b6 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.

Alternative photosynthetic electron transport pathways during anaerobiosis in the green alga Chlamydomonas 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.

A pyruvate formate lyase-deficient Chlamydomonas reinhardtii strain provides evidence for a link between fermentation and hydrogen production in green algae.

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.