Flavodiiron Proteins Research Articles

Diversity and complexity of flavodiiron NO/O2 reductases.

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-β-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.

Faster photosynthetic induction in tobacco by expressing cyanobacterial flavodiiron proteins in chloroplasts.

Plants grown in the field experience sharp changes in irradiation due to shading effects caused by clouds, other leaves, etc. The excess of absorbed light energy is dissipated by a number of mechanisms including cyclic electron transport, photorespiration, and Mehler-type reactions. This protection is essential for survival but decreases photosynthetic efficiency. All phototrophs except angiosperms harbor flavodiiron proteins (Flvs) which relieve the excess of excitation energy on the photosynthetic electron transport chain by reducing oxygen directly to water. Introduction of cyanobacterial Flv1/Flv3 in tobacco chloroplasts resulted in transgenic plants that showed similar photosynthetic performance under steady-state illumination, but displayed faster recovery of various photosynthetic parameters, including electron transport and non-photochemical quenching during dark-light transitions. They also kept the electron transport chain in a more oxidized state and enhanced the proton motive force of dark-adapted leaves. The results indicate that, by acting as electron sinks during light transitions, Flvs contribute to increase photosynthesis protection and efficiency under changing environmental conditions as those found by plants in the field.

Spectroscopy and DFT Calculations of a Flavo-diiron Enzyme Implicate New Diiron Site Structures.

Flavo-diiron proteins (FDPs) are non-heme iron containing enzymes that are widespread in anaerobic bacteria, archaea, and protozoa, serving as the terminal components to dioxygen and nitric oxide reductive scavenging pathways in these organisms. FDPs contain a dinuclear iron active site similar to that in hemerythrin, ribonucleotide reductase, and methane monooxygenase, all of which can bind NO and O2. However, only FDP competently turns over NO to N2O. Here, EPR and Mössbauer spectroscopies allow electronic characterization of the diferric and diferrous species of FDP. The exchange-coupling constant J (Hex = JS1·S2) was found to increase from +20 cm-1 to +32 cm-1 upon reduction of the diferric to the diferrous species, indicative of (1) at least one hydroxo bridge between the iron ions for both states and (2) a change to the diiron core structure upon reduction. In comparison to characterized diiron proteins and synthetic complexes, the experimental values were consistent with a dihydroxo bridged diferric core, which loses one hydroxo bridge upon reduction. DFT calculations of these structures gave values of J and Mössbauer parameters in agreement with experiment. Although the crystal structure shows a hydrogen bond between the iron bound aspartate and the bridging solvent molecule, the DFT calculations of structures consistent with the crystal structure gave calculated values of J incompatible with the spectroscopic results. We conclude that the crystal structure of the diferric state does not represent the frozen solution structure and that a mono-μ-hydroxo diferrous species is the catalytically functional state that reacts with NO and O2. The new EPR spectroscopic probe of the diferric state indicated that the diferric structure of FDP prior to and immediately after turnover with NO are flavin mononucleotide (FMN) dependent, implicating an additional proton transfer role for FMN in turnover of NO.

Opposite domination of cyclic and pseudocyclic electron flows in short-illuminated dark-adapted leaves of angiosperms and gymnosperms.

The present work was aimed to explain the recently reported higher O2-dependent electron flow capacity in gymnosperms than in angiosperms and to search for other differences in the electron transport processes by simultaneous characterization of the relative capacities of pseudocyclic (direct or Flavodiiron proteins (Flv)-mediated O2-reduction, Mehler(-like) reactions) and cyclic electron flows around photosystem I (CEF-PSI). To this end, a comparative multicomponent analysis was performed on the fluorescence decay curves of dark-adapted leaves after illumination with a 1-s saturating light pulse. In both gymnosperms and angiosperms, two or three exponential decay components were resolved: fast (t 1/21 ~ 170-260ms), middle (~1.0-2.3s), and slow (>4.2s). The sensitivity of the decay parameters (amplitudes A1-3, halftimes t 1/2 1-3) to the alternative electron flows was assessed using Arabidopsis pgr5 and ndhM mutants, defective in CEF-PSI, Synechocystis sp. PCC 6803 Δflv1 mutant, defective in Flv-mediated O2-photoreduction, different O2 concentrations, and methyl viologen treatment. A1 reflected the part of electrons involved in linear and O2-photoreduction pathways after PSI. The middle component appeared in pgr5 (but not in ndhM), in gymnosperms under low O2, and in Δflv1, and reflected limitations at the PSI acceptor side. The slow component was sensitive to CEF-PSI. The comparison of decay parameters provided evidence that Flv mediate O2-photoreduction in gymnosperms, which explains their higher O2-dependent electron flow capacity. The concomitant quantification of relative electrons branching in O2-photoreduction and CEF-PSI pathways under the applied non-steady-state photosynthetic conditions reveals that CEF-PSI capacity significantly exceeds that of O2-photoreduction in angiosperms while the opposite occurs in gymnosperms.

Diversity of strategies for escaping reactive oxygen species production within photosystem I among land plants: P700 oxidation system is prerequisite for alleviating photoinhibition in photosystem I.

In land plants, photosystem I (PSI) photoinhibition limits carbon fixation and causes growth defects. In addition, recovery from PSI photoinhibition takes much longer than PSII photoinhibition when the PSI core-complex is degraded by oxidative damage. Accordingly, PSI photoinhibition should be avoided in land plants, and land plants should have evolved mechanisms to prevent PSI photoinhibition. However, such protection mechanisms have not yet been identified, and it remains unclear whether all land plants suffer from PSI photoinhibition in the same way. In the present study, we focused on the susceptibility of PSI to photoinhibition and investigated whether mechanisms of preventing PSI photoinhibition varied among land plant species. To assess the susceptibility of PSI to photoinhibition, we used repetitive short-pulse (rSP) illumination, which specifically induces PSI photoinhibition. Subsequently, we found that land plants possess a wide variety of tolerance mechanisms against PSI photoinhibition. In particular, gymnosperms, ferns and mosses/liverworts exhibited higher tolerance to rSP illumination-induced PSI photoinhibition than angiosperms, and detailed analyses indicated that the tolerance of these groups could be partly attributed to flavodiiron proteins, which protected PSI from photoinhibition by oxidizing the PSI reaction center chlorophyll (P700) as an electron acceptor. Furthermore, we demonstrate, for the first time, that gymnosperms, ferns and mosses/liverworts possess a protection mechanism against photoinhibition of PSI that differs from that of angiosperms.

Flavodiiron Proteins Promote Fast and Transient O2 Photoreduction in Chlamydomonas.

During oxygenic photosynthesis, the reducing power generated by light energy conversion is mainly used to reduce carbon dioxide. In bacteria and archae, flavodiiron (Flv) proteins catalyze O2 or NO reduction, thus protecting cells against oxidative or nitrosative stress. These proteins are found in cyanobacteria, mosses, and microalgae, but have been lost in angiosperms. Here, we used chlorophyll fluorescence and oxygen exchange measurement using [18O]-labeled O2 and a membrane inlet mass spectrometer to characterize Chlamydomonas reinhardtii flvB insertion mutants devoid of both FlvB and FlvA proteins. We show that Flv proteins are involved in a photo-dependent electron flow to oxygen, which drives most of the photosynthetic electron flow during the induction of photosynthesis. As a consequence, the chlorophyll fluorescence patterns are strongly affected in flvB mutants during a light transient, showing a lower PSII operating yield and a slower nonphotochemical quenching induction. Photoautotrophic growth of flvB mutants was indistinguishable from the wild type under constant light, but severely impaired under fluctuating light due to PSI photo damage. Remarkably, net photosynthesis of flv mutants was higher than in the wild type during the initial hour of a fluctuating light regime, but this advantage vanished under long-term exposure, and turned into PSI photo damage, thus explaining the marked growth retardation observed in these conditions. We conclude that the C. reinhardtii Flv participates in a Mehler-like reduction of O2, which drives a large part of the photosynthetic electron flow during a light transient and is thus critical for growth under fluctuating light regimes.

Alternative electron transport mediated by flavodiiron proteins is operational in organisms from cyanobacteria up to gymnosperms.

Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.

The Liverwort, Marchantia, Drives Alternative Electron Flow Using a Flavodiiron Protein to Protect PSI.

The diffusion efficiency of oxygen in the atmosphere, like that of CO2, is approximately 104 times greater than that in aqueous environments. Consequently, terrestrial photosynthetic organisms need mechanisms to protect against potential oxidative damage. The liverwort Marchantia polymorpha, a basal land plant, has habitats where it is exposed to both water and the atmosphere. Furthermore, like cyanobacteria, M. polymorpha has genes encoding flavodiiron proteins (FLV). In cyanobacteria, FLVs mediate oxygen-dependent alternative electron flow (AEF) to suppress the production of reactive oxygen species. Here, we investigated whether FLVs are required for the protection of photosynthesis in M. polymorpha A mutant deficient in the FLV1 isozyme (ΔMpFlv1) sustained photooxidative damage to photosystem I (PSI) following repetitive short-saturation pulses of light. Compared with the wild type (Takaragaike-1), ΔMpFlv1 showed the same photosynthetic oxygen evolution rate but a lower electron transport rate during the induction phase of photosynthesis. Additionally, the reaction center chlorophyll in PSI, P700, was highly reduced in ΔMpFlv1 but not in Takaragaike-1. These results indicate that the gene product of MpFlv1 drives AEF to oxidize PSI, as in cyanobacteria. Furthermore, FLV-mediated AEF supports the production of a proton motive force to possibly induce the nonphotochemical quenching of chlorophyll fluorescence and suppress electron transport in the cytochrome b6/f complex. After submerging the thalli, a decrease in photosystem II operating efficiency was observed, particularly in ΔMpFlv1, which implies that species living in these sorts of habitats require FLV-mediated AEF.

Dissecting the Photoprotective Mechanism Encoded by the flv4-2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization.

In Synechocystis sp. PCC 6803, the flv4-2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4-2 operon mutants. In the ∆sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ∆sll0218-flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ∆sll0218 and ∆sll0218-flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218-lacking mutants. The YFP-tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA-defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ∆flv4-2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.

Flavodiiron proteins act as safety valve for electrons in Physcomitrella patens

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.

Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase

Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

Microoxic Niches within the Thylakoid Stroma of Air-Grown Chlamydomonas reinhardtii Protect [FeFe]-Hydrogenase and Support Hydrogen Production under Fully Aerobic Environment.

Photosynthetic hydrogen production in the microalga Chlamydomonas reinhardtii is catalyzed by two [FeFe]-hydrogenase isoforms, HydA1 and HydA2, both irreversibly inactivated upon a few seconds exposure to atmospheric oxygen. Until recently, it was thought that hydrogenase is not active in air-grown microalgal cells. In contrast, we show that the entire pool of cellular [FeFe]-hydrogenase remains active in air-grown cells due to efficient scavenging of oxygen. Using membrane inlet mass spectrometry, (18)O2 isotope, and various inhibitors, we were able to dissect the various oxygen uptake mechanisms. We found that both chlororespiration, catalyzed by plastid terminal oxidase, and Mehler reactions, catalyzed by photosystem I and Flavodiiron proteins, significantly contribute to oxygen uptake rate. This rate is considerably enhanced with increasing light, thus forming local anaerobic niches at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells.

Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803.

Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity.

The Flavodiiron Protein Flv3 Functions as a Homo-Oligomer During Stress Acclimation and is Distinct from the Flv1/Flv3 Hetero-Oligomer Specific to the O2 Photoreduction Pathway.

The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3. By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2. Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT.

Photosynthesis: Rewiring an angiosperm.

Flavodiiron proteins help to protect cyanobacteria from excess light through the dissipation of excess electrons. Introducing moss flavodiiron proteins into Arabidopsis raises its resilience to fluctuating light, illustrating the potential for augmenting higher plants with photosynthetic components from lower plants, algae and cyanobacteria.

Artificial remodelling of alternative electron flow by flavodiiron proteins in Arabidopsis

In photosynthesis, linear electron transport from water to nicotinamide adenine dinucleotide phosphate (NADP(+)) cannot satisfy the ATP/NADPH production stoichiometry required by the Calvin-Benson cycle. Cyclic electron transport (CET) around photosystem I (PSI) and pseudocyclic electron transport (pseudoCET) can produce ATP without the accumulation of NADPH. Flavodiiron proteins (Flv) are the main mediator of pseudoCET in photosynthetic organisms, spanning cyanobacteria to gymnosperms. However, their genes are not conserved in angiosperms. Here we explore the possibility of complementing CET with Flv-dependent pseudoCET in the angiosperm Arabidopsis thaliana. We introduced FlvA and FlvB genes from the moss Physcomitrella patens into both wild-type (WT) Arabidopsis and the proton gradient regulation 5 (pgr5) mutant, which is defective in the main pathway of CET. We measured rates of pseudoCET using membrane inlet mass spectrometry, along with several photosynthetic parameters. Flv expression significantly increased rates of pseudoCET in the mutant plants, particularly at high light intensities, and partially restored the photosynthetic phenotype. In WT plants, Flv did not compete with PGR5-dependent CET during steady-state photosynthesis, but did form a large electron sink in fluctuating light. We conclude that flavodiiron proteins can help to protect the photosystems in Arabidopsis under fluctuating light, even in the presence of CET.

The dual function of flavodiiron proteins: oxygen and/or nitric oxide reductases.

Flavodiiron proteins have emerged in the last two decades as a newly discovered family of oxygen and/or nitric oxide reductases widespread in the three life domains, and present in both aerobic and anaerobic organisms. Herein we present the main features of these fascinating enzymes, with a particular emphasis on the metal sites, as more appropriate for this special issue in memory of the exceptional bioinorganic scientist R. J. P. Williams who pioneered the notion of (metal) element availability-driven evolution. We also compare the flavodiiron proteins with the other oxygen and nitric oxide reductases known until now, highlighting how throughout evolution Nature arrived at different solutions for similar functions, in some cases adding extra features, such as energy conservation. These enzymes are an example of the (bioinorganic) unpredictable diversity of the living world.

Entamoeba thiol-based redox metabolism: A potential target for drug development

Amebiasis is an intestinal infection widespread throughout the world caused by the human pathogen Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its low efficacy against asymptomatic cyst carriers and emergence of resistance in other protozoa with similar anaerobic metabolism. Therefore, identification and characterization of specific targets is urgently needed to design new therapeutics for improved treatment against amebiasis. Toward this goal, thiol-dependent redox metabolism is of particular interest. The thiol-dependent redox metabolism in E. histolytica consists of proteins including peroxiredoxin, rubrerythrin, Fe-superoxide dismutase, flavodiiron proteins, NADPH: flavin oxidoreductase, and amino acids including l-cysteine, S-methyl-l-cysteine, and thioprolines (thiazolidine-4-carboxylic acids). E. histolytica completely lacks glutathione and its metabolism, and l-cysteine is the major intracellular low molecular mass thiol. Moreover, this parasite possesses a functional thioredoxin system consisting of thioredoxin and thioredoxin reductase, which is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In this review, we summarize and highlight the thiol-based redox metabolism and its control mechanisms in E. histolytica, in particular, the features of the system unique to E. histolytica, and its potential use for drug development against amebiasis.

Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation.

The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O2 in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O2 downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO2 levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H2 photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport.

The LysR-type transcription factor PacR is a global regulator of photosynthetic carbon assimilation in Anabaena.

Cyanobacteria perform water-splitting photosynthesis and are important primary producers impacting the carbon and nitrogen cycles at global scale. They fix CO2 through ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) and have evolved a distinct CO2 concentrating mechanism (CCM) that builds high CO2 concentrations in the vicinity of RuBisCo favouring its carboxylase activity. Filamentous cyanobacteria such as Anabaena fix CO2 in photosynthetic vegetative cells, which donate photosynthate to heterocysts that rely on a heterotrophic metabolism to fix N2 . CCM elements are induced in response to inorganic carbon limitation, a cue that exposes the photosynthetic apparatus to photodamage by over-reduction. An Anabaena mutant lacking the LysR-type transcription factor All3953 grew poorly and dies under high light. The rbcL operon encoding RuBisCo was induced upon carbon limitation in the wild type but not in the mutant. ChIP-Seq analysis was used to globally identify All3953 targets under carbon limitation. Targets include, besides rbcL, genes encoding CCM elements, photorespiratory pathway- photosystem- and electron transport-related components, and factors, including flavodiiron proteins, with a demonstrated or putative function in photoprotection. Quantitative reverse transcription polymerase chain reaction analysis of selected All3953 targets showed regulation in the wild type but not in the mutant. All3953 (PacR) is a global regulator of carbon assimilation in an oxygenic photoautotroph.