Interaction of flavodoxin with cyanobacterial thylakoids

Flavodoxin from the cyanobacterium Anabaena PCC 7119 has been shown to mediate, under illumination, the transfer of electrons from the thylakoidal membranes that were isolated from the same organism, to both the enzyme ferredoxin-NADP⁺ reductase and cytochrome c. Chemical cross-linking of ferredoxin or flavodoxin to the photosynthetic membranes provides a preparation that is active in cytochrome c photoreduction without the addition of external protein carrier. NADP⁺ photoreduction, albeit diminished, was observed only after addition of exogenous electron carrier protein. Immunoblotting analysis of the chemical adduct reveals that flavodoxin binds to a 10 kDa polypeptide subunit in the cyanobacterial Photosystem I which appears to act as its physiological partner in the electron transfer process.Abbreviations Fd ferredoxin - Fld flavodoxin - cyt c cytochrome c - EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide - PS I Photosystem I     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Evidence from directed mutagenesis that positively charged amino acids are necessary for interaction of nitrogenase with the [2Fe-2S) heterocyst ferredoxin (FdxH) from the cyanobacterium Anabaena sp., PCC7120

Sequence comparison of the heterocyst-type ferredoxin (FdxH) from Anabaena 7120 and type-I ferredoxins (PetF) from the same organism and other cyanobacteria revealed a group of positively charged residues characteristic for FdxH. Molecular modeling showed that these basic amino acids are clustered on the surface of FdxH. The corresponding domain of PetF contained acidic or nonpolar residues instead. To identify amino acids that are important for interaction with nitrogenase, we generated site-directed mutations in the fdxH gene and assayed the in vitro activity of the resulting recombinant proteins isolated from Escherichia coli. In addition to the point mutants, two chimeric proteins, FdxH : PetF and PetF : FdxH, were constructed containing the 58 N-terminal amino acids of one ferredoxin fused to the 40 C-terminal amino acids of the other. Exchange of lysines 10 and 11 of FdxH for the corresponding residues of PetF (glutamate 10 and alanine 11) resulted in a ferredoxin with greatly decreased affinity to nitrogenase. This indicates an important function of these basic amino acids in interaction with dinitrogenase reductase (NifH) from Anabaena. In addition we checked the reactivity of the recombinant ferredoxins with ferredoxin-NADP⁺ oxidoreductase (FNR) and photosystem I. The experiments with both the chimeric and point mutated ferredoxins showed that the C-terminal part of this protein determines its activity in NADP⁺ photoreduction.     

Immunoquantification of flavodoxin and ferredoxin from Scenedesmus vacuolatus (Chlorophyta) as iron-stress molecular markers

Quantification of the iron nutritional status of phytoplankton is of great interest not only for the study of the oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a molecular marker for iron stress, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin from Scenedesmus vacuolatus have been immunoquantified in cells grown under different iron nutritional conditions. Flavodoxin and ferredoxin levels correlate with the iron availability, and the calculated flavodoxin index can be used as an iron-stress marker. Other physiological parameters such as copper deficiency, heterotrophic or mixotrophic growth, nitrogen source and salt stress were also tested as potential factors influencing flavodoxin expression. Salt stress and heterotrophic growth conditions alter flavodoxin and ferredoxin expression. Once flavodoxin expression is repressed by iron (and severe deficiency alleviated), S.vacuolatus still increases its ferredoxin from 0·5 to 1·6 mol of ferredoxin per mole of ferredoxin-NADP⁺ reductase, and this ratio can be used for the evaluation of mild deficiency.     

Molecular characterization and overexpression of the petF gene from Synechococcus elongatus: Evidence for a second site of electrostatic interaction between ferredoxin and the PS I-D subunit

The petF gene from the cyanobacterium Synechococcus elongatus was isolated using the same gene from Synechocystis sp. PCC 6803 as a heterologous probe. The deduced primary sequence of the isolated single copy petF gene is identical to the primary sequence determined from the protein. Wild-type ferredoxin and a E93-95/Q93-95 mutant were overexpressed in E. coli and purified. Both types of ferredoxins are photoreduced by Photosystem I and can be cross-linked to the PsaD subunit of PS I, although with reduced affinity in case of the E93-95/Q93-95 mutant. These data indicate that the acidic patch of amino acids Glu94-95 of ferredoxin is most likely neither essential for the interaction of ferredoxin with PS I nor the only site of electrostatic contact with the PS I-D subunit. In contrast, NADP⁺photoreduction assays show drastically reduced rates in the presence of the E93-95/Q93-95 mutant ferredoxin, indicating that these residues play a crucial role in the interaction of ferredoxin with ferredoxin-NADP⁺reductase.     

Identification of the nifJ gene coding for pyruvate: ferredoxin oxidoreductase in dinitrogen-fixing cyanobacteria

A 329 bp DNA segment from both Anabaena variabilis and Anabaena PCC 7119 was amplified using the polymerase chain reaction (PCR). The sequences from the two cyanobacteria showed strong similarities to the corresponding part of the nifJ gene from Klebsiella pneumoniae and Enterobacter agglomerans. The present findings underline earlier results of enzymatic studies that heterocystous cyanobacteria possess a pyruvate: ferredoxin (flavodoxin) oxidoreductase (PFO). The nifJ gene segment could not be detected in the non-dinitrogenfixing, unicellular cyanobacterium Anacystis nidulans which is also in accord with previous findings from enzyme assays.     

Flavodoxin from the nitrogen-fixing cyanobacterium Anabaena PCC 7119

Flavodoxin has been isolated and purified from cultures of the cyanobacterium Anabaena cultivated in a low-iron medium. This flavoprotein has a molecular weight of 20,000 and contains 1 molecule of flavin mononucleotide per mol of protein. Various biochemical characteristics are reported including amino-acid composition, isoelectric point and the fluorescence properties of the apoprotein. The extinction coefficients and isosbestic points were determined for the oxidized and semiquinone forms of flavodoxin. The electron paramagnetic resonance spectrum of the semiquinone exhibited a spectral linewidth of 23 G, which is typical for a neutral flavoprotein semiquinone. Kinetic measurements give a rate constant of 9.6×10⁷ (M^-1 min^-1) for the reduction of flavodoxin in the photosynthetic electron-transport chain by the photosystem I and 6.6×10⁶ for the reaction in which flavodoxin is reduced by ferredoxin-NADP⁺ oxidoreductase. The Michaelis constant for electron donation to nitrogenase by reduced flavodoxin is 8.5 M.Abbreviations FMN flavin mononucleotide - FNR ferredoxin-NADP⁺ oxidoreductase - PSI photosystem I     

Chromosome locations of five genes encoding photosynthetic electron transfer proteins in Arabidopsis thaliana

The chromosome locations of nuclear genes encoding four photosynthetic electron transfer proteins have been determined by examining restriction fragment length polymorphisms in F₈recombinant inbred lines of Arabidopsis thaliana. The single-copy PetC gene encoding the chloroplast Rieske FeS protein was mapped to the top of chromosome 4, whereas the PetE and PetF genes encoding plastocyanin and ferredoxin, respectively, were mapped to different parts of chromosome 1. Two PetH genes encoding ferredoxin-NADP⁺oxidoreductase were mapped to the top of chromosome 1 and the bottom of chromosome 5.     

mrpA (all1838), a gene involved in alkali and Na sensitivity, may also have a role in energy metabolism in the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC7120 contains a gene, mrpA (all1838), which forms part of a seven gene-cluster (all1843–all1837) with significant sequence similarity to bacterial operons that putatively code for a multicomponent cation/proton antiporter involved in alkaline pH adaptation and salt resistance. We previously showed that growth and photosynthesis were inhibited in a strain mutated in mrpA, denoted as PHB11, particularly at alkaline pH. Here, we show that respiration was also impaired in the mutant independently of the external pH. In addition, at high pH, less ATP and vegetative cell ferredoxin were present in PHB11, which also showed lower levels of ferredoxin-NADP⁺ oxidoreductase (FNR). Ferredoxin and FNR are involved in the generation of reductant NADPH in cyanobacteria. These results suggest an energetic role of mrpA (and perhaps of the whole mrp-gene cluster) in Anabaena sp. PCC 7120 that is further supported by the significant similarity of putative Anabaena Mrp proteins to membrane subunits of complex I.     

Amino acid sequences of heterotrophic and photosynthetic ferredoxins from the tomato plant (Lycopersicon esculentum Mill.)

Several forms (isoproteins) of ferredoxin in roots, leaves, and green and red pericarps in tomato plants (Lycopersicon esculentum Mill.) were earlier identified on the basis of N-terminal amino acid sequence and chromatographic behavior (Green et al. 1991). In the present study, a large scale preparation made possible determination of the full length amino acid sequence of the two ferredoxins from leaves. The ferredoxins characteristic of fruit and root were sequenced from the amino terminus to the 30th residue or beyond. The leaf ferredoxins were confirmed to be expressed in pericarp of both green and red fruit. The ferredoxins characteristic of fruit and root appeared to be restricted to those tissue. The results extend earlier findings in demonstrating that ferredoxin occurs in the major organs of the tomato plant where it appears to function irrespective of photosynthetic competence.Abbreviations CBB Coomassie brilliant blue R-250 - Cm Carboxymethylated - Fd Ferredoxin - FNR ferredoxin-NADP⁺ oxidoreductase - FPLC Fast protein liquid chromatography - HPLC High performance liquid chromatography - rt root     

Stress response of transgenic tobacco plants expressing a cyanobacterial ferredoxin in chloroplasts

Expression of the chloroplast electron shuttle ferredoxin is induced by light through mechanisms that partially depend on sequences lying in the coding region of the gene, complicating its manipulation by promoter engineering. Ferredoxin expression is also down-regulated under virtually all stress situations, and it is unclear if light-dependent induction and stress-dependent repression proceed through the same or similar mechanisms. Previous reports have shown that expression of a cyanobacterial flavodoxin in tobacco plastids results in plants with enhanced tolerance to adverse environmental conditions such as drought, chilling and xenobiotics (Tognetti et al. in Plant Cell 18:2035–2050, 2006). The protective effect of flavodoxin was linked to functional replacement of ferredoxin, suggesting the possibility that tolerant phenotypes might be obtained by simply increasing ferredoxin contents. To bypass endogenous regulatory constraints, we transformed tobacco plants with a ferredoxin gene from Anabaena sp. PCC7120, which has only 53% identity with plant orthologs. The cyanobacterial protein was able to interact in vitro with ferredoxin-dependent plant enzymes and to mediate NADP⁺ photoreduction by tobacco thylakoids. Expression of Anabaena ferredoxin was constitutive and light-independent. However, homozygous lines accumulating threefold higher ferredoxin levels than the wild-type failed to show enhanced tolerance to oxidative stress and chilling temperatures. Under these adverse conditions, Anabaena ferredoxin was down-regulated even faster than the endogenous counterparts. The results indicate that: (1) light- and stress-dependent regulations of ferredoxin expression proceed through different pathways, and (2) overexpression of ferredoxin is not an alternative to flavodoxin expression for the development of increased stress tolerance in plants.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Cyanobacterial flavodoxin complements ferredoxin deficiency in knocked-down transgenic tobacco plants

Ferredoxins are the main electron shuttles in chloroplasts, accepting electrons from photosystem I and delivering them to essential oxido-reductive pathways in the stroma. Ferredoxin levels decrease under adverse environmental conditions in both plants and photosynthetic micro-organisms. In cyanobacteria and some algae, this decrease is compensated for by induction of flavodoxin, an isofunctional flavoprotein that can replace ferredoxin in many reactions. Flavodoxin is not present in plants, but tobacco lines expressing a plastid-targeted cyanobacterial flavodoxin developed increased tolerance to environmental stress. Chloroplast-located flavodoxin interacts productively with endogenous ferredoxin-dependent pathways, suggesting that its protective role results from replacement of stress-labile ferredoxin. We tested this hypothesis by using RNA antisense and interference techniques to decrease ferredoxin levels in transgenic tobacco. Ferredoxin-deficient lines showed growth arrest, leaf chlorosis and decreased CO(2) assimilation. Chlorophyll fluorescence measurements indicated impaired photochemistry, over-reduction of the photosynthetic electron transport chain and enhanced non-photochemical quenching. Expression of flavodoxin from the nuclear or plastid genome restored growth, pigment contents and photosynthetic capacity, and relieved the electron pressure on the electron transport chain. Tolerance to oxidative stress also recovered. In the absence of flavodoxin, ferredoxin could not be decreased below 45% of physiological content without fatally compromising plant survival, but in its presence, lines with only 12% remaining ferredoxin could grow autotrophically, with almost wild-type phenotypes. The results indicate that the stress tolerance conferred by flavodoxin expression in plants stems largely from functional complementation of endogenous ferredoxin by the cyanobacterial flavoprotein.     

Structure-function studies of [2Fe-2S] ferredoxins

The ability to overexpress [2Fe-2S] ferredoxins inEscherichia coli has opened up exciting research opportunities. High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegetative and heterocyst forms ofAnabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy. The electron delocalization in these proteins in their oxidized and reduced states has been studied by¹H,²H,¹³C, and¹⁵N NMR spectroscopy. Site-directed mutagenesis has been used to prepare variants of these ferredoxins. Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partnerAnabaena ferredoxin reductase. The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly. Electron transfer has been demonstrated with three of the four mutants. Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR. Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase. Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant).     

PetH is rate-controlling in the interaction between PetH, a component of the supramolecular complex with photosystem II, and PetF, a light-dependent electron transfer protein

Cyanobacterial PetH is similar to ferredoxin-NADP⁺ oxidoreductase (FNR) of higher plants and comprises 2 components, CpcD-like rod linker and FNR proteins. Here, I show that PetH controls the rate of the interaction with PetF (ferredoxin [Fd1]). Purified recombinant PetH protein, which cut off a CpcD-like rod linker domain, and Fd1 were used in detailed surface plasmon resonance analyses. The interaction between FNR and Fd1 chiefly involved extremely fast binding and dissociation reactions and the FNR affinity for Fd1 was stronger than the Fd1 affinity for FNR. The dissociation constant values were determined as approximately 93.65 μM (FNR) for Fd1 and 1.469 mM (Fd1) for FNR.     

An alternative plant-like cyanobacterial ferredoxin with unprecedented structural and functional properties

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = −440 mV, is lower than that of Fd1, Em = −372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP⁺ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Å resolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV–Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys₅₃ValAsnCys₅₆ of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.     

Complex formation by ferredoxin-NADP+ reductase with ferredoxin or NADP+

Complex formation by ferredoxin-NADP⁺ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.99.4) with ferredoxin was measured by the independent methods based on the changes of circular dichroism, fluorescence intensity and the chromatographic behavior on a Sephadex G-75 column of the two proteins after mixing. Complex formation between the flavoprotein and NADP⁺ was also detected from the changes of various optical properties of the protein. These experiments suggested that the optical changes accompanying the complex formation were due to a change of the chromophore group in ferredoxin-NADP⁺ reductase, but not due to that of ferredoxin.     

Ferredoxins as interchangeable redox components in support of MiaB,a radical S‐adenosylmethionine methylthiotransferase

MiaB is a member of the methylthiotransferase subclass of the radical S‐adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N⁶‐isopentenyl (i⁶A) group found at position 37 in several tRNAs to afford 2‐methylthio‐N⁶‐(isopentenyl)adenosine (ms²i⁶A). MiaB uses a reduced [4Fe–4S]⁺ cluster to catalyze a reductive cleavage of SAM to generate a 5′‐deoxyadenosyl 5′‐radical (5′‐dA?)—a required intermediate in its reaction—as well as an additional [4Fe–4S]²⁺ auxiliary cluster. In Escherichia coli and many other organisms, re‐reduction of the [4Fe–4S]²⁺ cluster to the [4Fe–4S]⁺ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin‐NADP⁺ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′‐dA? and ms²i⁶A production, indicating that only one hydrogen atom abstraction is required for the reaction.     

Ferredoxin and Ferredoxin-NADP Reductase from Photosynthetic and Nonphotosynthetic Tissues of Tomato

Ferredoxin and ferredoxin-NADP⁺ oxidoreductase (FNR) were purified from leaves, roots, and red and green pericarp of tomato (Lycopersicon esculentum, cv VFNT and cv Momotaro). Four different ferredoxins were identified on the basis of N-terminal amino acid sequence and charge. Ferredoxins I and II were the most prevalent forms in leaves and green pericarp, and ferredoxin III was the most prevalent in roots. Red pericarp of the VFNT cv yielded variable amounts of ferredoxins II and III plus a unique form, ferredoxin IV. Red pericarp of the Momotaro cv contained ferredoxins I, II, and IV. This represents the first demonstration of ferredoxin in a chromoplast-containing tissue. There were no major differences among the tomato ferredoxins in absorption spectrum or cytochrome c reduction activity. Two forms of FNR were present in tomato as judged by anion exchange chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FNR II had a lower apparent relative molecular weight, a slightly altered absorption spectrum, and a lower specific activity for cytochrome c reduction than FNR I. FNR II could be a partially degraded form of FNR I. The FNRs from the different tissues of tomato plants all showed diaphorase activity, with FNR II being more active than FNR I. The presence of ferredoxin and FNR in heterotrophic tissues of tomato is consistent with the existence of a nonphotosynthetic ferredoxin/FNR redox pathway to support the function of ferredoxin-dependent enzymes.