Efficiency of ferredoxins and flavodoxins as mediators in systems for hydrogen evolution.

1. The efficiencies of ferredoxins and flavodoxins from a range of sources as mediators in systems for hydrogen evolution were assessed. 2. In supporting electron transfer from dithionite to hydrogenase of the bacterium Clostridium pasteurianum, highest activity was shown by the ferredoxin from the cyanobacterium Chlorogloeopsis fritschii and flavodoxin from the bacterium Megasphaera elsdenii. The latter was some twenty times as active as comparable concentrations of Methyl Viologen. Ferredoxins from the cyanobacterium Anacystis nidulans and the red alga Porphyra umbilicalis also showed high activity. 3. In mediating electron transfer from chloroplast membranes to Clostridium pasteurianum hydrogenase the flavodoxin from Anacystis nidulans proved the most active with Nostoc strain MAC flavodoxin and Porphyra umbilicalis ferredoxin also being appreciably more active than other cyanobacterial and higher plant ferredoxins. 4. In both hydrogenase systems the ferredoxin and flavodoxin from the red alga Chondrus crispus and the ferredoxin from another red alga Gigartina stellata showed very low activity. 5. There appeared to be no apparent correlation of efficiency in supporting hydrogenase activity with midpoint redox potential (Em) of the mediators, though some correlation of Em with the efficiency of the mediators in supporting NADP+ photoreduction by chloroplasts, or pyruvate oxidation by a Clostridium pasteurianum system, was evident. 6. Activity of the mediators in the hydrogenase systems therefore primarily reflects differences in tertiary structure conferring differing affinities for the other components of the systems.     

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic,Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO₂-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.     

Cytochrome cM from synechocystis 6803. Detection in cells, expression in Escherichia coli, purification and physical characterization.

Based on DNA sequence data a novel c-type cytochrome, cytochrome cM, has been predicted to exist in the cyanobacterium Synechocystis 6803. The precursor protein consists of 105 amino acids with a characteristic heme-binding motif and a hydrophobic domain located at the N-terminal end that is proposed to act as either a signal peptide or a membrane anchor. For the first time we report the detection of cytochrome cM in Synechocystis 6803 using Western blot analysis. The soluble portion cytochrome cM has been overexpressed in Escherichia coli in two forms, one with a poly histidine tag to facilitate purification and one without such a tag. The overexpressed protein has been purified and shown to bind heme, exhibiting an absorption peak in the Soret band near 416 nm and a peak in the alpha band at 550 nm. The extinction coefficient of cytochrome cM is 23.2 +/- 0.5 mM-1.cm-1 for the reduced minus oxidized alpha band peak (550-535 nm). The isoelectric point of cytochrome cM is 5.6 (without the histidine tag), which is significantly lower than the pI of 7.2 predicted from the amino acid sequence. The redox midpoint potential of cytochrome cM expressed in E. coli is 151 +/- 5 mV (pH 7.1), which is quite low compared to other c-type cytochromes in which a histidine and a methionine residue serve as the axial ligands to the heme. This work opens the way for determining the three-dimensional structure of cytochrome cM and investigating its function in cyanobacteria.     

Characterization of a redox active cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin.

A covalent stoichiometric complex between photosystem I (PSI) and ferredoxin from the cyanobacterium Synechocystis sp. PCC 6803 was generated by chemical cross-linking. The photoreduction of ferredoxin, studied by laser flash absorption spectroscopy between 460 and 600 nm, is a fast process in 60% of the covalent complexes, which exhibit spectral and kinetic properties very similar to those observed with the free partners. Two major phases with t(1/2) <1 micros and approximately 10-14 micros are observed at two different pH values (5.8 and 8.0). The remaining complexes do not undergo fast ferredoxin reduction and 20-25% of the complexes are still able to reduce free ferredoxin or flavodoxin efficiently, thus indicating that ferredoxin is not bound properly in this proportion of covalent complexes. The docking site of ferredoxin on PSI was determined by electron microscopy in combination with image analysis. Ferredoxin binds to the cytoplasmic side of PSI, with its mass center 77 angstroms distant from the center of the trimer and in close contact with a ridge formed by the subunits PsaC, PsaD and PsaE. This docking site corresponds to a close proximity between the [2Fe- 2S] center of ferredoxin and the terminal [4Fe-4S] acceptor FII of PSI and is very similar in position to the docking site of flavodoxin, an alternative electron acceptor of PSI.     

Complex formation between ferredoxin and ferredoxin-NADP+ reductase from Anabaena PCC 7119: cross-linking studies.

Ferredoxin-NADP+ reductase and ferredoxin from the cyanobacterium Anabaena PCC 7119 have been covalently cross-linked by incubation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The covalent adduct, which shows a molecular mass consistent with a 1:1 stoichiometry of the two proteins, maintains nearly 60% of the NADPH-cytochrome c reductase activity of the enzyme saturated with ferredoxin and this value is considerably higher than when equimolar amounts of both proteins are assayed. No ternary complexes with Anabaena flavodoxin or horse heart cytochrome c were formed, suggesting that the binding site on the enzyme is the same for ferredoxin and flavodoxin and that ferredoxin-NADP+ reductase and cytochrome c bind at a common site on ferredoxin. In the noncovalent complex, titrated at pH 7, the oxidation-reduction potential of ferredoxin becomes 15 mV more negative and that of ferredoxin-NADP+ reductase 27 mV more positive compared to the proteins alone. When covalently linked, the midpoint potential of the enzyme has a value similar to that in the noncovalent complex, while the ferredoxin potential is 20 mV more positive compared to ferredoxin alone. The changes in redox potentials have been used to estimate the dissociation constants for the interaction of the different redox forms of the proteins, based on the value of 1.21 microM calculated for the oxidized noncovalent complex.     

Comparative characterization of ferredoxins from heterocysts and vegetative cells of Anabaena variabilis

The isolation and purification of a ferredoxin from heterocysts of Anabaena variabilis (ATCC 29413) is described. Heterocyst ferredoxin differs from vegetative cell ferredoxin in amino-acid composition, molecular weight, midpoint redox potential, optical and EPR spectra and in its immunological properties. The data confirm the view of a new ferredoxin synthesized in heterocysts for specific interaction with nitrogenase.     

Purification and characterization of ferredoxin-NADP+ reductase from the green alga Chlorella fusca

Ferredoxin-NADP⁺ reductase (FNR, EC I.18.1.2) from the green algae Chlorella fusca Shihira et Kraus 211–15, was purified to homogeneity. The molecular mass was 36.8 kDa as determined by SDS-polyacrylamide gel electrophoresis. The enzyme exhibits the typical spectrum of a flavoprotein with an absorption maximum at 459 nm and an A_273/459 ratio of 7.2. It contains one mol of FAD per mol of protein and the calculated extinction coefficient is 9.8 m M cm⁻¹. Four different forms of the purified enzyme were detected by isoelectric focusing (pI between 5.4 and 5.9), even when protease inhibitors were used during the first steps of the purification. Kinetic parameters were determined for several FNR-catalyzed reactions. NADP⁺ photoreduction gave comparable rates when either ferredoxin or flavodoxin was used.     

Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I reaction center of the cyanobacterium Synechocystis sp. PCC 6803 contains seven different polypeptide subunits. The subunit with a molecular mass of about 8 kDa was isolated, and the sequence of its amino-terminal residues was determined. Oligonucleotide probes corresponding to this sequence were used to isolate the gene encoding this subunit. The gene, termed as psaE, codes for a polypeptide with a mass of 8075 Da. It is present as a single copy in the genome and is transcribed as a monocistronic messenger. The amino acid sequence of the 8-kDa subunit deduced from the gene sequence shows high homology with the deduced amino acid sequence of subunit IV of photosystem I from spinach. The DNA fragment sequenced in these studies also contains two other unidentified major open reading frames. A stable deletion mutation for the psaE gene was generated by transforming Synechocystis sp. PCC 6803 with a cloned DNA in which the psaE gene for 8-kDa subunit was replaced by a gene conferring resistance to kanamycin. The mutant strain shows minor differences in growth under photoautotrophic conditions and in the photosystem I activity in comparison to the wild type.     

Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli

The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two different glutamate synthases whose genes, gltB and glsF (previously known as gltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-767). The glsF gene has been expressed in the glutamate auxotrophic Escherichia coli strain CLR207 RecA, but the corresponding protein does not complement the auxotrophy. The transformed strain showed ferredoxin-dependent glutamate synthase (Fd-GOGAT) activity, demonstrating the capability of E. coli for providing and correctly assembling both the iron-sulfur center and the flavin cofactor of the enzyme. Fd-GOGAT (GlsF) is correctly cleaved at Cys37 to form the mature enzyme in E. coli, as occurs with the large subunit of its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electrophoretic homogeneity, using as the main purification step a ferredoxin-affinity chromatography. The pure enzyme, with a molecular mass of about 180 kDa, shows an absorption spectrum characteristic of iron-sulfur flavoproteins. The analyses of the prosthetic groups indicate that Fd-GOGAT contains only one FMN, but no FAD, and one [3Fe-4S](+,0) cluster per molecule. Oxidation-reduction titration, using absorbance changes of the FMN group in the visible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent K(M) values similar to those of the native Synechocystis protein: 4.5 vs 3.5 microM, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 2-oxoglutarate, respectively. The addition of the reductant dithionite to the enzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxoglutarate, in the presence of glutamine.     

Heterogeneous Rieske proteins in the cytochrome b6f complex of Synechocystis PCC6803?

The completely sequenced genome of the cyanobacterium Synechocystis PCC6803 contains three open reading frames, petC1, petC2, and petC3, encoding putative Rieske iron-sulfur proteins. After heterologous overexpression, all three gene products have been characterized and shown to be Rieske proteins as typified by sequence analysis and EPR spectroscopy. Two of the overproduced proteins contained already incorporated iron-sulfur clusters, whereas the third one formed unstable aggregates, in which the FeS cluster had to be reconstituted after refolding of the denatured protein. Although EPR spectroscopy showed typical FeS signals for all Rieske proteins, an unusual low midpoint potential was revealed for PetC3 by EPR redox titration. Detailed characterization of Synechocystis membranes indicated that all three Rieske proteins are expressed under physiological conditions. Both for PetC1 and PetC3 the association with the thylakoid membrane was shown, and both could be identified, although in different amounts, in the isolated cytochrome b(6)f complex. The considerably lower redox potential determined for PetC3 indicates heterogeneous cytochrome b(6)f complexes in Synechocystis and suggests still to be established alternative electron transport routes.     

Crystal structure of heme oxygenase-1 from cyanobacterium Synechocystis sp. PCC 6803 in complex with heme.

Heme oxygenase (HO) catalyzes the oxidative degradation of heme utilizing molecular oxygen and reducing equivalents. In photosynthetic organisms, HO functions in the biosynthesis of such open-chain tetrapyrroles as phyto-chromobilin and phycobilins, which are involved in the signal transduction for light responses and light harvesting for photosynthesis, respectively. We have determined the first crystal structure of a HO-1 from a photosynthetic organism, Synechocystis sp. PCC 6803 (Syn HO-1), in complex with heme at 2.5 A resolution. Heme-Syn HO-1 shares a common folding with other heme-HOs. Although the heme pocket of heme-Syn HO-1 is, for the most part, similar to that of mammalian HO-1, they differ in such features as the flexibility of the distal helix and hydrophobicity. In addition, 2-propanol derived from the crystallization solution occupied the hydrophobic cavity, which is proposed to be a CO trapping site in rat HO-1 that suppresses product inhibition. Although Syn HO-1 and mammalian HO-1 are similar in overall structure and amino acid sequence (57% similarity vs. human HO-1), their molecular surfaces differ in charge distribution. The surfaces of the heme binding sides are both positively charged, but this patch of Syn HO-1 is narrow compared to that of mammalian HO-1. This feature is suited to the selective binding of ferredoxin, the physiological redox partner of Syn HO-1; the molecular size of ferredoxin is approximately 10 kDa whereas the size of NADPH-cytochrome P450 reductase, a reducing partner of mammalian HO-1, is approximately 77 kDa. A docking model of heme-Syn HO-1 and ferredoxin suggests indirect electron transfer from an iron-sulfur cluster in ferredoxin to the heme iron of heme-Syn HO-1.     

Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.     

Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp. PCC 6803 in vivo in ssr2016 deletion mutants generated either in a wild-type background or in a ndhB deletion mutant. Our results indicate that ssr2016 is required for FQR and that it operates in a parallel pathway to the NDH1 complex. The ssr2016 deletion mutants are high light sensitive, suggesting that FQR might be important in controlling redox poise under adverse conditions.     

Direct electron transfer of redox proteins at the bare glassy carbon electrode

A simple system is presented for the microscale, direct voltammetry of redox proteins, typically 25 micrograms, in the absence of mediators and/or modifiers. The sample consists of a droplet of anaerobic solution laid onto an oversized disc of nitric-acid-pretreated glassy carbon as the working electrode. Very reproducible, Nernstian responses are obtained with horse heart cytochrome c. The midpoint potential Em (pH 7.0) is dependent on the ionic strength, ranging from $293 mV in 1 mM potassium phosphate to $266 mV in 0.1 M phosphate. At fixed buffer and cytochrome concentrations the magnitude of the voltammetric response is found to be independent of pH over six pH units around neutrality. It is suggested that the response of the present system is not complicated by pH-dependent properties of the electrode surface around physiological pH and, therefore, that the use of this system is practical in biochemically oriented studies. Direct, quasi-reversible responses have also been obtained at pH 7.0 (5 mM phosphate) from Desulfovibrio vulgaris. Hildenborough strain, tetraheme cytochrome c3 (pI = 10.0 at 4 C; 3 X Em = -0.32 mV, Em = -0.26 V), and cytochrome c553 (pI = 9.3; Em = +60 mV), and from Megasphaera elsdenii rubredoxin (pI congruent to 3; Em = -353 mV). The latter protein absorbs onto the glassy carbon surface, thus forming a system with possible applications in the electrochemical study of ferredoxin-linked enzymes.     

Isolation and properties of the soluble c-type cytochromes of the dinoflagellate Peridinium cinctum

Four soluble cytochromes of the c type were isolated from the freshwater dinoflagellate Peridinium cinctum collected from Lake Kinneret, Israel. Cytochrome c with alpha-band maximum at 550 nm in the reduced state had a molecular mass of 10,200 Da, pI 7.4, and Em of 278 m V. This cytochrome was active in the respiratory chain of beef heart Keilin-Hartree particles. Cytochrome c-553 had a molecular mass of 13,200 Da, pI 4.9, and Em of 384 m V, and was active in light induced electron transport of Euglena gracilis chloroplast fragments. Cytochrome c-554 had a molecular mass of 13,500 Da, pI 4.4, and Em of 326 m V. This cytochrome was inactive in light induced electron transport but competed with cytochrome c-552 of Euglena in the assay. The acidic cytochrome c-557 was present in very small quantities. The properties of the soluble c-type cytochromes of P. cinctum are compatible with the classification of dinoflagellates as primitive eucaryotes.     

Cloning, sequence analysis and expression of Pseudoalteromonas elyakovii IAM 14594 gene (alyPEEC) encoding the extracellular alginate lyase.

A gene (alyPEEC) encoding an alginate lyase of Pseudoalteromonas elyakovii IAM 14594 was cloned using the plasmid vector pUC118 and expressed in Escherichia coli. Sequencing of a 3.0kb fragment revealed a 1,197bp open reading frame encoding 398 amino acid residues. The calculated molecular mass and isoelectric point of the alyPEEC gene product are 43.2 kDa and pI 5.29. A region G(165) to V(194) in the AlyPEEC internal sequence is identical to the N-terminal amino acid sequence of the previously purified extracellular alginate lyase of P. elyakovii, and the calculated molecular mass (25.4 kDa) and isoelectric point (pI 4.78) of the region resembled those of the purified enzyme. Expression of enzymically-active alginate lyase from alyPEEC required growth of recombinant E. coli in LB broth containing 50% (v/v) artificial seawater (ASW). Alginate lyase activity with broad substrate specificity was detected in both 42 and 30 kDa products. Subcloning of the region G(165) to N(398) of AlyPEEC corresponding to the 30 kDa protein confirmed that this region of the alyPEEC gene encoded the active site of the enzyme. A region A(32) to G(164) corresponding to about 13 kDa of the N-terminal region of AlyPEEC showed about 30% identity to a putative chitin binding domain of Streptomyces chitinases, but did not exhibit any catalytic activity.     

Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803.

NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate. The enzyme from the unicellular cyanobacterium Synechocystis sp. PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps. The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein. The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa. Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor. Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor. The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM). The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet. The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase. The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E. coli enzyme.     

Inactivation of ycf33 results in an altered cyclic electron transport pathway around photosystem I in Synechocystis sp. PCC6803

ycf33 encodes a small protein with a molecular mass of 7.5 kDa and is found from cyanobacteria to higher plants. A ycf33 deletion mutant was constructed in Synechocystis sp. PCC6803 and characterized. The mutant showed a higher phycobilisome/chlorophyll ratio than the wild type and a higher photosystem II/photosystem I fluorescence ratio measured at 77 K. Under photoautotrophic conditions, the growth rates were not much different from those of the wild type. Cyclic electron transport activities around photosystem I were not much different between the wild type and the mutant. However, the effects of diphenyleneiodonium, an inhibitor of flavoprotein, on cyclic electron transport in the mutant were different from those in the wild type; it was severely inhibited in the wild type but not much in the mutant. Together with the effects of nitrite, which accepts electrons from ferredoxin via nitrite reductase and those of HgCl2, it was suggested that the pathway of cyclic electron transport is altered in the mutant.