Comparative studies on two ferredoxins from the cyanobacterium Nostoc strain MAC.

Two ferredoxins were isolated from the cyanobacterium Nostoc strain MAC grown autotrophically in the light or heterotrophically in the dark. In either case approximately three times as much ferredoxin I as ferredoxin II was obtained. Both ferredoxins had absorption maxima at 276, 282 (shoulder), 330, 423 and 465 nm in the oxidized state, and each possessed a single 2 Fe-2S active centre. Their isoelectric points were approx. 3.2. The midpoint redox potentials of the ferredoxins differed markedly; that of ferredoxin I was --350mV and that of ferredoxin II was --445mV, at pH 8.0. The midpoint potential of ferredoxin II was unusual in being pH dependent. Ferredoxin I was most active in supporting NADP+ photoreduction by chloroplasts, whereas ferredoxin II was somewhat more active in pyruvate decarboxylation by the phosphoroclastic system of Clostridum pasteurianum. Though the molecular weights of the ferredoxins determined by ultracentrifugation were the same within experimetnal error, the amino acid compositions showed marked differences. The N-terminal amino acid sequences of ferredoxins I and II were determined by means of an automatic sequencer. There are 11--12 differences between the sequences of the first 32 residues. It appears that the two ferredoxins have evolved separately to fulfil different roles in the organism.     

Midpoint redox potentials of plant and algal ferredoxins.

Midpoint potentials of plant-type ferredoxins from a range of sources were measured by redox titrations combined with electron-paramagnetic-resonance spectroscopy. For ferredoxins from higher plants, green algae and most red algae, the midpoint potentials (at pH 8.0) were between --390 and --425 mV. Values for the major ferredoxin fractions from blue-green algae were less negative (between --325 and --390 mV). In addition, Spirulina maxima and Nostoc strain MAC contain second minor ferredoxin components with a different potential, --305 mV (the highest so far measured for a plant-algal ferrodoxin) for Spirulina ferrodoxin II, and --455 mV (the lowest so far measured for a plant-algal ferredoxin) for Nostoc strain MAC ferredoxin II. However, two ferredoxins extracted from a variety of the higher plant Pisum sativum (pea) had midpoint potentials that were only slightly different from each other. These values are discussed in terms of possible roles for the ferredoxins in addition to their involvement in photosynthetic electron transport.     

Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum.

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1 : 0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.     

Amino acid sequences of two ferredoxins from pokeweed, Phytolacca americana

The amino acid sequences of two ferredoxins isolated from pokeweed, Phytolacca americana, were determined. Tryptic peptides of maleyl-carboxymethyl-ferredoxin I and carboxymethyl-ferredoxin II were prepared and analyzed. The large peptides were further digested with staphylococcal protease and chymotrypsin. Ferredoxins I and II were composed of 96 and 98 amino acid residues, respectively. Though ferredoxin I lacks tryptophan and methionine, ferredoxin II contains both of them. In a comparison of the amino acid sequences with those of other higher plant ferredoxins, ferredoxin I is one residue shorter than others at the carboxyl-terminus and ferredoxin II one longer than others at the amino-terminus. Ferredoxins I and II differ in 23 sites from each other and in 27 to 37 sites from other higher plant ferredoxins. This suggests that duplication of the ferredoxin gene occurred after the divergence of pokeweed from other higher plants. A phylogenetic tree including all other ferredoxins was constructed.     

Two plant-type ferredoxins from a blue-green alga, Nostoc verrucosum

Two plant-type ferredoxins were isolated and purified from a blue-green alga, Nostoc verrucosum. They were separable by chromatography on a DEAE-cellulose column. The slow-moving band was designated ferredoxin I (Fd I) and the fast-moving band was ferredoxin II (Fd II). The ratio of the yield of ferredoxins I and II was about 1:0.84. Both ferredoxins had absorption spectra similar to those of plant-type ferredoxins. Two atoms of non-heme iron and two of labile sulfur were found per mol of both ferredoxin I and ferredoxin II. Their molecular weights were identical and estimated to be about 18 000 by a gel filtration method. The biochemical activities of these Nostoc ferredoxins were studied: the NADP photoreduction activity on one hand and the NADP-cytochrome c reductase activity on the other.     

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.     

Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution

Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003. Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation. Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms. Both contained two Cys clusters in their amino acid sequences. The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia. The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met. The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-[3Fe-4S] [4Fe-4S]-ferredoxin. Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type. The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium [Moreno-Vivian et al. (1989) J. Bacteriol. 171, 2591-2598] is discussed.     

Amino acid sequence of Synechocystis 6714 ferredoxin: a unique structural feature of unicellular blue-green algal ferredoxin

The amino acid sequence of ferredoxin from Synechocystis 6714, a unicellular blue-green alga, was determined by a combination of conventional methods. The ferredoxin was composed of 96 amino acid residues and lacked methionine and tryptophan. The sequence was as follows: Ala-Ser-Tyr-Thr-Val-Lys-Leu-Ile-Thr- Pro-Asp-Gly-Glu-Asn-Ser-Ile-Glu-Cys-Ser-Asp-Asp-Thr-Tyr-Ile-Leu-Asp-Ala-Ala- Glu-Glu-Ala-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys- Ala-Gly-Lys-Ile-Thr-Ala-Gly-Ser-Val-Asp-Gln-Ser-Asp-Gln-Ser-Phe-Leu-Asp-Asp- Asp-Gln-Ile-Glu-Ala-Gly-Tyr-Val-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Thr-Ile-Glu-Thr-His-Lys-Glu-Glu-Asp-Leu-Tyr. In an alignment of various ferredoxins with high homology from unicellular and filamentous blue-green algae, Synechocystis 6714 ferredoxin showed 4 gaps. Those between residues 9 and 10 and between residues 12 and 13 were unique for the ferredoxins from the unicellular algae Synechocystis 6714 and Aphanothece sacrum (ferredoxin I). Therefore, ferredoxins from unicellular algae were distinguishable from those of filamentous algae in terms of the presence of gaps. This feature appears to coincide with the phylogenetic division between the two types of blue-green algae.     

Amino acid sequence of a ferredoxin from Rhodymenia palmata, a red alga in the florideophyceae

The amino acid sequence of a [2Fe-2S] ferredoxin from a red alga, Rhodymenia palmata in the family Florideophyceae, was determined by conventional methods. The ferredoxin is composed of 97 amino acid residues having five cysteines, but lacking methionine and tryptophan. It possesses a number of structural features of particular interest. The amino acid sequence is compared with those previously determined for ferredoxins from two red algae in the family Bangiophyceae. Conclusions from a comparison of the structures, by noting features such as the presence of gaps in the sequences and by constructing a phylogenetic tree, were consistent with the proposed taxonomic relationship among these algae.     

Primary structure of the two (4 Fe-4 S) clusters ferredoxin from Desulfovibrio desulfuricans (strain Norway 4)

The primary structure of a ferredoxin isolated from D. desulfuricans Norway strain, which we called ferredoxin II (Fd II) has been elucidated. This ferredoxin is a dimer constituted of two identical subunits of molecular weight 6000. In ferredoxin II two (4 Fe-4 S) centers are present per subunit instead of one (Fe-S) center as is the case for the other ferredoxins isolated from Desulfovibrio and for Fd I from the same organism. The comparison of amino-acid sequences shows that ferredoxin II presents more homologies with clostridial type ferredoxin than with the ferredoxins from D. gigas and D. africanus.     

Plant-type and bacterial-type ferredoxins in a nitrogen-fixing cyanobacterium: Nostoc sp. strain PCC 73102

Abstract The cyanobacterium Nostoc sp. strain PCC 73102, cultured under nitrogen-fixing conditions, was investigated for the occurrence of ferrodoxins by SDS-PAGE/Western immunoblots using antisera directed against both a major plant-type and a bacterial-type ferredoxin purified from Anabaena variabilis . Immunocytological labelling and transmission electron microscopy were used to study the distribution of both types of ferredoxins in the Nostoc cells. SDS-PAGE/Western immunoblots revealed two proteins/polypeptides in the Nostoc strain, immunologically related to two soluble ferredoxins purified from Anabaena variabilis : the major plant-type ferredoxin (Fd I) and a bacterial-type ferredoxin (Fd III). Immunolocalization showed a uniform distribution of the plant-type and the bacterial-type ferredoxin in both the photosynthetic vegetative cells and in the nitrogen-fixing heterocysts, with no specific association with any subcellular inclusions. Using the particle analysis of an image processor, the labelling associated with the vegetative cells, expressed as number of gold particles per cell area, was found to be only slightly higher (1.2x) or almost twice as high (1.9x) compared to the heterocysts for the major plant-type and the bacterial-type ferredoxin, respectively.     

Crystalline Ferredoxin from a Blue-Green Alga, Nostoc sp.

Ferredoxin isolated from a blue-green alga, Nostoc sp., was purified and crystallized. The absorption spectrum of Nostoc ferredoxin had, in the oxidized state, peaks at 276, 331, 423, and 470 nm, a pattern characteristic of chloroplast-type ferredoxin. The 423:276 absorption ratio was 0.57. The midpoint oxidation-reduction potential of Nostoc ferredoxin was found to be –406 mV, at pH 7.5. Nostoc ferredoxin mediated the photoreduction of NADP by isolated Nostoc chromato-phores and spinach chloroplasts from which the native ferredoxin was removed. The molar ratio of Nostoc ferredoxin to chlorophyll a was about 1:50, a ratio higher than usually found in photosynthetic cells. The possible evolutionary significance of the properties of Nostoc ferredoxin compared with those of ferredoxins from other photosynthetic organisms is discussed.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A₂₇₈/A₄₉₀ is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids.

The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A₃₈₅/A₂₈₃ absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively.

The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A278/A490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A385/A283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.     

Purification, properties and complete amino acid sequence of the ferredoxin from a green alga, Chlamydomonas reinhardtii

The ferredoxin was purified from the green alga, Chlamydomonas reinhardtii. The protein showed typical absorption and circular dichroism spectra of a [2Fe-2S] ferredoxin. When compared with spinach ferredoxin, the C. reinhardtii protein was less effective in the catalysis of NADP+ photoreduction, but its activity was higher in the light activation of C. reinhardtii malate dehydrogenase (NADP). The complete amino acid sequence was determined by automated Edman degradation of the whole protein and of peptides obtained by trypsin and chymotrypsin digestions and by CNBr cleavage. The protein consists of 94 residues, with Tyr at both NH2 and COOH termini. The positions of the four cysteines binding the two iron atoms are similar to those found in other [2Fe-2S] ferredoxins. The primary structure of C. reinhardtii ferredoxin showed a great homology (about 80%) with ferredoxins from two other green algae.     

Ferredoxin from Aphanothece halophitica, a unicellular blue-green alga: close relationship to ferredoxins from filamentous blue-green algae and phylogenetic implications

The amino acid sequence of a ferredoxin from a unicellular blue-green alga, Aphanothece halophitica, was established by the conventional methods. Total number of residues was 98 lacking only tryptophan. A most probable phylogenetic tree was constructed for 19 algal ferredoxins on the basis of an amino acid difference matrix made from the sequence comparison. A. halophitica has been classified as a unicellular blue-green alga in the same genus to which Aphanothece sacrum belongs, but the tree indicates A. halophitica ferredoxin to be very close to those of the members of filamentous blue-green algae. The tree divides prokaryotic and eukaryotic algal ferredoxins into several groups, suggesting that the ferredoxin phylogenetic tree reflects the evolutionary trails of various algae, which is also reflected in the structural characteristics, particularly in the presence of gaps. Other notable features are presented in considering algal taxonomy.     

Amino acid sequence of a ferredoxin from Bumilleriopsis filiformis, a yellow-green alga: relationship with red algae, protoflorideophyceae, and filamentous blue-green algae

The amino acid sequence of a [2Fe-2S] ferredoxin isolated from Bumilleriopsis filiformis, a yellow-green alga, was determined by using conventional techniques. It consisted of 98 amino acid residues with a microheterogeneity at the amino-terminus: Ala/Glu-Thr-Tyr-Ser-Val-Thr-Leu-Val-Asn-Glu-Glu-Lys-Asn-Ile-Asn-Ala-Val- Ile- Lys-Cys-Pro-Asp-Asp-Gln-Phe-Ile-Leu-Asp-Ala-Ala-Glu-Glu-Gln-Gly-Ile-Glu- Leu- Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Val-Leu-Ser- Gly- Thr-Ile-Asp-Gln-Ser-Glu-Gln-Ser-Phe-Leu-Asp-Asp-Asp-Gln-Met-Gly-Ala-Gly- Phe- Leu-Leu-Thr-Cys-Val-Ala-Tyr-Pro-Thr-Ser-Asp-Cys-Lys-Val-Gln-Thr-His-Ala- Glu- Asp-Asp-Leu-Tyr. No prominent structural feature was noted in this ferredoxin in comparison with other homologous ferredoxins. From the structural comparison, B. filiformis was placed taxonomically close to filamentous blue-green algae and red algae.     

Ferredoxin and Formyltetrahydrofolate Synthetase: Comparative Studies with Clostridium acidiurici, Clostridium cylindrosporum, and Newly Isolated Anaerobic Uric Acid-Fermenting Strains

Six strains of Clostridium acidiurici and three strains of C. cylindrosporum were isolated from soil samples by enrichment culture with uric acid as the source of carbon, nitrogen, and energy. The newly isolated strains were characterized by their spore morphology and the amounts of glycine and formate formed by the fermentation of uric acid. The strains were easily identified as belonging to one species or the other on the basis of spore morphology and formate production. The crystal properties and spectra of the native ferredoxins of all the strains isolated and the amino acid composition and partial carboxy-terminal sequence of all their apoferredoxins were determined. All the ferredoxins were tested for cross-reactivity with antiserum to C. acidiurici ferredoxin by microcomplement fixation. Five of the six C. acidiurici strains, which had ferredoxins with amino acid compositions identical to that from C. acidiurici, also showed immunological identity (immunological distance = 0.0). These results suggest sequence identity. The one strain with a different amino acid composition failed to show complete cross-reactivity. Two of the three C. cylindrosporum strains have ferredoxin amino acid compositions identical to that from C. cylindrosporum. The third strain had a minimum of five differences in sequence. All C. cylindrosporum strains had ferredoxins that differed considerably from C. acidiurici strains (minimum of eight to nine differences), and none of these ferredoxins cross-reacted with antisera to C. acidiurici ferredoxin. Antisera were prepared to formyltetrahydrofolate synthetase from C. acidiurici and C. cylindrosporum, and all possible comparisons were made by using immunodiffusion and microcomplement fixation. There is more intraspecies variation in the synthetases than in the ferredoxins; however, the results suggest considerable interspecies differences in both proteins. These results suggest a low degree of genomic relatedness between the two species, which contrasts sharply with their apparent high degree of phenotypic similarity.     

Amino acid sequences of ferredoxin isoproteins from radish roots

Three ferredoxin isoproteins (R-Fd A, R-Fd B-1, and R-Fd B-2) were purified from white roots of radish (Raphanus sativus L. var. acantiformis cultivar Miyashige) and two isoproteins (L-Fd A and L-Fd B) from leaves. The amino acid sequences of three of them (L-Fd A, R-Fd B-1, and R-Fd B-2) were determined and compared with one another and with those of other higher plant ferredoxins so far studied. L-Fd A and R-Fd B-1 had heterogeneities at four and two amino acid sites, respectively. Two isoprotein (R-Fd B-1 and R-Fd B-2) were deduced to be expressed only in root tissue on the basis of sequence studies and amino acid compositions of all isoferredoxins isolated from the radish plant. The root ferredoxins sequenced in this study were similar to each other, but quite different from other higher plant ferredoxins, all of which were isolated from leaf tissue. The coupling activities of these ferredoxin isoproteins were measured in the NADP+-photoreduction system of radish chloroplasts and glutamate synthase [EC 1.4.7.1] systems isolated from radish leaf and root tissues. No distinctive physiological characteristics were observed among these isoferredoxins.