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 sequences of Nostoc strain MAC ferredoxins I and II

The amino acid sequences of ferredoxins I and II from a blue-green alga, Nostoc strain MAC were determined. This alga is able to grow autotrophically in the light or heterotrophically in the dark. Analyses of tryptic peptides of Cm-proteins by conventional methods including solid-phase Edman degradation gave the complete amino acid sequences. Both molecules consisted of 98 amino acid residues and 34 amino acid differences including two deletions were found between the two. Comparing these sequences with those of ferredoxins from Chlorogloeopsis fritschii and Synechocystis 6714, which are also capable of growing under both conditions, showed that Nostoc strain MAC ferredoxin II had unique amino acids around the [2Fe-2S] cluster. This finding provides a structural basis for explaining the different chemical and functional properties of Nostoc strain MAC ferredoxin II reported in a previous paper (Hutson et al. (1978) Biochem. J. 172, 465-477).     

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.     

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.     

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.     

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.     

Complete amino acid sequence of ferredoxin from Peridinium bipes (Dinophyceae)

The amino acid sequence of the major ferredoxin component isolated from a dinoflagellate, Peridinium bipes, was completely determined. Staphylococcus aureus V8 proteolytic, tryptic and chymotryptic peptides of Cm-ferredoxin were prepared and sequenced. The sequence was Phe-Lys-Val-Thr-Leu-Asp-Thr-Pro-Asp-Gly-Lys-Lys-Ser-Phe-Glu-Cys- Pro-Gly-Asp-Ser-Tyr-Ile-Leu-Asp-Lys-Ala-Glu-Glu-Glu-Gly-Leu-Glu-Leu-Pro- Tyr-Ser - Cys-Arg-Ala-Gly-Ser-Cys-Ser-Ser-Cys-Ala-Gly-Lys-Val-Leu-Thr-Gly-Ser-Ile- Asp-Gln - Ser-Asp-Gln-Ala-Phe-Leu-Asp-Asp-Asp-Gln-Gly-Gly-Asp-Gly-Tyr-Cys-Leu-Thr- Cys-Val - Thr-Tyr-Pro-Thr-Ser-Asp-Val-Thr-Ile-Lys-Thr-His-Cys-Glu-Ser-Glu-Leu. It was composed of 93 amino acid residues with 7 cysteine residues, the highest number found among the chloroplast-type ferredoxins so far sequenced. A cysteine residue was found for the first time at the 89th position in a chloroplast-type ferredoxin. Calculation of the numbers of amino acid differences among chloroplast-type ferredoxins indicates that the Peridinium ferredoxin is far divergent not only from higher plant ferredoxins but also from blue-green algal ferredoxins.     

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.     

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.     

New perspectives on bacterial ferredoxin evolution

Summary Recent evidence indicates that a gene transposition event occurred during the evolution of the bacterial ferredoxins subsequent to the ancestral intrasequence gene duplication. In light of this new information, the relationships among the bacterial ferredoxins were reexamined and an evolutionary tree consistent with this new understanding was derived. The bacterial ferredoxins can be divided into several groups based on their sequence properties; these include the clostridial-type ferredoxins, theAzotobacter-type ferredoxins, and a group containing the ferredoxins from the anaerobic, green, and purple sulfur bacteria. Based on sequence comparison, it was concluded that the amino-terminal domain of theAzotobacter-type ferredoxins, which contains the novel 3Fe3S cluster binding site, is homologous with the carboxyl-terminal domain of the ferredoxins from the anaerobic photosynthetic bacteria.A number of ferredoxin sequences do not fit into any of the groups described above. Based on sequence properties, these sequences can be separated into three groups: a group containingMethanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II, a group containingDesulfovibrio gigas ferredoxin andClostridium thermoaceticum ferredoxin, and a group containingDesulfovibrio africanus ferredoxin I andBacillus stearothermophilus ferredoxin. The last two groups differ from all of the other bacterial ferredoxins in that they bind only one FeS cluster per polypeptide, whereas the others bind two. Sequence examination indicates that the second binding site has been either partially or completely lost from these ferredoxins.Methanosarcina barkeri ferredoxin andDesulfovibrio desulfuricans ferredoxin II are of interest because, of all the ferredoxins whose sequences are presently known, they show the strongest evidence of internal gene duplication. However, the derived evolutionary tree indicates that they diverged from theAzotobacter-type ferredoxins well after the ancestral internal gene duplication. This apparent discrepancy is explained by postulating a duplication of one halfchain sequence and a deletion of the other halfchain. TheClostridium thermoaceticum andBacillus stearothermophilus groups diverged from this line and subsequently lost one of the FeS binding sites.It has recently become apparent that gene duplication is ubiquitous among the ferredoxins. Several organisms are now known to have a variety of ferredoxins with widely divergent properties. Unfortunately, in only one case are the sequences of more than one ferredoxin from the same organism known. Thus, although the major features of the bacterial ferredoxin tree are now understood, a complete bacterial phylogeny cannot be inferred until more sequence information is available.     

The evolution of ferredoxins

Ferredoxins are electron carrier proteins that contain active sites consisting of nonheme iron and inorganic sulfur. They are ubiquitous in living cells and are believed to be among the earliest redox proteins having appeared in primitive organisms. The small size of Ferredoxins allows their amino acid sequences to be determined with relative ease, and nearly a hundred primary structures have been elucidated over the past two decades. Most of these proteins belong to two distinct groups which have been used to construct phylogenetic trees of bacteria and oxygenic photosynthetic organisms respectively. A number of other Ferredoxins, however, seem to be unrelated to any of these two families of proteins and thus raise the problem of the origin of ferredoxins: are they all derived from a common ancestor, or have they appeared and evolved independently several times in the course of biological evolution? This issue is critical in view of the importance of Ferredoxins as evolutionary markers. There is evidence suggesting that presently known ferredoxins belong to at least five independent phyletic lines.     

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.     

Nuclear DNA codes for Nicotiana ferredoxin.

Ferredoxin was purified from 10 species of Nicotiana and spinach leaves. Fingerprints showed all to contain five major tryptic peptides. Some of the spinach peptides were different in RF and mobility from the Nicotiana peptides, but none of the Nicotiana ferredoxins had peptides which could distinguish one species of ferredoxin from another. Electrofocusing S-carbaminomethylcysteinyl ferredoxins showed spinach ferredoxin to have a more acidic and N. glutinosa ferredoxin a slightly more acidic isoelectric point than the other 9 Nicotiana species which were alike. Electro-focusing ferredoxin from the hybrid N. glutinosa female times N. glauca male resolved two bands or isozymes of ferredoxin, one corresponding to N. glutinosa, the other to N. glauca, the code for the latter having come from the DNA in the N. glauca pollen used to form the hybrid plant. N. glutinosa ferredoxin does not contain methionine and is different from N. tabacum and N. glauca ferredoxins which contain methionine. The N. glutinosa female times N. glauca male ferredoxin contained one-half the methionine found in N. glauca ferredoxin, thus confirming that some of the genetic information for ferredoxin in the hybrid was originally contained in the nuclear DNA of N. glauca.     

A Comparative Study of the Biological Activities of Two Molecular Species of Chloroplast-type Ferredoxins

Pairs of two molecular species of soluble chloroplast-type ferredoxins(Fd I and Fd II) from Nostoc muscorum and Aphanothece sacrumwere used to examine and compare the abilities of ferredoxinto substitute for spinach ferredoxin in the photoreduction ofNADP⁺ by spinach chloroplasts or N. muscorum membrane fragmentsand to link the reducing power of illuminated spinach chloroplaststo the Bacillus polymyxa nitrogenase system. Ferredoxins II of Nostoc and Aphanothece showed rather low activitiesin NADP⁺ photoreduction and nitrogenase system with spinachchloroplasts as the photosensitizer, compared to other ferredoxins.However, there was no difference between two ferredoxins (FdI and Fd II) from Nostoc in NADP⁺ photoreduction by photosyntheticmembrane fragments prepared from the same organism, N. muscorum. The biological significance of two molecular species of ferredoxinsin one organism could be ascribed to the different contributionof each ferredoxin to certain biological reactions in whichferredoxin functioned as an electron carrier. (Received November 4, 1980; Accepted January 9, 1981)     

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.     

The amino acid sequence of ferredoxin from Sambucus nigra

The amino acid sequence of the ferredoxin from Sambucus nigra consists of a single polypeptide chain of 97 amino acid residues, 5 of which are cysteine. The positions of the 4 cysteine residues which bind the iron atoms of the active centre are identical to those of other ferredoxins. Due to difficulties of obtaining pure protein, residues 87–90 have only been identified from the amino acid analysis of peptide C 10 and by homology with other higher plant ferredoxins.     

Electron transfer mechanism and interaction studies between cytochrome C3 and ferredoxin

Ferredoxin, cytochrome c3 and hydrogenase are specific partners of the sulfate reduction pathway of Desulfovibrio desulfuricans Norway and might be exemplary for electron exchange mechanism studies. Cytochrome c3 contains four low redox potential haems for 13 000 molecular weight. Two ferredoxins isolated from the same bacteria are dimers of 6 000 molecular weight per subunit (Ferredoxin I: one (4 Fe-4S) cluster per subunit, ferredoxin II: two (4 Fe-4 S) clusters per subunit). The amino acid sequence of ferredoxin I is reported and compared to the ferredoxin II sequence. The structural characteristics of ferredoxins and cytochrome c3 should allow a discussion on the nature of the interaction. 1H-NMR spectra of ferredoxin I and cytochrome c3 in the absence and presence of ferredoxin are presented.     

Effect of the ferredoxin electron donor on sunflower (Helianthus annuus) desaturases

Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants: light-regulated photosynthetic ferredoxins and heterotrophic ferredoxins whose activity is not influenced by light. Genes encoding the two forms of ferredoxin have been cloned and characterized in developing sunflower cotyledons. Here, these genes were overexpressed in Escherichia coli and they were purified by ion exchange and size exclusion chromatography to study their capacity to supply electrons to two different sunflower desaturases: soluble stearoyl-ACP desaturase from sunflower cotyledons, and membrane bound desaturase FAD7 expressed in yeast. In both cases photosynthetic ferredoxin was the form that promoted the strongest desaturase activity.     

Amino Acid Composition and Terminal Sequences of Ferredoxins from Two Photosynthetic Green Bacteria

The amino acid composition of ferredoxins from Chlorobium thiosulfatophilum 8327 and Chloropseudomonas ethylicum, like C. thiosulfatophilum Tassajara, resembled ferredoxins from nonphotosynthetic anaerobes rather than Chromatium; the terminal sequences, however, more closely resembled Chromatium ferredoxin.