Properties of a thermostable 4Fe-ferredoxin from the hyperthermophilic bacterium Thermotoga maritima

Abstract A ferredoxin has been purified from one of the most ancient and the most thermophilic bacteria known, Thermotoga maritima , which grous up to 90°C. The reduced protein ( M _r approx. 6300) contains a single S = 1 2 [4Fe 4S]¹⁺ cluster with complete cysteinyl ligation, and was unaffected after incubation at 95°C for 12 h. It functioned as an electron carrier for T. maritima pyruvate oxidoreductase. Remarkably, the properties and amino acid sequence of this hyperthermophilic bacterial protein are much more similar to those of ferredoxins from hyperthermophilic archaea, rather than ferredoxins from mesophilic and moderately thermophilic bacteria.     

Purification and characterization of a heat stable ferredoxin isolated fromClostridium thermocellum

A thermostable ferredoxin was purified from Clostridium thermocellum. The final preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate polyacrylamide gel and sedimentation equilibrium. It contains eight atoms of iron and eight acid-labile sulfur groups per molecule, the molecular weight is estimated to be 6 400 and the isoelectric point 3.35. Its amino-acid composition is characterized by the absence of histidine residues and the presence of eight cysteine residues. The absorption spectrum has a maximum at 390 nm with a molar absorption coefficient of 39 x 10(3) M1 cm-1, similar to that of other bacterial eight iron ferredoxins. The purified ferredoxin has high thermal stability, since the spectrophotometric absorption of the protein at 390 nm did not change after one hour at 70 degrees C and only thirty five per cent of absorbance were lost after one hour at 80 degrees C. With regard to the electron carrier activity, the stability is slightly higher, only twenty five per cent of the activity were lost after one hour at 80 degrees C. During pyruvate oxidation, ferredoxin functions in the transfer of electrons to hydrogenase and also in the back reaction during pyridine nucleotide reduction by a ferredoxin -NAD oxidoreductase using hydrogen as electron donor.     

Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.

Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.     

Characterization of two soluble ferredoxins as distinct from bound iron-sulfur proteins in the photosynthetic bacterium Rhodospirillum rubrum.

In an earlier investigation (Shanmugam, K. T., Buchanan, B. B., and Arnon, D. I. (1972) Biochim. Biophys. Acta 256, 477-486) the extraction of ferredoxin from Rhodospirillum rubrum cells with the aid of a detergent (Triton X-100) and acetone revealed the existence of two types of ferredoxin (I and II) and led to the conclusion that both are membrane-bound. In the present investigation, ferredoxin and acid-labile sulfur analyses of photosynthetic membranes (chromatophores) and soluble protein extracts of the photosynthetic bacteria R. rubrum and Rhodopseudomonas spheroides showed that ferredoxins I and II are primarily components of the soluble protein fraction. After their removal, washed R. rubrum chromatophores were found to contain a considerable amount of tightly bound iron-sulfur protein(s), as evidenced by acid-labile sulfur and electron paramagnetic resonance analyses. Thus, like all other photosynthetic cells examined to date, R. rubrum cells contain both soluble ferredoxins and iron-sulfur proteins tightly bound to photosynthetic membranes. The molecular weights of ferredoxins I and II from photosynthetically grown R. rubrum cells were found to be 8,800 and 14,500, respectively. Using these molecular weights, the molar extinction coefficients at 390 nm for ferredoxins I and II were determined to be 30.3 and 17.2 mM-1 CM-1, respectively. Ferredoxin I contains 8 non-heme iron and 8 acid-labile sulfur atoms per molecule; ferredoxin II contains 4 non-heme iron and 4 acid-labile sulfur atoms per molecule. Ferredoxin I was found only in photosynthetically grown cells whereas ferredoxin II was present in both light- and dark-grown cells. Ferredoxin II from both light- and dark-grown cells has the same molecular weight (14,500) and absorption spectrum and has 4 iron and 4 acid-labile sulfur atoms per molecule. Low temperature electron paramagnetic resonance spectra of oxidized and photoreduced ferredoxins I and II from R. rubrum were recorded. The EPR spectrum of oxidized ferredoxin II exhibited a single resonance line at g = 2.012. Oxidized ferredoxin I, however, exhibited a spectrum that may arise from the superimposition of two resonance lines near g = 2.012. Photoreduced ferredoxin II displayed a rhombic EPR spectrum with a g value of 1.94. Photoreduced ferredoxin I exhibited a similar EPR spectrum at a temperature of 16 K, but when the temperature was lowered to 4.5 K the spectrum of ferredoxin I changed. This temperature-dependent spectrum may result from a weak spin-spin interaction between two iron-sulfur clusters. These results are consistent with the conclusion that R. rubrum ferredoxins I and II are, respectively, 8 iron/8 sulfur and 4 iron/4sulfur proteins.     

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.     

Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents

Ferredoxin, flavodoxin, and rubredoxin were purified to homogeneity from Clostridium formicoaceticum and characterized. Variation of the iron concentration of the growth medium caused substantial changes in the concentrations of ferredoxin and flavodoxin but not of rubredoxin. The ferredoxin has a molecular weight of 6,000 and is a four iron-four sulfur protein with eight cysteine residues. The spectrum is similar to that of other ferredoxins. The molar extinction coefficients are 22.6 X 10(3) and 17.6 X 10(3) at 280 and 390 nm, respectively. From 100 g wet weight of cells grown with 3.6 microM iron and with 40 microM iron, 5 and 20 mg offerredoxin were isolated, respectively. The molecular weight of rubredoxin is 5,800 and it contains one iron and four cysteines. The UV-visible absorption spectrum is dissimilar to those of other rubredoxins in that the 373 nm absorption peak is quite symmetric, lacking the characteristic 350-nm shoulder found in other rubredoxins. The flavodoxin is a 14,500-molecular-weight protein which contains 1 mol of flavin mononucleotide per mol of protein. It forms a stable, blue semiquinone upon light irradiation in the presence of EDTA or during enzymatic reduction. When cells were grown in low-iron medium, flavodoxin constituted at least 2% of the soluble cell protein; however, it was not detected in extracts of cells grown in high-iron medium. The rubredoxin and ferredoxin expressed during growth in low-iron and high-iron media are identical as judged by iron, inorganic sulfide, and amino acid analysis, as well as light absorption spectroscopy.     

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.     

Ferredoxin from Halobacterium of the Dead Sea — Mössbauer and EPR spectra and comparison with mössbauer spectrum of whole cells

Recoil-free measurements were carried out on a 2 Fe-ferredoxin, which was isolated and purified from an extreme halophile, Halobacterium of the Dead Sea. The spectrum of this ferredoxin in the oxidized state at 82 K is a superposition of two quadrupole doublets, representing two non-equivalent Fe³⁺ sites of equal intensity. The spectrum of the reduced ferredoxin is consistent with the presence of two pure classes of iron atoms, ferric (lower isomer shift) and ferrous (higher isomer shift). Interpretations of the recoil-free spectra are discussed. Mössbauer measurements were also carried out on frozen whole bacterial cells and the resulting spectrum was found to be quite different from that observed in the isolated ferredoxin. Tentative conclusions are reached concerning the localization of this ferredoxin in the cytosol of the Halobacteria.The EPR spectrum of the reduced ferredoxin obtained at 24 K exhibits rhombic symmetry with the following g values: 1.894, 1.984 and 2.07. These values are similar to those obtained with 2 Fe-ferredoxins of the plant type, except that the g _y and g _z values are somewhat higher. Both from the EPR and Mössbauer data, it is deduced that the spin relaxation times in reduced halophilic ferredoxins are faster than in the reduced plant ferredoxins.     

Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis.

The pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis is an extrinsic protein bound to the hydrogenosomal membrane. It has been solubilized and purified to homogeneity, principally by salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by O2 and the irreversible loss of thiamin pyrophosphate. It is a dimeric enzyme of overall Mr 240,000 and subunit Mr 120,000. The enzyme contains, per mol of dimer, 7.3 +/- 0.3 mol of iron and 5.9 +/- 0.9 mol of acid-labile sulphur, suggesting the presence of two [4Fe-4S] centres, and 0.47 mol of thiamin pyrophosphate. The absorption spectrum of the enzyme is characteristic of a non-haem iron protein. The pyruvate: ferredoxin oxidoreductase from T. vaginalis is therefore broadly similar to the 2-oxo acid: ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, except that it is membrane-bound.     

Keto-acid oxidoreductases in the anaerobic protozoa.

In anaerobes, decarboxylation of pyruvate is executed by the enzyme pyruvate:ferredoxin oxidoreductase, which donates electrons to ferredoxin. The pyruvate:ferredoxin oxidoreductase and its homologues utilise many alternative substrates in bacterial anaerobes. The pyruvate:ferredoxin oxidoreductase from anaerobic protozoa, such as Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica have retained this diversity in usage of alternative keto acids for energy production utilising a wide variety of substrates. In addition to this flexibility, both T. vaginalis and G. duodenalis have alternative enzymes that are active in metronidazole-resistant parasites and that do not necessarily involve donation of electrons to characterized ferredoxins. Giardia duodenalis has two oxoacid oxidoreductases, including pyruvate:ferredoxin oxidoreductase and T. vaginalis has at least three. These alternative oxoacid oxidoreductases apparently do not share homology with the characterized pyruvate:ferredoxin oxidoreductase in either organism. Independently, both G. duodenalis and T. vaginalis have retained alternative oxoacid oxidoreductase activities that are clearly important for the survival of these parasitic protists.     

A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum.

A ferredoxin containing only one [Fe4S4] cluster was purified from Clostridium thermoaceticum. It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25. Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1). The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two [Fe4S4] clusters, and is similar to other ferredoxins with only one [Fe4S4] cluster. The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C. It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum. NADPH is used in the synthesis of acetate from CO2 in this organism.     

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.     

A new plant-type ferredoxin from halobacteria.

A stable, 2Fe-type ferredoxin has been prepared from Halobacterium halobium and purified by chromatography. A similar ferredoxin was also found in three other Halobacteria. The ferredoxin is present in large amounts-about 1 percent of the total soluble protein. From amino acid composition a molecular weight of 14800 +/- 200 was calculated. The ferredoxin was found to contain two atoms each of iron and sulphide. The midpoint redox potential of the protein is about -345 mV. The electron paramagnetic resonance spectrum of the reduced form shows much similarity to plant and algal ferredoxins with gx = 1.90, gy = 1.97 and gz = 2.07. The same similarity is observed in the optical absorption, optical rotatory dispersion and circular dichroism spectra. However it does not seem to mediate electron transport in the NADP-photoreduction system of chloroplasts. Extracts of the bacterial cells catalyze the reduction of the ferredoxin by NADH.     

Effects of specific reagents and urea on the reactivity of non-heme iron and thiol groups of pea and corn ferredoxins]

The reactivities of the SH-groups of pea and corn ferredoxins were found to be different. One or two SH-groups in the molecule of pea ferredoxin and one SH-group in the molecule of corn ferredoxin are readily available for the thyol group specific reagents. Four SH-groups of both ferredoxins are completely masked, i. e. available for the thyol reagents only after protein denaturation in the presence of urea. The rates of SH-group interaction with the sulfhydryl reagents in corn ferredoxin are lower than those in pea ferredoxin. The non-haem iron of pea ferredoxin interacts with the complex formers far more rapidly as compared to corn ferredoxin. The ferredoxins tested differ in the amount of iron atoms. The latter require the presence of oxygen for their complete interaction with the complex formers.     

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.     

Purification and characterization of a 7Fe ferredoxin from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii.

A ferredoxin (Fd) was purified from a thermophilic hydrogen-oxidizing bacterium, Bacillus schlegelii. This ferredoxin was a monomer with apparent molecular weight of 13,000 and contained 7 mol Fe/mol ferredoxin. The oxidized ferredoxin showed the characteristic EPR spectrum for [3Fe-4S]1+ (1.2 spin/mol Fd). This signal disappeared upon reduction with dithionite and new signals due to [3Fe-4S]0 and [4Fe-4S]1+ (0.7 spin/mol Fd) appeared. The quantitation of EPR signals and the iron content reveal that B. schlegelii ferredoxin contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster. The ferredoxin has the characteristic distribution of cysteines (-Cys8-X7-Cys16-X3-Cys20-Pro-) for 7Fe ferredoxins in the N-terminus.     

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.