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PubMed1993

Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus.

A new ferredoxin, called FdIII, has been isolated and purified from the photosynthetic bacterium Rhodobacter capsulatus. Its complete amino acid sequence has been determined. The FdIII polypeptide consists of 100 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry. In its native form, FdIII is a homodimer as deduced from molecular sieve chromatography and non-denaturing polyacrylamide gel electrophoresis, as well as cross-linking experiments. The dimeric ferredoxin was found to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur atoms, consistent with the presence of four [4Fe-4S] clusters/molecule. The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm. FdIII was fully reduced by excess dithionite at pH 8.0 or photochemically using 5-deazaflavin. Anaerobic oxidative titration of reduced FdIII with thionin indicated that each FdIII monomer exchanges two electrons. Exposure of FdIII to air resulted in a rapid and irreversible oxidative denaturation of the Fe-S clusters. The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer. EPR analysis of partially reduced FdIII (approximately 20% reduction) revealed a complex set of signals which was interpreted as being the resulting sum of the contribution of two distinct paramagnetic centres. Based on its biochemical and spectroscopic properties, it is concluded that FdIII is a dimeric ferredoxin containing four [4Fe-4S] clusters. The synthesis of FdIII occurs only under growth conditions allowing the derepression of nif genes. Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electron donor to nitrogenase.

Amino Acid Sequence↗
PubMed1979

[Porton uptake by isolated chloroplasts during cyclic and non-cyclic electron transport catalyzed by photosystem I in the presence of ferredoxin].

Proton uptake by isolated chloroplasts during cyclic electron transport in the presence of ferredoxin and under NADP+ reduction from the ascorbate--TMPD donor pair under anaerobic conditions was studied. It was found that during cyclic transport the proton uptake is less intensive than under NADP+ reduction. In the presence of ATP the proton uptake is increased in the first case and is decreased in the second one. During cyclic transport in the presence of gramicidin D the proton uptake is completely suppressed and under NADP+ reduction is decreased down to 0,08--0,09 mk equiv H+ per mg of chlorophyll, irrespective of ferredoxin or NADP+ concentrations. The role of ferredoxin NADP+ reductase in a proton uptake by thylakoids is discussed.

Adenosine Triphosphate↗
PubMed1995

Existence of two ferredoxin-glutamate synthases in the cyanobacterium Synechocystis sp. PCC 6803. Isolation and insertional inactivation of gltB and gltS genes.

The first two genes of ferredoxin-dependent glutamate synthase (Fd-GOGAT) from a prokaryotic organism, the cyanobacterium Synechocystis sp. PCC 6803, were cloned in Escherichia coli. Partial sequencing of the cloned genomic DNA, of the 6.3 kb Hind III and 9.3 kb Cla I fragments, confirmed the existence of two different genes coding for glutamate synthases, named gltB and gltS. The gltB gene was completely sequenced and encodes for a polypeptide of 1550 amino acid residues (M(r) 168,964). Comparative analysis of the gltB deduced amino acid sequence against other glutamate synthases shows a higher identity with the alfalfa NADH-GOGAT (55.2%) than with the corresponding Fd-GOGAT from the higher plants maize and spinach (about 43%), the red alga Antithamnion sp. (42%) or with the NADPH-GOGAT of bacterial source, such as Escherichia coli (41%) and Azospirillum brasilense (45%). The detailed analysis of Synechocystis gltB deduced amino acid sequence shows strongly conserved regions that have been assigned to the 3Fe-4S cluster (CX5CHX3C), the FMN-binding domain and the glutamine-amide transferase domain. Insertional inactivation of gltB and gltS genes revealed that both genes code for ferredoxin-dependent glutamate synthases which were nonessential for Synechocystis growth, as shown by the ferredoxin-dependent glutamate synthase activity and western-blot analysis of the mutant strains.

Amino Acid Oxidoreductases↗
PubMed1997

Purification and characterization of pyruvate:ferredoxin oxidoreductase from Hydrogenobacter thermophilus TK-6.

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an alphabetagammadelta-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron acceptors in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80 degrees C. The pH optimum was 7.6-7.8. The apparent Km values for pyruvate and coenzyme A at 70 degrees C were 3.45 mM and 54 microM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t50%) at 70 degrees C was approximately 8 h.

Amino Acid Sequence↗
PubMed1988

Purification and properties of ferredoxin-NADP+ oxidoreductase from the nitrogen-fixing cyanobacteria Anabaena variabilis.

The isolation and characterization of ferredoxin-NADP+ -oxidoreductase from Anabaena variabilis, a nitrogen-fixing, filamentous cyanobacterium, is described. Purified enzyme was obtained in four steps with a 55% yield and 300-fold purification utilizing chromatographic separations on DEAE-cellulose and Cibacron Blue-Sepharose columns. The enzyme is quite similar but not identical to the spinach enzyme as judged by isoelectric focusing, molecular weight determination, and amino acid composition. N-terminal sequence analysis allowed identification of 28 of the first 33 residues. Alignment with the corresponding sequences from spinach and Spirulina FNR preparations was possible. A higher degree of homology was found with the Spirulina enzyme than with the spinach enzyme. Small differences with the spinach enzyme were also shown by absorption and circular dichroism spectral measurements. Oxidation-reduction potential measurements of the bound FAD coenzyme show an Em = -320 mV at pH 7 for the two-electron process. Complex formation between the reductase and ferredoxin from the same organism was observed by difference absorption spectroscopy with a Kd = 4 microM. Similar Kd and difference absorption properties were observed on complex formation with spinach ferredoxin.

Amino Acid Sequence↗
PubMed1994

Transient kinetic and oxidation-reduction studies of spinach ferredoxin:nitrite oxidoreductase.

The oxidation-reduction midpoint potentials for the two prosthetic groups of the chloroplast-located, ferredoxin-dependent nitrite reductase of spinach leaves have been determined by spectroelectrochemical titrations and cyclic voltammetry. The average of the results obtained by the two techniques are Em = -290 mV for the siroheme group and Em = -365 mV for the [4Fe-4S] cluster. The value obtained for the [4Fe-4S] cluster is substantially more positive than values obtained previously in experiments which utilized electron paramagnetic resonance spectroscopy at cryogenic temperatures to monitor the reduction state of the cluster. Laser flash photolysis experiments have been used to monitor electron transfer from reduced ferredoxin to nitrite reductase and have provided the first evidence for electron transfer between the two prosthetic groups of the enzyme. The effect of ionic strength on the observed kinetics has provided support for the proposal that electrostatic interactions between ferredoxin and nitrite reductase play an important role in the reaction mechanism.

Kinetics↗
PubMed1994

Characterization of an ancestral type of pyruvate ferredoxin oxidoreductase from the hyperthermophilic bacterium, Thermotoga maritima.

The hyperthermophilic bacterium, Thermotoga maritima, is a strict anaerobe that grows up to 90 degrees C by carbohydrate fermentation. We report here on its pyruvate ferredoxin oxidoreductase (POR), the enzyme that catalyzes the oxidation of pyruvate to acetyl-CoA, the terminal oxidation step in the conversion of glucose to acetate. T. maritima POR was purified to electrophoretic homogeneity under strictly anaerobic conditions. It has a molecular weight of 113,000 and comprises four dissimilar subunits with M(r) values of approximately 43,000, 34,000, 23,000, and 13,000. It contains thiamine pyrophosphate (TPP) and at least two ferredoxin-type [4Fe-4S] clusters per molecule, as determined by iron analysis and EPR spectroscopy. CoASH was absolutely required for pyruvate oxidation activity, while the addition of TPP was stimulatory. The apparent Km values at 80 degrees C for pyruvate, CoASH, and TPP were 14.5, 0.34, and 0.043 mM, respectively, and the corresponding apparent Vm values ranged from 154 to 170 mumol of pyruvate oxidized/min/mg (units/mg). The apparent Km and Vm values for T. maritima ferredoxin, the proposed physiological electron carrier for POR, were 26 microM and 280 units/mg, respectively. POR did not use 2-oxoglutarate, phenyl pyruvate, or indolyl pyruvate as substrates. The enzyme was extremely thermostable: the temperature optimum for pyruvate oxidation was above 90 degrees C, and the time for a 50% loss of activity (t50%) at 80 degrees C (under anaerobic conditions) was 15 h. The enzyme was also very sensitive to inactivation by oxygen, with a t50% in air at 25 degrees C of 70 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

Individual Reduction Potentials of the Iron Ions in Fe(2)S(2) and High-Potential Fe(4)S(4) Ferredoxins.

The DelPhi program package has been used to confirm that the span in reduction potentials among high-potential Fe(4)S(4) ferredoxins must be mainly ascribed to the net protein charges arising from acidic and basic residues. Subsequently, the order of the individual reduction potentials of the iron ions in Fe(2)S(2) ferredoxins as found from NMR spectroscopy was explained mainly on the basis of different solvation contributions to the electrostatic potential. The individual reduction potentials of the iron ions in high-potential Fe(4)S(4) ferredoxins, again available from NMR spectroscopy, are only qualitatively reproduced. It is proposed that the protein triggers a distortion in the cluster which would be a further contribution to the electrochemical inequivalence of the individual iron ions.

Journal Article↗
PubMed2005

Spectroscopic characterization of site-specific [Fe(4)S(4)] cluster chemistry in ferredoxin:thioredoxin reductase: implications for the catalytic mechanism.

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe(4)S(4)](2+) cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequential one-electron steps using a [Fe(2)S(2)](2+/+) ferredoxin as the electron donor. In this work, we report on spectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the active site of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of the active-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfide intermediate. The results reveal novel site-specific Fe(4)S(4)-cluster chemistry in oxidized, one-electron-reduced, and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe(4)S(4) cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active site to accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues, cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues that form the active-site disulfide to yield a [Fe(4)S(4)](3+) cluster with two cysteinate ligands at a unique Fe site. The most intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol contains an unprecedented electron-rich [Fe(4)S(4)](2+) cluster comprising both valence-delocalized and valence-localized Fe(2+)Fe(3+) pairs. These results provide molecular level insights into the catalytic mechanism of FTR, and two viable mechanisms are proposed.

Binding Sites↗
PubMed2004

Structural Basis of Redox Signaling in Photosynthesis: Structure and Function of Ferredoxin:thioredoxin Reductase and Target Enzymes.

The role of the ferredoxin:thioredoxin system in the reversible light activation of chloroplast enzymes by thiol-disulfide interchange with thioredoxins is now well established. Recent fruitful collaboration between biochemists and structural biologists, reflected by the shared authorship of the paper, allowed to solve the structures of all of the components of the system, including several target enzymes, thus providing a structural basis for the elucidation of the activation mechanism at a molecular level. In the present Review, these structural data are analyzed in conjunction with the information that was obtained previously through biochemical and site-directed mutagenesis approaches. The unique 4Fe-4S cluster enzyme ferredoxin:thioredoxin reductase (FTR) uses photosynthetically reduced ferredoxin as an electron donor to reduce the disulfide bridge of different thioredoxin isoforms. Thioredoxins in turn reduce regulatory disulfides of various target enzymes. This process triggers conformational changes on these enzymes, allowing them to reach optimal activity. No common activation mechanism can be put forward for these enzymes, as every thioredoxin-regulated protein undergoes specific structural modifications. It is thus important to solve the structures of the individual target enzymes in order to fully understand the molecular mechanism of the redox regulation of each of them.

Journal Article↗
PubMed2004

How is Ferredoxin-NADP Reductase Involved in the NADP Photoreduction of Chloroplasts?

NADP photoreduction of chloroplasts was discovered in 1951, and subsequent research was conducted to elucidate the enzymatic mechanisms involved in this reaction. In 1963, ferredoxin-NADP reductase (FNR; EC 1.18.1.2, ferredoxin-NADP oxidoreductase) was isolated and purified to a crystalline form. Because the reaction mechanism of ferredoxin-NADP reducing system was clarified in the isolated enzyme system, it was generally thought that the role of FNR in the NADP photoreduction of chloroplasts had been fully elucidated. However, the results of a reconstitution study using the crystallized FNR and the depleted grana, from which 'built-in' FNR had been eliminated, showed that the NADP photoreducing activity of reconstituted FNR was much lower than the original physiological activity, and as a result, more studies had to be continued. In 1985, a protein factor called 'connectein' was discovered, and it was shown that this new protein binds with two FNR molecules to form an FNR-connectein complex. Then in 1991, the FNR-connectein complex was formed using purified connectein and FNR, and after eliminating 'built-in' FNR, the reconstituted complex was bound to the depleted grana having reduced NADP photoreducing activity. The results showed that NADP photoreducing activity of the reconstituted system was comparable to the original physiological activity. This proved that the FNR-connectein complex, which binds to a specific site on the surface of thylakoid membrane, is functionally responsible for NADP photoreduction in chloroplasts.

Journal Article↗
PubMed2004

Structure and function of plant-type ferredoxins.

The plant-type ferredoxins (Fds) are the [2Fe-2S] proteins that function primarily in photosynthesis; they transfer electrons from photoreduced Photosystem I to ferredoxin NADP(+) reductase in which NADPH is produced for CO(2) assimilation. In addition, Fds partition electrons to various ferredoxin-dependent enzymes not only for assimilations of inorganic nitrogen and sulfur and N(2) fixation but also for regulation of CO(2) assimilation cycle. Although Fds are small iron-sulfur proteins with molecular weight of 11 KDa, they are expected to interact with surprisingly many enzymes. Several Fd isoforms were found in non-photosynthetic cells as well as Fds in photosynthetic cells, leading to the recognition that they have differentiated physiological roles. In a quarter of century, X-ray crystallography and NMR spectroscopy provided wealth of structural data, which shed light on the structure-function relationship of the plant-type Fds and gave structural basis for the biochemical and spectroscopic properties so far accumulated. Thus the structural studies of Fds have come to a new era in which different roles of Fds and interactions with various enzymes are clarified on the basis of the tertiary and quaternary structures, although they are premature at present. This article reviews briefly the structures of the plant-type Fds together with their functions, properties, and interactions with Fd related enzymes. Lastly the folding motif of Fd, that has grown to be a large family by including many functionally unrelated proteins, is noted.

Journal Article↗
PubMed1990

Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex.

Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.

Animals↗
PubMed2001

Role of a highly conserved YPITP motif in 2-oxoacid:ferredoxin oxidoreductase: heterologous expression of the gene from Sulfolobus sp.strain 7, and characterization of the recombinant and variant enzymes.

2-Oxoacid:ferredoxin oxidoreductase from Sulfolobus sp. strain 7, an aerobic and thermoacidophilic crenoarchaeon, catalyses the coenzyme A-dependent oxidative decarboxylation of pyruvate and 2-oxoglutarate, a cognate Zn-7Fe-ferredoxin serving as an electron acceptor. It comprises two subunits, a (632 amino acids) and b (305 amino acids). To further elucidate its structure and function, we constructed a gene expression system. The wild-type recombinant enzyme was indistinguishable from the natural one in every criterion investigated. A series of variants was constructed to elucidate the role of the YPITP-motif (residues 253-257) in subunit a, which is conserved universally in the 2-oxoacid:ferredoxin oxidoreductase (OFOR) family. Single amino-acid replacements at Y253 and P257 by other amino acids caused a drastic loss of enzyme activity. T256, the hydroxyl group of which has been proposed to be essential for binding of the 2-oxo group of the substrate in the Desulfovibrio africanus enzyme, was unexpectedly replaceable with Ala, the kcat and Km for 2-oxoglutarate being approximately 33% and approximately 51%, respectively, as compared with that of the wild-type enzyme. Replacement at other positions resulted in a significant decrease in the kcat of the reaction while the Km for 2-oxoacid was only slightly affected. Thus, the YPITP-motif is essential for the turnover of the reaction rather than the affinity toward 2-oxoacid.

Amino Acid Motifs↗
PubMed1966

Photosynthetic Electron Transport Chain of Chlamydomonas reinhardi. V. Purification and Properties of Cytochrome 553 and Ferredoxin.

Cytochrome 553 and ferredoxin were isolated and purified from acetone powders prepared from intact cells of the wild-type strain of Chlamydomonas reinhardi. Purification was achieved by ion exchange chromatography on DEAE cellulose and gel filtration on Sephadex G-75.Cytochrome 553 could be oxidized with postassium ferricyanide and reduced with sodium ascorbate. The absorption maxima of the cytochrome in reduced form were found to be alpha-band 552.5 nm, beta-band 522.5 nm, and Soret band 416.5 nm. The normal redox potential was +0.37 volt, and the molecular weight was estimated to be 12,000 +/- 2000. The cytochrome closely resembled that of the c-type cytochromes extracted from Euglena and Porphyra. When cultures were harvested after they had entered stationary phase, the cytochrome could be extracted in soluble form, and in some cases a yield of one cytochrome per 1000 chlorophyll molecules could be obtained.Ferredoxin showed absorption maxima at 278, 330, 420 and 460 nm. Thus it resembles the ferredoxin extracted from spinach. The molecular weight of the ferredoxin was estimated to be 15,000 +/- 2000.

Journal Article↗
PubMed1981

Purification and properties of two 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium.

Pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase were obtained from cell-free extracts of Halobacterium halobium as homogeneous proteins after ammonium sulfate precipitation, salting-out chromatography with ammonium sulfate on unsubstituted agarose, gel filtration and chromatography on hydroxyapatite. The respective molecular weights are 256000 and 248000. Both enzymes consist of two sets of non-identical subunits of Mr 86000 and 42000 in the case of the pyruvate-degrading enzyme and of 88000 and 36000 in the case of the 20 -oxogluatarate-degrading enzyme. Analyses indicate that an intact enzyme molecule contains two [4 Fe-4S]2 + (2 + , 1+) clusters and two molecules of thiamin diphosphate. Flavin nucleotides, lipoic acid and pantetheine are absent. Thus the enzymes are very similar to the 2-oxoacid:ferredoxin oxidoreductases from fermentative and photosynthetic anaerobes described previously, but are clearly different from the 2-oxoacid dehydrogenase multienzyme complexes which commonly occur in anaerobic organisms.

Amino Acids↗
PubMed1981

The catalytic mechanism of 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium. One-electron transfer at two distinct steps of the catalytic cycle.

The catalytic cycle of the 2-oxoacid:ferredoxin oxidoreductases from Halobacterium halobium was investigated. The first step is binding of the 2-oxoacid to the enzyme followed by decarboxylation and transfer of one electron to the [4Fe-4S] cluster of the functional unit. The cluster is then reoxidized by ferredoxin or, in the absence of the physiological electron acceptor, by oxygen. In the resulting stable enzyme-intermediate radical the decarboxylation product of the 2-oxoacid remains tightly bound until reaction with coenzyme A caused formation of acyl-CoA and concomitant transfer of the second electron to the cluster, which again is reoxidized by ferredoxin or oxygen. After purification, part of the enzyme molecules still contain the intermediate radical. Enzyme preparations either free of radical or containing enhanced amounts are obtained by treatment with coenzyme A or 2-oxoacid, respectively. Whenever the radical is present in an enzyme molecule the respective binding site for the 2-oxoacid is blocked.

Binding Sites↗
PubMed1995

Amino acid sequence of spinach ferredoxin:thioredoxin reductase catalytic subunit and identification of thiol groups constituting a redox-active disulfide and a [4Fe-4S] cluster.

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

Amino Acid Sequence↗