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PubMed2003

Open questions in ferredoxin-NADP+ reductase catalytic mechanism.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one-electron donors (ferredoxin, flavodoxin) to redox-based metabolisms in plastids, mitochondria and bacteria. The plant-type reductase is also the basic prototype for one of the major families of flavin-containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site-directed mutagenesis, steady-state and transient kinetic experiments, spectroscopy and X-ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced-fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery-Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.

Bacterial Proteins↗
PubMed1969

The biosynthesis of ferredoxin in a cell-free system.

The investigation reported in this communication was concerned with the biosynthesis of ferredoxin in a cell-free system. A cell-free system, with the ability to incorporate amino acids into peptides and proteins, was developed from Cl. pasteurianum, and its requirements were established. After the incorporation of L-alanine-C(14), ferredoxin-C(14) was isolated in crystalline form with the aid of added carrier ferredoxin. Amino acid analyses and absorption spectra strongly indicated that the isolated ferredoxin-C(14), crystallized three times, was essentially free from impurities. Furthermore, experiments with Fe(59) strongly indicated that the iron may combine with the apoprotein after the latter is completely synthesized and released from the polysomes.

Alanine↗
PubMed1975

Regulation of ferredoxin-catalyzed photosynthetic phosphorylations.

Under aerobic conditions that are likely to prevail in chloroplasts in vivo, the optimal concentration of ferredoxin for cyclic photophosphorylation was found to be equal to that required for NADP reduction and about one-tenth of that needed for cyclic photophosphorylation under anaerobic conditions. In the presence of ferredoxin and NADP, cyclic photophosphorylation operated concurrently with noncyclic photophosphorylation, producing an ATP: NADPH ratio of about 1.5. The effective operation of ferredoxin-catalyzed cyclic photophosphorylation by itself required a curtailment of the electron flow from water which was accomplished experimentally by the use of either an inhibitor or far-red monochromatic light. An unexpected discovery was that the operation of cyclic photophosphorylation by itself was also regulated by a back reaction of NADPH and ferredoxin with two components of chloroplast membranes, component C550 and cytochrome b559. The significance of these findings to photosynthesis in vivo is discussed.

Aerobiosis↗
PubMed1991

Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.

Amino Acid Sequence↗
PubMed1998

The two [4Fe-4S] clusters in Chromatium vinosum ferredoxin have largely different reduction potentials. Structural origin and functional consequences.

The 2[4Fe-4S] ferredoxin from Chromatium vinosum arises as one prominent member of a recently defined family of proteins found in very diverse bacteria. The potentiometric circular dichroism titrations of the protein and of several molecular variants generated by site-directed mutagenesis have established that the reduction potentials of the two clusters differ widely by almost 200 mV. This large difference has been confirmed by electrochemical methods, and each redox transition has been assigned to one of the clusters. The unusually low potential center is surprisingly the one that displays a conventional CX1X2CX3X4C (Xn, variable amino acid) binding motif and a structural environment similar to that of clusters having less negative potentials. A comparison with other ferredoxins has highlighted factors contributing to the reduction potential of [4Fe-4S] clusters in proteins. (i) The loop between the coordinating cysteines 40 and 49 and the C terminus alpha-helix of C. vinosum ferredoxin cause a negative, but relatively moderate, shift of approximately 60 mV for the nearby cluster. (ii) Very negative potentials, below -600 mV, correlate with the presence of a bulky side chain in position X4 of the coordinating triad of cysteines. These findings set the framework in which previous observations on ferredoxins can be better understood. They also shed light onto the possible occurrence and properties of very low potential [4Fe-4S] clusters in less well characterized proteins.

Chromatium↗
PubMed2000

Essential role of a single arginine of photosystem I in stabilizing the electron transfer complex with ferredoxin.

PsaE is one of the photosystem I subunits involved in ferredoxin binding. The central role of arginine 39 of this 8-kDa peripheral polypeptide has been established by a series of mutations. The neutral substitution R39Q leads to a 250-fold increase of the dissociation constant K(d) of the photosystem I-ferredoxin complex, as large as the increase induced by PsaE deletion. At pH 8.0, this K(d) value strongly depends on the charge of the residue substituting Arg-39: 0.22 microM for wild type, 1.5 microM for R39K, 56 microM for R39Q, and more than 100 microM for R39D. The consequences of arginine 39 substitution for the titratable histidine were analyzed as a function of pH. The K(d) value of R39H is increased 140 times at pH 8.0 but only 5 times at pH 5.8, which is assigned to the protonation of histidine at low pH. In the mutant R39Q, the association rate of ferredoxin was decreased 3-fold compared with wild type, whereas an 80-fold increase is calculated for the dissociation rate. We propose that a major contribution of PsaE is to provide a prominent positive charge at position 39 for controlling the electrostatic interaction and lifetime of the complex with ferredoxin.

Arginine↗
PubMed2001

Iron-sulfur cluster assembly: characterization of IscA and evidence for a specific and functional complex with ferredoxin.

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon. The product of one member of this operon, IscA, has been overexpressed, purified, and characterized. It can assemble an air-sensitive [2Fe-2S] cluster as shown by UV-visible and resonance Raman spectroscopy. The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the [2Fe-2S] holoferredoxin. These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation. This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.

Bacterial Proteins↗
PubMed2000

Spectroscopic investigation of selective cluster conversion of archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7.

Archaeal zinc-containing ferredoxin from Sulfolobus sp. strain 7 contains one [3Fe-4S] cluster (cluster I), one [4Fe-4S] cluster (cluster II), and one isolated zinc center. Oxidative degradation of this ferredoxin led to the formation of a stable intermediate with 1 zinc and approximately 6 iron atoms. The metal centers of this intermediate were analyzed by electron paramagnetic resonance (EPR), low temperature resonance Raman, x-ray absorption, and (1)H NMR spectroscopies. The spectroscopic data suggest that (i) cluster II was selectively converted to a cubane [3Fe-4S](1+) cluster in the intermediate, without forming a stable radical species, and that (ii) the local metric environments of cluster I and the isolated zinc site did not change significantly in the intermediate. It is concluded that the initial step of oxidative degradation of the archaeal zinc-containing ferredoxin is selective conversion of cluster II, generating a novel intermediate containing two [3Fe-4S] clusters and an isolated zinc center. At this stage, significant structural rearrangement of the protein does not occur. We propose a new scheme for oxidative degradation of dicluster ferredoxins in which each cluster converts in a stepwise manner, prior to apoprotein formation, and discuss its structural and evolutionary implications.

Chromatography, Agarose↗
PubMed1975

A combined procedure for preparation of plastocyanin, ferredoxin and CF1.

Homogeneous preparations of ferredoxin, plastocyanin, and chloroplast coupling factor (CF1) have been isolated from spinach by a combined procedure in which supernatants from preparation of chloroplasts are used for isolation of ferredoxin and the chloroplasts serve as the source of plastocyanin. The proteins were purified by DEAE-cellulose chromatography and gel filtration, after precipitation with acetone in the case of ferredoxin or release from membranes in the case of plastocyanin. The proteins obtained by this procedure are pure, as evidenced by absorption ratios (ferredoxin, A420/A276 = 0.47-0.48; plastocyanin, A278/A597 = 1.2) and by the fact that both proteins migrate as single bands on polyacrylamide gels in the presence of sodium dodecyl sulfate.

Chloroplasts↗
PubMed1998

Modeling of Entamoeba histolytica ferredoxin.

The E. histolytica ferredoxin (EHFXD) is an iron-sulphur protein and is important in the control of E. histolytica because it donates an electron to and activates metronidazole (4) the most effective anti-amoebic drug. The knowledge of the three-dimensional structure of EHFXD can help to assist in rational design of anti-amebic drugs. Homology modeling of EHFXD has been done by using the knowledge of crystal structure of homologous Ferredoxin structures. It has been shown that EHFXD is more likely to contain 2[4Fe-4S] clusters and has a pseudodiad symmetrical fold. The fold is stabilised by hydrophobic patches as well as by intramolecular hydrogen bonds. The importance of two Leu residues in the N- and C-termi in bridging the two clusters has been emphasised. The electrostatic properties of the surface of EHFXD were examined and compared with that of other ferredoxins. It was observed that large regions of negative as well as positive electrostatic potential is a common feature of the [4Fe-4S] containing ferredoxins which might explain their biological role as both electron acceptor and electron donor molecules.

Amino Acid Sequence↗
PubMed1989

Ferredoxin and rubredoxin from Butyribacterium methylotrophicum: complete primary structures and construction of phylogenetic trees.

Complete amino acid sequences of ferredoxin and rubredoxin from Butyribacterium methylotrophicum, a methylotrophic hetero-acetogen, were determined by combination of protease digestion, Edman degradation, carboxypeptidase digestion, and/or partial acid hydrolysis. The ferredoxin was composed of 55 amino acids with a molecular weight of 5,732 excluding iron and sulfur atoms and showed a typical 2[4Fe-4S]-type ferredoxin sequence with an internal repeat at the 14-23 and 42-51 positions. The rubredoxin was composed of 53 amino acids with a molecular weight of 5,672 excluding iron atom and showed a sequence similar to those of other anaerobic rubredoxins. The sequences were compared to those of corresponding proteins from six different bacteria to construct phylogenetic trees, which showed essentially the same topology. The relationships between the ferredoxin sequences from this bacterium and those of Clostridium thermoaceticum and Methanosarcina barkeri, both of which possess a carbonyl-dependent acetyl-CoA metabolic system, are also discussed.

Amino Acid Sequence↗
PubMed1982

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 Sequence↗
PubMed1994

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.

Amino Acid Sequence↗
PubMed2001

Maize non-photosynthetic ferredoxin precursor is mis-sorted to the intermembrane space of chloroplasts in the presence of light.

Preprotein translocation across the outer and inner envelope membranes of chloroplasts is an energy-dependent process requiring ATP hydrolysis. Several precursor proteins analyzed so far have been found to be imported into isolated chloroplasts equally well in the dark in the presence of ATP as in the light where ATP is supplied by photophosphorylation in the chloroplasts themselves. We demonstrate here that precursors of two maize (Zea mays L. cv Golden Cross Bantam) ferredoxin isoproteins, pFdI and pFdIII, show distinct characteristics of import into maize chloroplasts. pFdI, a photosynthetic ferredoxin precursor, was efficiently imported into the stroma of isolated maize chloroplasts both in the light and in the dark. In contrast pFdIII, a non-photosynthetic ferredoxin precursor, was mostly mis-sorted to the intermembrane space of chloroplastic envelopes as an unprocessed precursor form in the light but was efficiently imported into the stroma and processed to its mature form in the dark. The mis-sorted pFdIII, which accumulated in the intermembrane space in the light, could not undergo subsequent import into the stroma in the dark, even in the presence of ATP. However, when the mis-sorted pFdIII was recovered and used for a separate import reaction, pFdIII was capable of import into the chloroplasts in the dark. pFNRII, a ferredoxin-NADP+ reductase isoprotein precursor, showed import characteristics similar to those of pFdIII. Moreover, pFdIII exhibited similar import characteristics with chloroplasts isolated from wheat (Pennisetum americanum) and pea (Pisum sativum cv Alaska). These findings suggest that the translocation of precursor proteins across the envelope membranes of chloroplasts may involve substrate-dependent light-regulated mechanisms.

Adenosine Triphosphate↗
PubMed2004

Crystallization of 4-hydroxybenzoyl-CoA reductase and the structure of its electron donor ferredoxin.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a central enzyme in the metabolism of phenolic compounds in anaerobic bacteria. The enzyme catalyzes the reductive removal of the phenolic hydroxyl group from 4-hydroxybenzoyl-CoA, yielding benzoyl-CoA and water. 4-HBCR belongs to the xanthine oxidase (XO) family of molybdenum enzymes which occur as heterodimers, (alphabetagamma)(2). 4-HBCR contains two molybdopterins, four [2Fe-2S] and two [4Fe-4S] clusters and two FADs. A low-potential Allochromatium vinosum-type ferredoxin containing two [4Fe-4S] clusters serves as an in vivo electron donor for 4-HBCR. In this work, the oxygen-sensitive proteins 4-HBCR and the ferredoxin (TaFd) from Thauera aromatica were crystallized under anaerobic conditions. 4-HBCR crystallized with PEG 4000 and MPD as precipitant diffracted to about 1.6 A resolution and the crystals were highly suitable for X-ray structure analysis. Crystals of TaFd were obtained with (NH(4))(3)PO(4) as precipitant and revealed a solvent content of 77%, which is remarkably high for a small soluble protein. The structure of TaFd was solved at 2.9 A resolution by the molecular-replacement method using the highly related structure of the ferredoxin (CvFd) from A. vinosum as a model. Structural changes between the two ferredoxins around the [4Fe-4S] cluster can be correlated with their different redox potentials.

Crystallography, X-Ray↗
PubMed2001

High stability of a ferredoxin from the hyperthermophilic archaeon A. ambivalens: involvement of electrostatic interactions and cofactors.

The ferredoxin from the thermophilic archaeon Acidianus ambivalens is a small monomeric seven-iron protein with a thermal midpoint (T(m)) of 122 degrees C (pH 7). To gain insight into the basis of its thermostability, we have characterized unfolding reactions induced chemically and thermally at various pHs. Thermal unfolding of this ferredoxin, in the presence of various guanidine hydrochloride (GuHCl) concentrations, yields a linear correlation between unfolding enthalpies (DeltaH[T(m)]) and T(m) from which an upper limit for the heat capacity of unfolding (DeltaC(P)) was determined to be 3.15 +/- 0.1 kJ/(mole * K). Only by the use of the stronger denaturant guanidine thiocyanate (GuSCN) is unfolding of A. ambivalens ferredoxin at pH 7 (20 degrees C) observed ([GuSCN](1/2) = 3.1 M; DeltaG(U)[H(2)O] = 79 +/- 8 kJ/mole). The protein is, however, less stable at low pH: At pH 2.5, T(m) is 64 +/- 1 degrees C, and GuHCl-induced unfolding shows a midpoint at 2.3 M (DeltaG(U)[H(2)O] = 20 +/- 1 kJ/mole). These results support that electrostatic interactions contribute significantly to the stability. Analysis of the three-dimensional molecular model of the protein shows that there are several possible ion pairs on the surface. In addition, ferredoxin incorporates two iron-sulfur clusters and a zinc ion that all coordinate deprotonated side chains. The zinc remains bound in the unfolded state whereas the iron-sulfur clusters transiently form linear three-iron species (in pH range 2.5 to 10), which are associated with the unfolded polypeptide, before their complete degradation.

Archaeal Proteins↗
PubMed1975

The active species of 'CO2' utilized by reduced ferredoxin:CO2 oxidoreductase from Clostridium pasteurianum.

Reduced ferredoxin:CO2 oxidoreductase (CO2 reductase) from Clostridium pasteurianum catalyzes the reduction of 'CO2' to formate with reduced ferredoxin, an isotopic exchange between 'CO2' and formate in the absence of ferredoxin, and the oxidation of formate to 'CO2' with oxidized ferredoxin. The active species of 'CO2', i.e. CO2 or HCO3 (H2CO3), utilized by the enzyme was determined. The method employed for the species identification was that of Copper et al. (1968). Both 'CO2' reduction to formate and the exchange reaction were studied. Data were obtained which are compatible with those expected if CO2 is the active species. The V and the dissociation constant Ks of the enzyme - CO2 complex in dependence of pH were determined from initial velocity studies of the exchange reaction. V was found to be only slightly affected by pH between 5.5 and 7.5. Ks was markedly dependent on pH; the constant increased with decreasing pH from 0.2 mM at pH 7.5 to 3 mM at pH 5.5.

Aldehyde Oxidoreductases↗
PubMed1978

Oxygen activation in isolated chloroplasts. Mechanism of ferredoxin-dependent ethylene formation from methionine.

Low-potential electron acceptors of photosystem I of chloroplast lamellae produce superoxide anions (0-2) and hydrogen peroxide by autoxidation, but have no effect on ethylene formation from methionine; equimolar amounts of ferredoxin are less active in photosynthetic O-2 and H2O2 production but strongly stimulate ethylene production from methionine. 2. Ten to fifty units of superoxide dismutase inhibit fifty to two hundred units of superoxide dismutase stimulate ethylene formation from methionine by chloroplast lamellae in the presence of ferredoxin. This stimulation is stronger at pH 7.0 than at pH 7.8. Catalase inhibits ethylene formation from methionine. 3. Pulse-radiolytic production of nitrite (NO-2) from hydroxylamine, initiated by hydroxyl radicals (.OH) or O-2, shows no difference in the presence or absence of ferredoxin, nor do the decay kinetics of O2. 4. From the above observations and from model reactions (xanthine/xanthine oxidase; iron salts in the presence of H2O2), it is concluded that reduced ferredoxin in the presence of H2O2 forms a Fenton-type oxidizing species for methionine, generating ethylene in the presence of pyridoxal phosphate. 5. Inhibitory effects of both superoxide dismutase and catalase in oxygen-dependent reactions need not necessarily indicate the participation of the 'Haber-Weiss' reaction.

Chloroplasts↗