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.     

Exchange integral for a variety of tetranuclear ferredoxins.

The temperature dependence of EPR spectra of oxidized [4Fe-4S](-1,-2) ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S](-2,-3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm-1; Center S-3 from mung bean, 53 cm-1; the [4Fe-4S](-1,-2) ferredoxin from Thermus thermophilus, 78 cm-1; Center N-2 of NADH ubiquinone reductase, 83 cm-1. Increasing axial distortion in the EPR spectra of the [4Fe-4S](-1,-2), ferrodoxins was associated with higher energy differences. Center N-2, a [Fe-4S](-2,-3) iron-sulfur cluster does not fit this relationship.     

New insights into the thermostability of bacterial ferredoxins: high-resolution crystal structure of the seven-iron ferredoxin from Thermus thermophilus

The crystal structure of the seven-iron ferredoxin from Thermus thermophilus (FdTt) has been determined at 1.64 A resolution, allowing us to unveil the common mechanisms of thermostabilization within "bacterial-type" ferredoxins. FdTt and other homologous thermophilic seven-iron ferredoxins are smaller than their mesophilic counterparts. Thermostabilizing features are optimized in a minimal structural and functional unit, with an extensive cross-linking of secondary structure elements mediated by improved polar and hydrophobic interactions. Most of the potentially stabilizing features are focused on the vicinity of the functional [3Fe-4S] cluster. The structural [4Fe-4S] cluster is shielded in thermophilic FdTt by an increased number of polar interactions involving the two N-terminal residues. Comparisons with the hyperthermostable ferredoxin from Thermotoga maritima reveal that (1) a reduction in the number of non-glycine residues in strained conformations, (2) improved polar interactions within the common iron-sulfur cluster binding (betaalphabeta)2 motif, and (3) an optimized charge distribution at the protein surface, constitute a common strategy for increasing the thermal stability of these ferredoxins.     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

Sulredoxin: a novel iron-sulfur protein of the thermoacidophilic archaeon Sulfolobus sp. strain 7 with a Rieske-type [2Fe-2S] center.

A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called "sulredoxin." Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79; average g-factor [gav] = 1.90) which is remarkably similar to that of Thermus thermophilus respiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. O. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Münck, J. Biol. Chem. 259:124-133, 1984) and distinctively different from those of the plant-type ferredoxins (gav = 1.96). Sulredoxin, which is the first Rieske-type [2Fe-2S] protein isolated from an archaeal species, does not function as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase. Whether sulredoxin is derived from the archaeal membrane-bound respiratory Rieske-type FeS center (gy = 1.91) is the subject of further investigation.     

Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus

The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy. The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced [3Fe-xS] clusters. MCD magnetization data are consistent with an S = 2 ground state for both reduced [3Fe-xS] clusters, but indicate differences in the rhombicity of the zero-field splittings. The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced [3Fe-xS] cluster in A. vinelandii ferredoxin I. Spectroscopic studies of anaerobically isolated A. vinelandii ferredoxin I do not support the hypothesis that the [3Fe-xS] cluster arises as a result of aerial oxidative damage to a [4Fe-4S] cluster during isolation. The possibility that two distinct forms of [3Fe-xS] clusters can exist in A. vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH. The results reveal two distinct and interconvertible forms of the reduced [3Fe-xS] cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

EXAFS studies of the isolated bovine heart Rieske [2Fe-2S]1+(1+,2+) cluster

Recently the involvement of one or, more likely, two nitrogen-ligands in the Rieske-type [2Fe-2S] cluster has been reported based on the chemical assay and various spectroscopic analyses, such as EPR, M?ssbauer, ENDOR, and resonance Raman, of isolated Thermus thermophilus HB-8 protein by Fee and his collaborators. Similarly, the presence of at least one nitrogen ligand was shown in the mitochondrial Rieske [2Fe-2S] cluster. We have conducted EXAFS studies of the Rieske [2Fe-2S] protein isolated from the cytochrome bc1 complex of bovine heart mitochondria. Standard analysis could not distinguish one or two nitrogen ligands per cluster. However, one nitrogen and three cysteine ligands per cluster was found to be, possibly, a better solution in more comprehensive analysis procedures.     

Exchange integral for a variety of tetranuclear ferredoxins

The temperature dependence of EPR spectra of oxidized [4Fe-4S1]^{(?1, ?2)} ferredoxins (previously designated HiPIP) and a reduced [4Fe-4S1]^(?2,?3) ferredoxin have been analyzed so as to determine the energy of a low-lying excited electronic state. The values obtained were: Center S-3 from beef heart, 44 cm^?1; Center S-3 from mung bean, 53 cm^?1; the [4Fe-4S1]^(?1,?2) ferredoxin from Thermus thermophilus, 78 cm^?1; Center N-2 of NADH ubiquinone reductase, 83 cm^?1. Increasing axial distortion in the EPR spectra of the [4Fe-4S1]^(?1,?2) ferredoxins was associated with higher energy differences. Center N-2, a [4Fe-4S1]^(?2,?3) iron-sulfur cluster does not fit this relationship.     

Characterization of recombinant Desulfovibrio gigas ferredoxin.

Dg ferredoxin gene was cloned using the polymerase chain reaction (PCR), inserted into vector pT7-7, and overexpressed in Escherichia coli (E. coli) grown in aerobic media. The recombinant protein is a dimer and contains a [3Fe-4S] cluster per monomer. EPR and (1)H NMR data of recombinant and wild-type protein are compared.     

Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

A gene encoding the exact sequence of Clostridium pasteurianum 2[4Fe-4S] ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli. The synthetic gene is efficiently expressed in E. coli and its product has been purified and characterized. The N-terminal sequence is identical to that of the protein isolated from C. pasteurianum and the recombinant ferredoxin contains the exact amount of [4Fe-4S] clusters (2 per monomer) expected for homogeneous holoferredoxin. It displays reduction potential and kinetic parameters as electron donor to C. pasteurianum hydrogenase I identical to those determined for the native ferredoxin. All of these properties demonstrate that the 2[4Fe-4S] ferredoxin expressed in E. coli is identical to the parent clostridial protein.     

Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus.

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-'edge' carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0' = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.     

Characterization of the selenium-substituted 2 [4Fe-4Se] ferredoxin from Clostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from Clostridium pasteurianum have been replaced by selenium. The substitution is readily carried out by incubating the apoferredoxin with excess amounts of Fe3+, selenite, and dithiothreitol under anaerobic conditions. The UV-visible absorption spectrum of the Se-substituted ferredoxin, the core extrusion of its active sites, and analyses of its iron and selenium contents show that it contains two [4Fe-4Se] clusters. The Se-substituted ferredoxin is considerably less resistant to oxygen or to acidic and alkaline pH than the native ferredoxin: the half-lives of the former are 20-500 times shorter than those of the latter. The native ferredoxin and the Se-substituted ferredoxin display similar kinetic properties when used as electron donors to the hydrogenase from C. pasteurianum. It is of note, however, that the Km and Vmax values are lower for the 2[4Fe-4Se] ferredoxin than for the 2[4Fe-4S] ferredoxin. Reductive and oxidative titrations with dithionite and with thionine, respectively, show that both ferredoxins are two-electron carriers. The redox potentials of the ferredoxins have been measured by equilibrating them with the H2/H+ couple via hydrogenase: values of -423 and -417 mV have been found for the 2[4Fe-4S] ferredoxin and 2[4Fe-4Se] ferredoxin, respectively. Ferredoxins containing both chalcogenides in their [4Fe-4X] (X = S, Se) clusters have been prepared by reconstitution reactions involving mixtures of sulfide and selenide: the latter experiments show that sulfide and selenide are equally reactive in the incorporation of [4Fe-4X] (X = S, Se) sites into ferredoxin. The present report, together with former studies, establishes the general feasibility of the Se/S substitution in [2Fe-2S] and in [4Fe-4S] clusters of proteins and of synthetic analogues.     

Characterization and cloning of an extremely thermostable, Pyrococcus furiosus-type 4Fe ferredoxin from Thermococcus profundus.

An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid sequence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100 degrees C, while the half-life of the aerobically-purified ferredoxin is 10 h at 100 degrees C. Both the anaerobically- and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.     

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.     

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6–7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g=2.01 which is typical of [3Fe-4S]¹⁺ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g=1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]¹⁺ clusters at much higher yields (0.4–0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. grisues ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.     

DNA binding studies of ruthenium(II) complexes containing asymmetric tridentate ligands

Absorption spectroscopy, fluorescence spectroscopy and viscosity measurements have been used to characterize the DNA binding of [Ru(tpy)(dppt)](2+) (tpy=2,2':6',2"-terpyridine, dppt=3-(1,10-phenanthrolin-2-yl)-5,6-diphenyl-as-triazine), [Ru(tpy)(pta)](2+) (pta=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]acenaphthylene) and [Ru(tpy)(ptp)](2+) (ptp=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]-phenanthrene). The results indicate that [Ru(tpy)(pta)](2+) and [Ru(tpy)(ptp)](2+) bind with CT-DNA in an intercalative mode, while [Ru(tpy)(dppt)](2+) binds with DNA by partial intercalation. The ligand planarity of the complex has a significant effect on DNA binding affinity increases in the order [Ru(tpy)(dppt)](2+)<[Ru(tpy)(pta)](2+)<[Ru(tpy)(ptp)](2+).     

A novel ferredoxin-dependent glutamate synthase from the hydrogen-oxidizing chemoautotrophic bacterium Hydrogenobacter thermophilus TK-6

Glutamate synthases are classified according to their specificities for electron donors. Ferredoxin-dependent glutamate synthases had been found only in plants and cyanobacteria, whereas many bacteria have NADPH-dependent glutamate synthases. In this study, Hydrogenobacter thermophilus, a hydrogen-oxidizing chemoautotrophic bacterium, was shown to possess a ferredoxin-dependent glutamate synthase like those of phototrophs. This is the first observation, to our knowledge, of a ferredoxin-dependent glutamate synthase in a nonphotosynthetic organism. The purified enzyme from H. thermophilus was shown to be a monomer of a 168-kDa polypeptide homologous to ferredoxin-dependent glutamate synthases from phototrophs. In contrast to known ferredoxin-dependent glutamate synthases, the H. thermophilus glutamate synthase exhibited glutaminase activity. Furthermore, this glutamate synthase did not react with a plant-type ferredoxin (Fd3 from this bacterium) containing a [2Fe-2S] cluster but did react with bacterial ferredoxins (Fd1 and Fd2 from this bacterium) containing [4Fe-4S] clusters. Interestingly, the H. thermophilus glutamate synthase was activated by some of the organic acids in the reductive tricarboxylic acid cycle, the central carbon metabolic pathway of this organism. This type of activation has not been reported for any other glutamate synthases, and this property may enable the control of nitrogen assimilation by carbon metabolism.     

Crystallization and preliminary analysis of oxidized, recombinant, heterocyst [2Fe-2S] ferredoxin from Anabaena 7120.

The [2Fe-2S] ferredoxin produced in the heterocyst cells of Anabaena 7120 plays a key role in nitrogen fixation, where it serves as an electron acceptor from various sources and an electron donor to nitrogenase. Crystals of recombinant heterocyst ferredoxin, coded for by the fdx H gene from Anabaena 7120 and overproduced in Escherichia coli, have been grown from ammonium sulfate solutions and are suitable for high resolution X-ray crystallographic analysis. They belong to the hexagonal space group P6(1) or P6(5) with unit cell dimensions of a = b = 44.2 A and c = 80.6 A. The crystals contain one molecule per asymmetric unit and diffract to a nominal resolution of 1.6 A. The molecular structure of this heterocyst ferredoxin is of special interest in that 4 of the 22 amino acid positions thought to be absolutely conserved in nonhalophilic ferredoxins are different and, based on amino acid sequence alignments, three of these positions are located in the metal-cluster binding loop. Consequently, a high-resolution X-ray analysis of this [2Fe-2S] ferredoxin, and subsequent three-dimensional comparisons with other known ferredoxin models, will provide new insight into structure/function relationships for this class of redox proteins.