Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

A [4Fe4S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions $\text{a = 32.96}\text{A}\text{?}\text{, b = 37.83}\text{A}\text{?}\text{, c = 39.82}\text{A}\text{?}\text{, α = 118.1 °, β = 104.2 °}\text{and}\text{γ = 89.7 °}$. The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Fe4S] cluster.     

Primary structure of a 7Fe ferredoxin from Streptomyces griseus

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the FeS clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two FeS clusters are conserved.     

Iron-sulfur clusters and cysteine distribution in a ferredoxin from Azotobactervinelandii

In 80% dimethyl sulfoxide/H₂O, $\text{Azotobacter}$ ferredoxin FeS clusters can be extruded with benzene thiol. The extruded clusters have an absorption spectra maximum at 458 nm which is characteristic of 4Fe4S centers. The amino terminal sequence of the $\text{Azotobacter}$ ferredoxin has 7 of the 8 Cys residues at residue numbers 8, 11, 16, 20, 24, 39 and 42. Except for Cys 24, all of these residues can be correlated to homologous Cys residues in other bacterial ferredoxins. Although two thirds of the first 45 residues are identical to or conservative replacements for the first 43 residues of other bacterial ferredoxins, the insertion of Cys-24 indicates a major change in the environment of one of the two 4Fe4S clusters.     

Isolation and characterization of a rubredoxin and an (8Fe-8S) ferredoxin from Desulfuromonas acetoxidans

A two cluster (4Fe4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins.The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an absorbance ratio of $\text{A}{}_{385}\text{A}{}_{283}\text{= 0.74}$. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C.The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.     

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.     

A new membrane iron-sulfur flavoprotein of the mitochondrial electron transfer system the entrance point of the fatty acyl dehydrogenation pathway?

An iron-sulfur (FeS) protein has been purified from beef heart mitochondria by following its EPR signal after reduction, which is characteristic of a ferredoxin-type FeS protein (g_x = 1.886; g_y = 1.939; g_z = 2.086). The signal intensity corresponds to one unpaired spin for 4 to 5 Fe atoms. The light absorption spectrum indicates the presence of flavin. Fe, labile S, and FAD are released by acid at a ratio of approximately 4:4:1. Neither prosthetic group of the protein is reduced by NADH, NADPH, succinate, glycerol-3-phosphate or dihydroorotate. The FeS group is, however, reduced with a half-time of ～5 msec, when the protein is mixed with an equivalent amount of electron transferring flavoprotein (ETF) of the β-oxidation cycle, prereduced with an acyl CoA dehydrogenase and a saturated fatty acyl CoA. In the presence of the two added flavoproteins the behavior of the flavin of the FeS flavoprotein could not be determined. Complexes I–III are not reduced by reduced ETF under analogous conditions. The low field EPR resonance [“center 5”, Ohnishi $\text{et al}$. (1972), Biochem. Biophys. Res. Commun. $\text{46}$, 1631–1638] of the protein is readily observed in whole tissue, mitochondria and sonic fragments from all species we have examined. Therefore, the protein appears to be a universal constituent of mitochondrial electron transfer systems.     

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe2S] ferredoxins isolated from various plants and algae comprise 93–99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be ProTyrSerCysArgAlaGlyAlaCysSerThrCysAlaGly ProLeuLeuThr CysVal which should have a [2Fe2S] cluster with a low redox potential     

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe–4S] Cluster Chemistry

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe–2S] ferredoxin as the electron donor and an active site comprising a [4Fe–4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM–FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM–FTR using absorption, EPR, electron–nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe–4S]²⁺ cluster to yield a unique type of S= 1/2 [4Fe–4S]³⁺ cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe–4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe–4S]-mediated cleavage of the CoM–S–S–CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.     

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.     

Replacement of sulfide by selenide in the [4Fe-4S] clusters of the ferredoxin fromClostridium pasteurianum

The sulfur atoms of the two [4Fe-4S] clusters present in the ferredoxin from $\text{C. pasteurianum}$ have been replaced by selenium. The optical absorption spectrum of the Se-ferredoxin is slightly different from the spectrum of the native protein, but it displays the characteristic features of [4Fe-4X] ( X = S, Se) clustors. The reduced Se-ferredoxin can reduce hydrogenase, and the oxidized Se-ferredoxin can be reduced by hydrogenase in the presence of molecular hydrogen. This is the first report of sulfide substitution by selenide in an iron-sulfur protein containing [4Fe-4S] active sites.     

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from<Emphasis Type="Italic"> Acidaminococcus fermentans</Emphasis>

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as ?405 mV and ?340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.     

Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe–2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe–2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe–S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)^?1min^?1 for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)^?1 min^?1 for Pux. This difference mainly arises from weak CYP199A2–PuxB binding (K _m 34.3 vs. 0.45 μM for Pux) rather than slow electron transfer (k _cat 19.1 vs. 37.9 s^?1 for Pux). Comparison of the 2.0-Å-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin–P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe–2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.     

Low-temperature magnetic circular dichroism evidence for the conversion of four-iron-sulphur clusters in a ferredoxin from Clostridium pasteurianum into three-iron-sulphur clusters

Oxidation of the 8Fe ferredoxin from Clostridium pasteurianum with potassium ferricyanide, followed by purification on Sephadex G-25 and DE-23 cellulose columns, gives a protein with an intense EPR signal at g 2.01. The low-temperature magnetic circular dichroism (MCD) spectra of this species are different from those of the oxidized high-potential iron protein from Chromatium but identical with the spectra of ferredoxin II from Desulphovibrio gigas. On reduction of the ferricyanide-treated ferredoxin with sodium dithionite only a weak EPR signal with g factors of 2.05, 1.94 and 1.89 is obtained. The low-temperature MCD spectra are strongly temperature dependent with a form similar to those of dithionite-reduced D. gigas ferredoxin II. The MCD magnetization curves are dominated by a species with ground-state effective g factors of $\text{g}{}_{\mathrm{?}}$ 8.0 and g_⊥ 0.0, which are also similar to those determined recently by low-temperature MCD spectroscopy for D. gigas ferredoxin II. The MCD characteristics are quite different from those of dithionite-reduced ferredoxin from Cl. pasteurianum, untreated with ferricyanide. This establishes the close similarity of the iron-sulphur clusters in ferricyanide-treated Cl. pasteurianum ferredoxin and in D. gigas ferredoxin II. The latter is known to contain a single 3Fe centre, similar to that observed in ferredoxin I from Azotobacter vinelandii by X-ray crystallography. Therefore, it is concluded that the [4Fe-4S] clusters of Cl. pasteurianum ferredoxin are converted to 3Fe clusters on oxidation with ferricyanide.     

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus

A [3Fe–4S]^1+/0 ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe–4S]^1+/0, with an unusually low reduction potential of ?650 mV, determined by cyclic voltammetry at pH 7.6. [¹H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe–4S]^1+/0 centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe–4S]^2+/1+ cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism.The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T_m) for thermally induced unfolding is 102±2 °C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 °C). The iron–sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.     

Redox reactions of the hydrogenase-cytochrome C3 system from Desulfovibrio gigas with the synthetic analogue of ferredoxin active sites [Fe4S4 (SCH2CH2OH)4]2−

The synthetic cluster [Fe₄S₄ (SCH₂CH₂OH)₄]^2? and ferredoxin I from $\text{Desulfovibrio}\text{gigas}$ have been comparatively reacted in the presence of hydrogen with two enzyme components isolated from the $\text{D.}\text{gigas}$ electron carrier system: hydrogenase and cytochrome c₃. The reactions have been followed by spectrophotometric and manometric methods. No reduction is detected unless both enzymes are simultaneously present. The easy reduction in the presence of hydrogenase-cytochrome c₃ combination takes place in two stages corresponding each to one equivalent of reduction. The first stage is reversible while the second is not and yields a super reduced species deriving from the cluster complex.     

Magnetic interaction of nickel(III) and the iron-sulphur cluster in hydrogenase from Chromatium vinosum

Upon partial reduction of hydrogenase from Chromatium vinosum with ascorbate plus phenazine methosulphate, EPR signals due to Ni(III) and a [3Fe-xS] cluster appear simultaneously and with equal intensities. Since the intact enzyme shows no $\text{S =}\text{1}\text{2}$ signals, it is concluded that Ni(III) and a [4Fe-4S]³⁺ cluster interact magnetically in such a way as to prevent the detection of the two paramagnets as individual $\text{S =}\text{1}\text{2}$ systems. This interaction is thought to be the origin of a signal in which Fe is involved and which is not due to an $\text{S =}\text{1}\text{2}$ system (Albracht, S.P.J., Albrecht-Ellmer, K.J., Schmedding, D.J.M. and Slater, E.C. (1982) Biochim. Biophys. Acta 681, 330–334). A variable fraction of the enzyme preparation shows signals due to Ni(III) and a [3Fe-xS] cluster with equal intensities without any further treatment. These are thought to be derived from irreversibly inactivated enzyme molecules. The enzyme contains no selenium.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of < 5 μs and ≈ 50 μs). We believe that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll.We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below ?450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD⁺ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Insight into the protein and solvent contributions to the reduction potentials of [4Fe–4S]2+/+ clusters: crystal structures of the Allochromatium vinosum ferredoxin variants C57A and V13G and the homologous Escherichia coli ferredoxin

The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe–4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-Å resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E°). This is better illustrated by V13G AlvinFd (high exposure, E° = ?594 mV) and EcFd (low exposure, E° = ?675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180–250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe–4S] ferredoxins on their electrochemical properties.