7Fe Ferredoxin Research Articles

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe–4S] and one [4Fe–4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S] + 1/0 (E 0′ = − 158 ± 5 mV); [4Fe–4S] + 2/+1 (E 0′ = − 474 ± 5 mV) and [3Fe–4S] 0/− 2 (E 0′ = − 660 ± 5 mV). The effect of pH on these signals showed that the reduced [3Fe–4S] 0 cluster has a p Kʹ red ′ = 5.1 ± 0.1, the [4Fe–4S] + 2/+1 centre is pH independent, and the [3Fe–4S] 0/−2 reduction is accompanied by the binding of two protons. The ability of the [3Fe–4S] 0 cluster to be converted into a new [4Fe–4S] cluster was proven. The redox potential of the original [4Fe–4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift ( ca. 70 mV) of the redox potential of the original centre. Being most [Fe–S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.

Characterization of a 2[4Fe-4S] Ferredoxin Obtained by Chemical Insertion of the Fe-S Clusters into the Apoferredoxin II from Rhodobacter Capsulatus

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.

Mycobacterium tuberculosis Senses Host-Derived Carbon Monoxide during Macrophage Infection

Mycobacterium tuberculosis (MTB) expresses a set of genes known as the dormancy regulon in vivo. These genes are expressed in vitro in response to nitric oxide (NO) or hypoxia, conditions used to model MTB persistence in latent infection. Although NO, a macrophage product that inhibits respiration, and hypoxia are likely triggers in vivo, additional cues could activate the dormancy regulon during infection. Here, we show that MTB infection stimulates expression of heme oxygenase (HO-1) by macrophages and that the gaseous product of this enzyme, carbon monoxide (CO), activates expression of the dormancy regulon. Deletion of macrophage HO-1 reduced expression of the dormancy regulon. Furthermore, we show that the MTB DosS/DosT/DosR two-component sensory relay system is required for the response to CO. Together, these findings demonstrate that MTB senses CO during macrophage infection. CO may represent a general cue used by pathogens to sense and adapt to the host environment.

Purification and characterization of the tungsten enzyme aldehyde:ferredoxin oxidoreductase from the hyperthermophilic denitrifier Pyrobaculum aerophilum

A tungsten-containing aldehyde:ferredoxin oxidoreductase (AOR) has been purified to homogeneity from Pyrobaculum aerophilum. The N-terminal sequence of the isolated enzyme matches a single open reading frame in the genome. Metal analysis and electron paramagnetic resonance (EPR) spectroscopy indicate that the P. aerophilum AOR contains one tungsten center and one [4Fe-4S](2+/1+) cluster per 68-kDa monomer. Native AOR is a homodimer. EPR spectroscopy of the purified enzyme that has been reduced with the substrate crotonaldehyde revealed a W(V) species with g(zyx) values of 1.952, 1.918, 1.872. The substrate-reduced AOR also contains a [4Fe-4S](1+) cluster with S=3/2 and zero field splitting parameters D=7.5 cm(-1) and E/D=0.22. Molybdenum was absent from the enzyme preparation. The P. aerophilum AOR lacks the amino acid sequence motif indicative for binding of mononuclear iron that is typically found in other AORs. Furthermore, the P. aerophilum AOR utilizes a 7Fe ferredoxin as the putative physiological redox partner, instead of a 4Fe ferredoxin as in Pyrococcus furiosus. This 7Fe ferredoxin has been purified from P. aerophilum, and the amino acid sequence has been identified using mass spectrometry. Direct electrochemistry of the ferredoxin showed two one-electron transitions, at -306 and -445 mV. In the presence of 55 microM ferredoxin the AOR activity is 17% of the activity obtained with 1 mM benzyl viologen as an electron acceptor.

Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase.

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD-NADPH and FADH2-NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one-electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe-2S] iron-sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 microM(-1) x s(-1) (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH-ferredoxin oxidoreductase.

Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry.

Rapid responses of biological [4Fe-4S] clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions. Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that [3Fe-4S] clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control. Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two [4Fe-4S](2+/+) clusters at similar potentials. Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from [3Fe-4S] species (+/0 and 0/2- couples). The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored. Optimal [3Fe-4S] formation (in excess over [4Fe-4S]) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein. Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired. The [3Fe-4S]:[4Fe-4S] ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that [3Fe-4S](+) resists further oxidative damage. Oxidative disassembly involves successive one-electron oxidations of [4Fe-4S] (i.e., 2+ --> 3+ --> 4+), with [3Fe-4S](+) being a relatively stable byproduct, that is, not an intermediate. Disassembly of [3Fe-4S] in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2-. In the presence of Fe(2+), the 0 level is stabilized by conversion back to [4Fe-4S](2+/+). By pulsing in the presence of Zn(2+), the [3Fe-4S] clusters that are formed are trapped rapidly as their Zn adducts.

Protein film cryovoltammetry: demonstrations with a 7Fe ([3Fe–4S] + [4Fe–4S]) ferredoxin

Low-temperature protein film voltammetry, demonstrated here by studies of Fe–S clusters in the 7Fe ferredoxin from Thermoplasma acidophilum, offers a novel way to investigate active site redox reactions, allowing resolution of complex transformations and transient states, and providing an immediate and sensitive gauge of solvent effects.

Fast-scan cyclic voltammetry of protein films on pyrolytic graphite edge electrodes: characteristics of electron exchange.

The rapid electron-exchange characteristics of metalloproteins adsorbed at a pyrolytic graphite "edge" electrode have been studied by analog dc cyclic voltammetry at scan rates up to 3000 V s-1. The voltammetry of four proteins, azurin (a "blue" copper protein) and three 7Fe ferredoxins, reveals oxidation and reduction peaks that display only modest increases in width and peak separation as the scan rate is raised. This is indicative of a substantially homogeneous population of noninteracting centers which undergo rapid electron exchange with the electrode. Both the Butler--Volmer and Marcus models have been tested. The electrochemical kinetics, as reflected by k0 (the rate at zero overpotential), are too fast to allow the determination of reorganization energies by this method. Nonetheless, the rapid and energetically coherent nature of the electron transfer enables the cyclic oxidation and reduction of protein redox centers to be examined on a time scale sufficiently short to recognize coupled processes occurring in the millisecond time domain, which are characteristic of the protein under investigation. Two of the ferredoxins display increasingly asymmetric voltammetry as the scan rate is increased, which is attributed to the coupling of electron transfer to conformational (or orientational) changes. For azurin, the use of higher electrolyte concentrations enables studies to be made at scan rates up to 3000 V s-1, from which a standard electron-transfer rate constant in the region of 5000 s-1 is obtained. At these high scan rates, azurin still shows very symmetrical voltammograms but with peak shapes displaying a more gradual decrease in current, at increasing overpotential, than is predicted using realistic values of the reorganization energy. The ability to measure even faster rate constants and access coupled reactions occurring in shorter time domains is likely to be limited by complex processes occurring on the graphite surface.

Kinetics and Mechanism of Redox-Coupled, Long-Range Proton Transfer in an Iron−Sulfur Protein. Investigation by Fast-Scan Protein-Film Voltammetry

Fast-scan protein-film voltammetry has been used to study and deconvolute the kinetics of proton-coupled electron transfer to and from the [3Fe−4S] cluster in Azotobacter vinelandii Ferredoxin I (Av Fd I). This crystallographically defined 7Fe ferredoxin contains a [3Fe−4S] cluster which takes up a proton when in its reduced (0) state. Results conform to a model for stepwise electron and proton transfers. Electron transfer to the cluster drives its protonation, whereas the rate of reoxidation of [3Fe−4S]0-H+ is controlled by the rate of release of the proton. The cluster is buried and inaccessible to solvent water molecules; an aspartate (D15) is located on the protein surface but otherwise proton transfer occurs across an aprotic barrier. The D15N mutant, in which this aspartate is replaced by asparagine, has also been studied. Rate constants for proton transfer in each direction and the accompanying energetics (reduction potentials and interdependent pK values of the cluster and aspartate) have been determined simultaneously by modeling voltammetric peak positions (measured over a range of scan rate and pH) using numerical (digital) simulation. For comparison, rates of proton-gated oxidation were measured independently using stopped-flow spectrophotometry. Proton transfer between the cluster and bulk water occurs over 100 times faster in the native protein than in D15N, and the kinetics are described by a mechanism in which the proton is transferred using the aspartate carboxylate. Lack of an H/D isotope effect suggests that this group acts as a short-range courier. The system provides an intriguing functional model for redox-driven proton pumps, being both well-defined at the molecular level and amenable to detailed study by film voltammetry.

Structure of Azotobacter vinelandii 7Fe ferredoxin at 1.35 Å resolution and determination of the [Fe-S] bonds with 0.01 Å accuracy

The crystal structure of Azotobacter vinelandii ferredoxin I (FdI) at 100 K has been refined at 1.35 Å resolution by full matrix block diagonal least-squares methods with anisotropic temperature factors for all non-hydrogen atoms and with hydrogen atoms included in the model. Fe-S bonds within the [3Fe-4S] + and [4Fe-4S] 2+ clusters of the protein are determined with an accuracy of at least 0.01 Å. Analysis of metric parameters reveals greater variation in bonds and angles within the [3Fe-4S] + cluster than in the [4Fe-4S] 2+ cluster, whereas the opposite is true regarding the cysteine Sγ atoms ligating to the two [Fe-S] cores. The [3Fe-4S] + core is asymmetrically distorted by the protein matrix but relatively uniformly ligated by its three Cys ligands; in contrast the tetrahedral [4Fe-4S] 2+ core is relatively symmetric but non-uniformily ligated by its four Cys ligands, three of which occur in a conserved CysxxCysxxCys residue motif. Comparison of the [3Fe-4S] + clusters in FdI and Desulfovibrio gigas ferredoxin II, refined at 1.7 Å resolution, indicates that within the limit of accuracy of the two refinements the cuboidal core is differently distorted in the two proteins. Comparison of the [3Fe-4S] + core in FdI with the structure of a reduced [Fe 3S 4] o synthetic analog † indicates that the protein-bound cluster displays distortions not intrinsic to the core itself. Nevertheless, both [3Fe-4S] + and [Fe 3S 4] o cores have metric features consistent with expected trends due to net charge on Fe and valency of S, and both exhibit a splayed configuration with respect to their three μ 2S atoms in the absence of a fourth Fe. Comparison of the[4Fe-4S] 2+ cluster in FdI with the structures of [Fe 4S 4] 2+ synthetic analogs shows that the protein bound and synthetic cubanes are very similar in geometric parameters, including the presence of tetragonal distortion in the FdI cluster common to this oxidation state.

A stable intermediate product of the archaeal zinc-containing 7Fe ferredoxin from Sulfolobus sp. strain 7 by artificial oxidative conversion

Irreversible conversion of the purified zinc-containing 7Fe ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was found to occur under aerobic conditions at pH 5.0 and at 4°C. This process accompanied a substantial increase of the electron paramagnetic resonance signal attributed to a [3Fe-4S] 1+ cluster, and the converted form containing ∼6 Fe/Zn (mol/mol) had a net charge different from that of the native 7Fe ferredoxin. These data provide evidence for the formation of a stable intermediate product of the archaeal ferredoxin having two [3Fe-4S] clusters and a zinc center by artificial oxidative degradation. This further explains the discrepancy that a zinc center and two [3Fe-4S] clusters (but not a zinc center and one [3Fe-4S] cluster plus one [4Fe-4S] cluster) are observed in the crystal structure at pH 5.0.

Electrochemical Potential and pH Dependences of [3Fe-4S] ↔ [M3Fe-4S] Cluster Transformations (M = Fe, Zn, Co, and Cd) in Ferredoxin III from Desulfovibrio africanus and Detection of a Cluster with M = Pb

The 7Fe ferredoxin from Desulfovibrio africanus contains a reactive 3Fe cluster which incorporates Fe and other metals to form cubanes [M3Fe-4S] having non-cysteine ligation to the added metal (M). These reactions are addressed at controlled potentials in protein molecules adsorbed on a graphite electrode, thereby facilitating an appraisal of factors that control cluster interconversions. Electrochemical and spectroscopic methods have been used to establish connectivities among interconversions with different M, explore the influence of pH, and determine reactivities of specific cluster oxidation levels. Formation of clusters with M = Fe, Zn, and Co depends on ionization of a group with pK = 5.5 + 0.3, thus supporting congruence among products and suggesting common involvement of aspartate-14 which replaces a cysteine normally present in [4Fe-4S] binding motifs. The influence of potential is complex: rapid and reversible interconversion (M = Fe, Zn) occurs only between the states [M3Fe-4S]2+ and [3Fe-4S]...

The D13C variant of Bacillus schlegelii 7Fe ferredoxin is an 8Fe ferredoxin as revealed by 1H-NMR spectroscopy

The N-terminal cluster binding motif Cys 8XXXXXXXCys 16....Cys 49 of Bacillus schlegelii 7Fe ferredoxin, which provides the ligands to the [Fe 3S 4] + cluster, was modified by the mutation Asp 13→Cys. The mutant D13C is expressed in Escherichia coli as an 8Fe ferredoxin, with NMR properties similar to those of clostridial-type ferredoxins. The full assignment of the hyperfine shifted resonances indicates that Cys 13 serves as ligand to the new fourth iron atom in the N-terminal cluster despite the atypical binding sequence CysXXXXCysXXCys....Cys. The Cα–Cβ–S–Fe dihedral angles of all cysteine ligands to the two [Fe 4S 4] 2+ clusters of the D13C variant are similar to those observed in other 8Fe and 4Fe ferredoxins.

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Identification of a Palindromic Sequence That Is Responsible for the Up-regulation of NAPDH-Ferredoxin Reductase in a Ferredoxin I Deletion Strain of Azotobacter vinelandii

Azotobacter vinelandii ferredoxin I (AvFdI) is one member of a class of 7Fe ferredoxins found in a variety of organisms that are all capable of aerobic growth. Disruption of the fdxA gene, which encodes AvFdI, leads to overexpression of its redox partner, NADPH-ferredoxin reductase (FPR). In this study the mechanism of FdI-mediated regulation of FPR was investigated. Northern analysis has shown that regulation is at the level of fpr transcription, the start site for transcription has been identified, and it is preceded by a canonical sigma 70-type bacterial promoter. Gel mobility shift assays show that there is a putative regulatory protein in A. vinelandii that binds specifically upstream of the -35 region. That protein is not AvFdI. A palindromic sequence was identified as a putative binding site, and randomization of that sequence completely eliminates binding of the putative regulatory protein. A luciferase reporter gene was placed under control of the A. vinelandii fpr promoter and introduced into wild type and FdI- strains of A. vinelandii. Luciferase activity was enhanced 7-fold in the FdI- mutant relative to the wild type. Alteration of the palindromic sequence reduced the luciferase levels in the FdI- strain to those of the wild type, demonstrating that FdI regulates FPR through the palindrome and that the reaction is an activation rather than a repression. The identified palindrome is approximately 50% identical to the SoxS binding site upstream of Escherichia coli fpr, suggesting that A. vinelandii may have a SoxS-like regulatory system and that the function of FdI might be to specifically inactivate that system.

3Fe-4S] [4Fe-4S] cluster interconversion in Desulfovibrio africanus ferredoxin III: properties of an Asp14 --> Cys mutant.

The 8Fe ferredoxin III from Desulfovibrio africanus is a monomeric protein which contains two [4Fe-4S]2+/1+ clusters, one of which is labile and can readily and reversibly lose one Fe under oxidative conditions to yield a [3Fe-4S]1+/0 cluster. This 4Fe cluster has an S = 3/2 ground sping state insteaed of S = 1/2 in the reduced +1 state [George, Armstrong, Hatchikian and Thomson (1989) Biochem. J. 264, 275-284]. The co-ordination to this cluster is unusual in that an aspartate (Asp14, D14, is found where a cysteine residue normally occurs. Using a mutant protein obtained from the overexpression in Escherichia coli of a synthetic gene in which Asp14, the putative ligand to the removable Fe, has been changed to Cys, we have studied the cluster interconversion properties of the labile cluster. Analysis by EPR and magnetic-circular-dichroism spectroscopies showed that the Asp14 --> Cys (D14C) mutant contains two [4Fe-4S]2+/1+ clusters, both with S = 1/2 in the reduced state. Also, unlike in native 8Fe D. africanus ferredoxin III, the 4Fe <--> 3Fe cluster interconversion reaction was found to be sluggish and did not go to completion. It is inferred that the reversibility of the reaction in the native protein is due to the presence of the aspartate residue at position 14 and that this residue might protect the [3Fe-4S] cluster from further degradation.

Characterization of a 7Fe Ferredoxin Isolated from the Marine DenitrifierPseudomonas nauticaStrain 617: Spectroscopic and Electrochemical Studies

A 7Fe ferredoxin, isolated from the marine denitrifierPseudomonas nauticastrain 617, was characterized. The NH2-terminal sequence analysis, performed until residue number 56, shows a high similarity with the 7Fe ferredoxins isolated fromAzotobacter vinelandii, Pseudomonas putida,andPseudomonas stutzeri.EPR and NMR spectroscopies identify the presence of both [3Fe-4S] and [4Fe-4S] clusters, with cysteinyl coordination. The electrochemical studies on [Fe-S] clusters show that a fast diffusion-dominated electron transfer, promoted by Mg(II), takes place between the ferredoxin and the glassy carbon electrode. Square wave voltammetry studies gave access to the electrosynthesis of a 4Fe center formed within the [3Fe-4S] core. The [3Fe-4S] cluster exhibited two reduction potentials at −175 and −680 ± 10 mV and the [4Fe-4S] cluster was characterized by an unusually low reduction potential of −715 ± 10 mV, at pH 7.6.

Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus.

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

Novel Redox Chemistry of [3Fe−4S] Clusters: Electrochemical Characterization of the All-Fe(II) Form of the [3Fe−4S] Cluster Generated Reversibly in Various Proteins and Its Spectroscopic Investigation in Sulfolobus acidocaldarius Ferredoxin

The novel “hyper-reduced” form of protein-bound [3Fe−4S] clusters, which is two electron equivalents below the normal reduced form [3Fe−4S]0 and thus formally composed entirely of Fe(II) subsites, has been characterized by electrochemistry and by EPR, MCD, and UV/visible spectroscopy. The two-electron reduction of [3Fe−4S]0 has been studied for a range of proteins, in particular the 7Fe ferredoxins from Sulfolobus acidocaldarius, Desulfovibrio africanus, and Azotobacter vinelandii. In each case, the reaction is chemically reversible, the product is surprisingly inert, and the pH-dependent reduction potential is in the region of −700 mV vs SHE at pH 7, regardless of the identity of the protein. Protein film voltammetry of three different ferredoxins investigated in detail over a wide pH range shows that the novel species denoted as [3Fe−4S]2- is formed by a cooperative two-electron reduction of [3Fe−4S]0 and there is a net uptake of three protons relative to the all-Fe(III) state [3Fe−4S]1+. The protons ar...

The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.