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Electrochemical and spectroscopic characterization of the conversion of the 7Fe into the 8Fe form of ferredoxin III from Desulfovibrio africanus. Identification of a [4Fe-4S] cluster with one non-cysteine ligand.

ABSTRACT: Desulfovibrio africanus ferredoxin III is a protein (Mr 6585) containing one [3Fe-4S]1+,0 and one [4Fe-4S]2+,1+ core cluster when aerobically isolated. The amino acid sequence contains only seven cysteine residues, the minimum required to ligand these two clusters. Cyclic voltammery by means of direct electrochemistry at a pyrolytic-graphite-'edge' electrode promoted by neomycin shows that, when reduced, the [3Fe-4S]0 centre reacts rapidly with Fe(II) ion to form a [4Fe-4S]2+ cluster. The latter, which can be reduced at a redox potential similar to that of the other [4Fe-4S] cluster, must include non-thiolate ligation. We propose that the carboxylate side chain of aspartic acid-14 is the most likely candidate, since this amino acid occupies the position of a cysteine residue in the sequence typical of an 8Fe ferredoxin. The magnetic properties at liquid-He temperature of this novel cluster, studied by low-temperature magnetic-c.d. and e.p.r. spectroscopy, are diamagnetic in the oxidized state and S = 3/2 in the one-electron-reduced state. This cluster provides a plausible model for the ligation states of the [4Fe-4S]1+ core in the S = 3/2 cluster of the iron protein of nitrogenase and in Bacillus subtilis glutamine:phosphoribosyl pyrophosphate amidotransferase.

SUBMITTER: George SJ

PROVIDER: S-EPMC1133573 | biostudies-other | 1989 Nov

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1133572 | biostudies-other

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

Project description: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.

| S-EPMC1218320 | biostudies-other

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

Project description:The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

| S-EPMC21691 | biostudies-literature

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

Project description: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.

| S-EPMC1217053 | biostudies-other

Azotobacter chroococcum 7Fe ferredoxin. Two pH-dependent forms of the reduced 3Fe clusters and its conversion to a 4Fe cluster.

Project description:Ferredoxin from Azotobacter chroococcum has been studied by low-temperature magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy. When aerobically isolated ferredoxin contains a [3Fe-4S] and [4Fe-4S] cluster. Anaerobic treatment with dithionite in the presence of ethanediol reduces the [3Fe-4S] cluster to give two spectroscopically distinct forms RI and RII which are reversibly interconvertible with a pKa approximately 7.5. The higher-pH form, RII, has a high affinity for ferrous ion and converts readily to a [4Fe-4S]1+ cluster, scavenging iron from the medium. The presence of the iron chelator EDTA inhibits this conversion.

| S-EPMC1144420 | biostudies-other

Spectroscopic studies on the [4Fe-4S] cluster in adenosine 5'-phosphosulfate reductase from Mycobacterium tuberculosis.

Project description:Mycobacterium tuberculosis adenosine 5'-phosphosulfate reductase (MtAPR) is an iron-sulfur protein and a validated target to develop new antitubercular agents, particularly for the treatment of latent infection. The enzyme harbors a [4Fe-4S](2+) cluster that is coordinated by four cysteinyl ligands, two of which are adjacent in the amino acid sequence. The iron-sulfur cluster is essential for catalysis; however, the precise role of the [4Fe-4S] cluster in APR remains unknown. Progress in this area has been hampered by the failure to generate a paramagnetic state of the [4Fe-4S] cluster that can be studied by electron paramagnetic resonance spectroscopy. Herein, we overcome this limitation and report the EPR spectra of MtAPR in the [4Fe-4S](+) state. The EPR signal is rhombic and consists of two overlapping S = ½ species. Substrate binding to MtAPR led to a marked increase in the intensity and resolution of the EPR signal and to minor shifts in principle g values that were not observed among a panel of substrate analogs, including adenosine 5'-diphosphate. Using site-directed mutagenesis, in conjunction with kinetic and EPR studies, we have also identified an essential role for the active site residue Lys-144, whose side chain interacts with both the iron-sulfur cluster and the sulfate group of adenosine 5'-phosphosulfate. The implications of these findings are discussed with respect to the role of the iron-sulfur cluster in the catalytic mechanism of APR.

| S-EPMC3020729 | biostudies-literature

Reversible cycling between cysteine persulfide-ligated [2Fe-2S] and cysteine-ligated [4Fe-4S] clusters in the FNR regulatory protein.

Project description:Fumarate and nitrate reduction (FNR) regulatory proteins are O(2)-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S](2+)-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S](2+) cluster undergoes a rapid conversion to a [2Fe-2S](2+) cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S](2+) and [2Fe-2S](2+) clusters in Escherichia coli FNR. Selective (34)S labeling of the bridging sulfides in the [4Fe-4S](2+) cluster-bound form of FNR facilitated identification of resonantly enhanced Cys(32)S-(34)S stretching modes in the resonance Raman spectrum of the O(2)-exposed [2Fe-2S](2+) cluster-bound form of FNR. This result indicates O(2)-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S](2+) cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O(2) exposure of [4Fe-4S](2+)-FNR. The [4Fe-4S](2+) cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S](2+) cluster by anaerobic incubation with DTT and Fe(2+) ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O(2)-exposed forms of O(2)-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O(2) sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.

| S-EPMC3465412 | biostudies-literature

Draft Genome Sequence for Desulfovibrio africanus Strain PCS.

Project description:Desulfovibrio africanus strain PCS is an anaerobic sulfate-reducing bacterium (SRB) isolated from sediment from Paleta Creek, San Diego, CA. Strain PCS is capable of reducing metals such as Fe(III) and Cr(VI), has a cell cycle, and is predicted to produce methylmercury. We present the D. africanus PCS genome sequence.

| S-EPMC3624683 | biostudies-literature

Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.

Project description:The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1-ISCA2-IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1-ISCA2-IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factor-assisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion.

| S-EPMC7456137 | biostudies-literature

Characterization of [4Fe-4S]2+, [4Fe-4Se]2+ and hybrid (S, Se) clusters in Clostridium pasteurianum ferredoxin. A resonance Raman study.

Project description:Resonance Raman spectra of native 2[4Fe-4S] ferredoxin from Clostridium pasteurianum and of its selenium-substituted analogue 2[4Fe-4Se] are compared. The experimental conditions used in this study included low temperature (approx. 25K) and the absence of a glass sample cell, thus ensuring high signal-to-noise ratios. The spectra of the 2[4Fe-4S] and 2[4Fe-4Se] ferredoxins display similar numbers of bands, but the resonance Raman patterns differ largely, except for two bands observed at 353 cm-1 and 365 cm-1 in spectra of the native ferredoxin, which are only moderately shifted upon S----Se substitution and are attributed to Fe-S(cysteine) stretching modes. The activities of the latter modes, enhanced by 457.9 nm excitation, are nearly equal in both ferredoxins. These data were used to demonstrate the presence of hybrid clusters [4Fe-(4-n)S-nSe] (n = 1, 2, 3) in a ferredoxin the active sites of which had been reconstituted in the presence of both S and Se.

| S-EPMC1153550 | biostudies-other

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