Azotobacter chroococcum 7Fe ferredoxin. Two pH-dependent forms of the reduced 3Fe clusters and its conversion to a 4Fe cluster.
ABSTRACT: 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.
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.
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.
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.
Project description: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.
Project description:The D14C variant of Pyrococcus furiosus ferredoxin provides an extraordinary framework to investigate a [3Fe-4S] cluster at two oxidation levels and compare the results to its physiologic [4Fe-4S] counterpart in the very same protein. Our spectroscopic and computational study reveals vibrational property changes related to the electronic and structural aspects of both Fe-S clusters.
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.
Project description:The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.
Project description:Azotobacter vinelandii ferredoxin I is a small protein that contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. Recently the x-ray crystal structure has been redetermined and the fdxA gene, which encodes the protein, has been cloned and sequenced. Here we report the site-directed mutation of Cys-20, which is a ligand of the [4Fe-4S] cluster in the native protein, to alanine and the characterization of the protein product by x-ray crystallographic and spectroscopic methods. The data show that the mutant protein again contains one [4Fe-4S] cluster and one [3Fe-4S] cluster. The new [4Fe-4S] cluster obtains its fourth ligand from Cys-24, a free cysteine in the native structure. The formation of this [4Fe-4S] cluster drives rearrangement of the protein structure.
Project description:Reactions of the [3Fe-4S] cluster and various metallated [M3Fe-4S] adducts co-ordinated in the ferredoxin from the hyperthermophile Pyrococcus furiosus have been studied by protein-film voltammetry, bulk-solution voltammetry, solution kinetics and magnetic CD (MCD). The [3Fe-4S] cluster exhibits two couples, [3Fe-4S]+/0 and [3Fe-4S]0/2-. Film voltammetry is possible over a wide pH range (2-8), revealing that the [3Fe-4S]+/0 couple shows a complex pH dependence with pKred1=2.8, pKox=4.9 and pKred2=6.7. From MCD, pKred1 corresponds with protonation of [3Fe-4S]0 to give a spectroscopically distinct species, as reported for ferredoxins from Azotobacter and Sulfolobus. The status of the disulphide/disulphydryl entity makes no significant difference to the data (given for the -S-S- form). Formation of the hyper-reduced [3Fe-4S]2- state is observed, requiring 3H+ for the overall 3e- reduction of [3Fe-4S]+, the change therefore being electroneutral. By comparison with the ferredoxin from Desulfovibrio africanus, uptake of Fe(II) and other M(II) by [3Fe-4S]0 to give [M3Fe-4S] clusters is slow (t1/2>10 min at room temperature, slower still if the protein is adsorbed on the electrode), whereas reaction with Tl(I) to produce [Tl3Fe-4S] is very rapid (t1/2<<1 s), suggesting that co-ordination of Tl does not require reorganization of the protein structure. Rates of formation of [3Fe-4S] from [M3Fe-4S] adducts increase sharply at high potentials, showing that metal release involves a labile 'super-oxidized' [M3Fe-4S]3+ state.
Project description:All radical <i>S</i>-adenosylmethionine (radical-SAM) enzymes, including the noncanonical radical-SAM enzyme diphthamide biosynthetic enzyme Dph1-Dph2, require at least one [4Fe-4S](Cys)<sub>3</sub> cluster for activity. It is well-known in the radical-SAM enzyme community that the [4Fe-4S](Cys)<sub>3</sub> cluster is extremely air-sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1-Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe-4S] cluster is easily degraded into a [3Fe-4S] cluster. Remarkably, the small iron-containing protein Dph3 donates one Fe atom to convert the [3Fe-4S] cluster in Dph1-Dph2 to a functional [4Fe-4S] cluster during the radical-SAM enzyme catalytic cycle. This mechanism to maintain radical-SAM enzyme activity in aerobic environments is likely general, and Dph3-like proteins may exist to keep other radical-SAM enzymes functional in aerobic environments.