Three-iron clusters in iron-sulfur proteins

Contents. 1. Introduction and history. 2. Characteristic spectroscopic features of 3Fe clusters. 1. General considerations. 2. M?ssbauer spectroscopy. 3. Magnetic circular dichroism (MCD) spectroscopy. 4. Electron paramagnetic resonance (EPR) spectroscopy. 5. Resonance Raman (RR) spectroscopy. 6. Extended X-ray fine-structure (EXAFS) spectroscopy. 3. Results of X-Ray diffraction studies. 4. Proteins containing or showing features characteristic of 3Fe clusters 1. Overview. 2. Ferredoxin I of Azotobacter vinelandii. 3. Ferredoxin II of Desulfovibrio gigas. 4. Aconitase from beef heart. 5. Other observations and considerations relevant to 3Fe clusters or cluster interconversions 1. Oxidative degradation of [4Fe-4S] clusters to 3Fe clusters. 2. Extrusion studies on 3Fe clusters. 3. Reconstitution of 3Fe clusters. 4. Disposition of iron ligands in cluster interconversions. 6. Do all 3Fe clusters have the same structure? Evidence for [3Fe-4S] clusters. 7. Are 3Fe clusters artifacts or biologically significant structures?     

The spatial relationships and structure of the binuclear iron-sulfur clusters in succinate dehydrogenase.

Two binuclear iron-sulfur clusters (designated S-1 and S-2) are present in succinate dehydrogenase in approximately equal concentration to that of flavin. The large difference in their midpoint potentials (0 and -400 mV, respectively, in the soluble enzyme) permits the acquisition of individual electron paramagnetic resonance spectra characterized by nearly identical rhombic g tensors (gz = 2.025, gy = 1.93, gx = 1.905). Spin-coupling between the two centers is manifested by broadening and splitting of spectra of reconstitutively active and inactive succinate dehydrogenase, respectively, as the temperature is lowered; relief of power saturation of Center S-1 spectra on reduction of Center S 2; and observation of half-field ("delta ms = 2") signals in the dithionite-reduced enzyme. Saturation behavior of fully reduced dehydrogenase is consistent with the presence of S-1 and S-2 at equivalent concentrations/molecule. Simulation of the spin-coupled spectra, assuming dipolar interaction, provides information on molecular structure. Electron paramagnetic resonance spectra of the enzyme in 80% dimethylsulfoxide are nearly identical to the characteristic binuclear spectra obtained with adrenodoxin. These data provide additional evidence for binuclear structure of both Center S-1 and S-2. The extremely fast relaxation of Center S-2 at low temperatures would imply either an anomalously small value of J or an alternative relaxation mechanism, possibly due to the coupling between S-1 and S-2.     

On the interpretation of electron paramagnetic resonance spectra of binuclear iron-sulfur proteins

A method is described for the interpretation of electron paramagnetic resonance spectra of reduced binuclear iron-sulfur proteins. The g_y values for any protein can be analyzed so that both the symmetry and the extent of covalency at the paramagnetic site can be parameterized. These parameters can be related to the chemical composition of the paramagnetic center, the protein-dependent charge delocalization of the unpaired electron, and the geometric arrangement at the reduced iron atom. These analyses may ultimately be used to rationalize certain aspects of the redox potentials of the various iron-sulfur proteins.     

Structure and electrochemistry of proteins harboring iron-sulfur clusters of different nuclearities. Part IV. Canonical,non-canonical and hybrid iron-sulfur proteins

A plethora of proteins are able to express iron-sulfur clusters, but have a clear picture of the different types of proteins and the different iron-sulfur clusters they harbor it is not easy.In the last five years we have reviewed structure/electrochemistry of metalloproteins expressing: (i) single types of iron-sulfur clusters (namely: {Fe(Cys)₄}, {[Fe₂S₂](Cys)₄}, {[Fe₂S₂](Cys)₃(X)} (X?=?Asp, Arg, His), {[Fe₂S₂](Cys)₂(His)₂}, {[Fe₃S₄](Cys)₃}, {[Fe₄S₄](Cys)₄} and {[Fe₄S₄](Cys)₃(nonthiolate ligand)} cores); (ii) metalloproteins harboring iron-sulfur centres of different nuclearities (namely: [4Fe-4S] and [2Fe-2S], [4Fe-4S] and [3Fe-4S], and [4Fe-4S], [3Fe-4S] and [2Fe-2S] clusters. Our target is now to review structure and electrochemistry of proteins harboring canonical, non-canonical and hybrid iron-sulfur proteins.     

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.     

Tetranuclear and binuclear iron-sulfur clusters in succinate dehydrogenase: a method of iron quantitation by formation of paramagnetic complexes.

Soluble succinate dehydrogenase contains 8 atoms of iron, 8 atoms of acid labile sulfur and one covalently bound FAD per molecule; however, the distribution of iron and sulfur has not been well established. An iron counting method was devised in which electron spin resonance detectable complexes containing one iron each were formed with NO and cysteine and complex formation was measured during the gradual dissociation of the iron-sulfur clusters. In addition, a method described by Cammack was used to provide independent evidence. Both methods point to the existence of two binuclear clusters and one tetranuclear iron-sulfur cluster in the succinate dehydrogenase molecule.     

Insights into properties and energetics of iron-sulfur proteins from simple clusters to nitrogenase

Some of the principal physical features of iron-sulfur clusters in proteins are analyzed, including metal-ligand covalency, spin polarization, spin coupling, valence delocalization, valence interchange and small reorganization energies, with emphasis on recent spectroscopic and theoretical work. The current state of structural, spectroscopic, and computational knowledge for the iron-sulfur clusters in the nitrogenase iron and iron-molybdenum proteins is examined by comparison and contrast to 'simpler' ironclusters. The differing interactions of the nitrogenase iron and iron-molybdenum clusters compared with those of other iron-sulfur clusters with the protein and solvent environment are also explored.     

Estimation of labile sulfide in iron-sulfur proteins.

Lowry's method (1) for protein determination is subject to interference from the nonionic detergent Triton X-100 (2,3) which is used in high concentrations (1–5%) to solubilize membrane proteins or enzymes (4–6) and structural acidic proteins (7). Hartree (3) could reduce the errors caused by 0.1% Triton X-100 by a modification of Lowry's method. However, when protein solutions containing 0.2% or more of the detergent are mixed with the Folin-Ciocalteu reagent (1) a precipitate forms that interferes with the assay. We could reduce this interference to an insignificant level either by centrifuging the precipitate and incorporating Triton X-100 in both the reagent blank and standards, or by removing the detergent prior to the assay. This report presents two simple procedures for the Lowry assay of dilute protein samples containing 1–5% Triton X-100.     

On the iron-sulfur clusters in the complex redox enzyme dihydropyrimidine dehydrogenase.

Porcine liver dihydropyrimidine dehydrogenase is a homodimeric iron-sulfur flavoenzyme that catalyses the first and rate-limiting step of pyrimidine catabolism. The enzyme subunit contains 16 atoms each of nonheme iron and acid-labile sulfur, which are most likely arranged into four [4Fe-4S] clusters. However, the presence and role of such Fe-S clusters in dihydropyrimidine dehydrogenase is enigmatic, because they all appeared to be redox-inactive during absorbance-monitored titrations of the enzyme with its physiological substrates. In order to obtain evidence for the presence and properties of the postulated four [4Fe-4S] clusters of dihydropyrimidine dehydrogenase, a series of EPR-monitored redox titrations of the enzyme under a variety of conditions was carried out. No EPR-active species was present in the enzyme 'as isolated'. In full agreement with absorbance-monitored experiments, only a small amount of neutral flavin radical was detected when the enzyme was incubated with excess NADPH or dihydrouracil under anaerobic conditions. Reductive titrations of dihydropyrimidine dehydrogenase with dithionite at pH 9.5 and photochemical reduction at pH 7.5 and 9.5 in the presence of deazaflavin and EDTA led to the conclusion that the enzyme contains two [4Fe-4S]2+,1+ clusters, which both exhibit a midpoint potential of approximately -0.44 V (pH 9.5). The two clusters are most likely close in space, as demonstrated by the EPR signals which are consistent with dipolar interaction of two S = 1/2 species including a half-field signal around g approximately 3.9. Under no circumstances could the other two postulated Fe-S centres be detected by EPR spectroscopy. It is concluded that dihydropyrimidine dehydrogenase contains two [4Fe-4S] clusters, presumably determined by the C-terminal eight-iron ferredoxin-like module of the protein, whose participation in the enzyme-catalysed redox reaction is unlikely in light of the low midpoint potential measured. The presence of two additional [4Fe-4S] clusters in dihydropyrimidine dehydrogenase is proposed based on thorough chemical analyses on various batches of the enzyme and sequence analyses. The N-terminal region of dihydropyrimidine dehydrogenase is similar to the glutamate synthase beta subunit, which has been proposed to contain most, if not all, the cysteinyl ligands that participate in the formation of the [4Fe-4S] clusters of the glutamate synthase holoenzyme. It is proposed that the motif formed by the Cys residues at the N-terminus of the glutamate synthase beta subunit, which are conserved in dihydropyrimidine dehydrogenase and in several beta-subunit-like proteins or protein domains, corresponds to a novel fingerprint that allows the formation of [4Fe-4S] clusters of low to very low midpoint potential.     

Characterization of the iron-sulfur clusters in ferredoxin from acetate-grown Methanosarcina thermophila.

Ferredoxin from Methanosarcina thermophila is an electron acceptor for the CO dehydrogenase complex which decarbonylates acetyl-coenzyme A and oxidizes the carbonyl group to carbon dioxide in the pathway for conversion of the methyl group of acetate to methane (K. C. Terlesky and J. G. Ferry, J. Biol. Chem. 263:4080-4082, 1988). Resonance Raman spectroscopy and electron paramagnetic resonance spectroelectrochemistry indicated that the ferredoxin contained two [4Fe-4S] clusters per monomer of 6,790 Da, each with a midpoint potential of -407 mV. A [3Fe-4S] species, with a midpoint potential of +103 mV, was also detected in the protein at high redox potentials. Quantitation of the [3Fe-4S] and [4Fe-4S] centers revealed 0.4 and 2.1 spins per monomer, respectively. The iron-sulfur clusters were unstable in the presence of air, and the rate of cluster loss increased with increasing temperature. A ferredoxin preparation, with a low spin quantitation of [4Fe-4S] centers, was treated with Fe2+ and S2-, which resulted in an increase in [4Fe-4S] and a decrease in [3Fe-4S] clusters. The results of these studies suggest the [3Fe-4S] species may be an artifact formed from degradation of [4Fe-4S] clusters.     

Non-redox roles for iron-sulfur clusters in enzymes

In recent years a number of enzymes have been discovered which, contrary to prior expectations, contain FeS clusters but do not participate in redox reactions. In all cases but one, where the FeS cluster in these enzymes has been identified, it is a [4Fe-4S] cluster. In mammalian aconitase a single Fe atom of the [4Fe-4S] cluster participates in catalysis of hydration-dehydration reactions by direct ligation to the substrates. A number of hydrolyases containing FeS clusters have now been identified. In Bacillus subtilis glutamine phosphoribosyl-pyrophosphate amidotransferase the [4Fe-4S] cluster is essential for the active structure of the enzyme, but probably does not participate directly in catalysis. Rather, the cluster may serve as part of a mechanism of oxidative inactivation of the enzyme in vivo, which is followed by its intracellular degradation. The role played by a [4Fe-4S] cluster in Escherichia coli endonuclease III is at present completely unknown. Thus, a number of novel roles for FeS clusters in enzymology and protein structure have been discovered, and more novel findings must be anticipated.     

Valence electron delocalization in polynuclear iron-sulfur clusters

 This commentary assesses the evidence for valence electron delocalization in dinuclear and polynuclear iron-sulfur clusters. We outline a simple Hamiltonian model that contains the important physical interactions and briefly review the experimental and computational tools that can be used to distinguish between valence electron delocalization and electron trapping and to assess likely magnitudes of resonance interactions. Received: 12 January 1996 / Accepted: 23 January 1996     

Repair of oxidized iron-sulfur clusters in Escherichia coli

The [4Fe-4S]2+ clusters of dehydratases are rapidly damaged by univalent oxidants, including hydrogen peroxide, superoxide, and peroxynitrite. The loss of an electron destabilizes the cluster, causing it to release its catalytic iron atom and converting the cluster initially to an inactive [3Fe-4S]1+ form. Continued exposure to oxidants in vitro leads to further iron release. Experiments have shown that these clusters are repaired in vivo. We sought to determine whether repair is mediated by either the Isc or Suf cluster-assembly systems that have been identified in Escherichia coli. We found that all the proteins encoded by the isc operon were critical for de novo assembly, but most of these were unnecessary for cluster repair. IscS, a cysteine desulfurase, appeared to be an exception: although iscS mutants repaired damaged clusters, they did so substantially more slowly than did wild-type cells. Because sulfur mobilization should be required only if clusters degrade beyond the [3Fe-4S]1+ state, we used whole cell EPR to visualize the fate of oxidized enzymes in vivo. Fumarase A was overproduced. Brief exposure of cells to hydrogen peroxide resulted in the appearance of the characteristic [3Fe-4S]1+ signal of the oxidized enzyme. When hydrogen peroxide was then scavenged, the enzyme activity reappeared within minutes, in concert with the disappearance of the EPR signal. Thus it is unclear why IscS is required for efficient repair. The iscS mutants grew poorly, allowing the possibility that metabolic defects indirectly slow the repair process. Our data did indicate that damaged clusters decompose beyond the [3Fe-4S]1+ state in vivo when stress is prolonged. Under the conditions of our experiments, mutants that lacked other repair candidates--Suf proteins, glutathione, and NADPH: ferredoxin reductase--all repaired clusters at normal rates. We conclude that the mechanism of cluster repair is distinct from that of de novo assembly and that this is true because mild oxidative stress does not degrade clusters in vivo to the point of presenting an apoenzyme to the de novo cluster-assembly systems.     

A role for iron-sulfur clusters in DNA repair

The presence of 4Fe-4S clusters in enzymes involved in DNA repair has posed the question of the role of these intricate cofactors in damaged DNA recognition and repair. It is particularly intriguing that base excision repair glycosylases that remove a wide variety of damaged bases, and also have vastly different sequences and structures, have been found to contain this cofactor. The accumulating biochemical and structural evidence indicates that the region supported by the cluster is intimately involved in DNA binding, and that such binding interactions impact catalysis of base removal. Recent evidence has also established that binding of the glycosylases to DNA facilitates oxidation of the [4Fe-4S](2+) cluster to the [4Fe-4S](3+) form. Notably, the measured redox potentials for a variety of 4Fe-4S cluster-containing glycosylases are remarkably similar. Based on this DNA-mediated redox behavior, it has been suggested that this property may be used to enhance the activity of these enzymes by facilitating damaged DNA location.     

Biogenesis of iron-sulfur proteins in plants

Iron-sulfur (Fe-S) clusters are ubiquitous prosthetic groups required to sustain fundamental life processes. The assembly of Fe-S clusters and insertion into polypeptides in vivo has recently become an area of intense research. Many of the genes involved are conserved in bacteria, fungi, animals and plants. Plant cells can carry out both photosynthesis and respiration - two processes that require significant amounts of Fe-S proteins. Recent findings now suggest that both plastids and mitochondria are capable of assembling Fe-S proteins using assembly machineries that differ in biochemical properties, genetic make-up and evolutionary origin.