This class of proteins comprises several types of proteins and enzymes that contain iron atoms coordinated by sulfur atoms in clusters of different types (Fig. 1). The simplest FeS proteins are the rubredoxins (1Fe, no inorganic sulfur) and the ferredoxins (2Fe, 3Fe and 4Fe). In more complex FeS proteins, other prosthetic groups (FAD, FMN, heme, Mo, Ni, etc.) are present, in addition to the iron-sulfur cluster(s). Thus, FeS proteins fulfill several roles: electron transfer and storage, catalysis, structural, and regulatory functions (1, 2).
1. High Potential Electron Carriers: Rubredoxins, HiPIPs, and Rieske Proteins
Figure 1. Schematic structures of iron-sulfur clusters found in FeS proteins as determined by X-ray crystallography studies: (a) the Fe(S-Cys)4 unit in rubredoxin; (b) the 2Fe-2S cluster in eukaryotic ferredoxins and in Rieske proteins; (c) the cubane-like 4Fe-4S structure of bacterial ferredoxins, HiPIPS, IRPs, and active aconitase; (d) the cuboidal 3Fe-4S structure found in some bacterial ferredoxins, in inactive aconitase, and in some complex redox enzymes; (e) the P cluster of dinitrogenase; (f) the FeMoCo cofactor of dinitrogenase.
The simplest of all FeS proteins is rubredoxin, containing a single Fe atom ligated by a four-cysteine-residue unit (Fig. 1a), and no inorganic sulfide, in a polypeptide of 50 to 60 amino acid residues. Rubredoxins are found in strictly anaerobic bacteria and archea (Clostridia, Desulfovibriacee, Pyrococcales) and have oxidation/reduction potentials (+30 to 60 mV, relative to the hydrogen electrode) that appear to be too high to be relevant for bacterial metabolism. Although assumed to function as electron carriers, the redox partners of rubredoxins in anaerobes have not been identified. Recently, a rubredoxin-oxygen oxidoreductase was found to function in vitro at the end of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in Desulfovibrio gigas. There is another protein in D. gigas containing a single Fe(Cys)4 unit in each of its two small subunits, named desulforedoxin. Rubredoxin-like proteins that contain two Fe(Cys)4 clusters are also found in Pseudomonacee. These aerobic rubredoxins transfer electrons to enzymes that catalyze the oxidation of alkanes.
Photosynthetic microorganisms such as Chromatium spp and Rhodospira spp contain the so-called HiPIPs (high-potential iron-sulfur proteins). These proteins contain a single, cysteine-coordinated 4Fe-4S cubane-like structure (Fig. 1c), but these protein structures favor the high-potential 3+/2+ transition of the metal cluster (reduction potentials +90 to +450mV), rather than the 2+/1+ transition typical of [4Fe-4S] clusters in bacterial ferredoxins.
Among the high-potential FeS proteins of the mitochondrial and photosynthetic electron transfer chains, a singularity is represented by the so-called "Rieske" protein in the cytochromes b-^ and b6-f complexes. These "Rieske" proteins contain a 2Fe-2S cluster with two histidine and two cysteine ligands in a unique binding motif (Fig. 1b). The same type of coordination is found in other FeS proteins, most notably in ferrochelatase (found in the intermembrane space of mitochondria and required for inserting iron in tetrapyrroles) and in some bacterial dioxygenases.
2. Complex FeS Proteins
The redox properties of FeS clusters are utilized in multielectron transfer systems, which can be made more efficient by combining donors and acceptors into a single, often multimeric structure. This evolutionary advantage led to the appearance of complex FeS proteins, in which 4Fe-4S or 2Fe-2S clusters are combined with other inorganic or organic cofactors.
Desulfovibriacee and some Clostridia also contain what apparently amounts to a natural chimeric protein, in which a rubredoxin-like domain is linked to a domain that contains a binuclear, oxo-bridged iron center, such as those found in ribonucleotide reductase or in hemerythrin—hence the trivial names of these proteins: rubrerythrin and nigerythrin. Rubrerythrin was found to have ferroxidase activity, and an oxygen-scavenging function was hypothesized for these proteins. Yet another combination of metallic clusters has been found in D. gigas desulfoferrodoxin, a protein in which a desulforedoxin site is associated with an iron site having nitrogen ligands. No biological function is known for desulforedoxin or desulfoferrodoxin.
Nitrogenase components 1 and 2 represent the best-known examples of combinations of different and unusual metal-sulfur centers. In the dimeric component 2 (dinitrogenase reductase), a 4Fe-4S cluster (Fig. 1c) is bound at the dimer interface. Each of the subunits provides two of the coordinating cysteine residues, and the cluster structure and properties are modified upon adenylylation of the dimer. The a2b2 nitrogenase component 1 (the true dinitrogenase) contains a number of the so-called P-clusters (derived from 4Fe-4S cubanes) (Fig. 1e), again harbored jointly by adjacent subunits. In the same intersubunit fashion, dinitrogenase also binds FeMoCo, the metal cluster at which the multielectronic reduction of dinitrogen to ammonia occurs. FeMoCo is a modified double cubane structure, in which one of the corners is occupied by a Mo rather than a Fe atom, and is coordinated also by histidine and by an isocitrate molecule (Fig. 1f). Another elementary process in bacterial physiology is hydrogen evolution, which uses an enzyme containing an Ni-FeS cluster, along with 4Fe-4S clusters.
FeS proteins containing multiple iron centers and organic cofactors (most typically flavins and pterins) abound in the electron transport chains of both prokaryotes and eukaryotes. These membrane-bound enzymes are typically made up of several subunits, with a variety of arrangements, and types of clusters and cofactors spanning a broad range of reduction potential values, often modulated through the binding of suitable effectors. Also, an active role for FeS clusters in energy transduction has been proposed. Soluble complex iron-sulfur enzymes play key roles in bacterial metabolism (glutamate synthase or anaerobic ribonucleotide reductase) and in the assimilation pathways of inorganic nutrients in plants (as in sulfite, nitrate, and nitrite reductases), often in conjunction with unique cofactors. A mammalian soluble protein of great complexity is xanthine oxidase, containing flavin, iron-sulfur, and a molybdopterin cofactor. Formylmethanofuran dehydrogenases are FeS proteins found in methylotrophs, which contain Mo or W and a pterin dinucleotide.
3. FeS Proteins Having No Electron-Transfer Function
There is a remarkable structural similarity between the Fe(Cys) 4 unit found in rubredoxin-like proteins and the Zn(Cys) 4 unit found in DNA-binding zinc fingers, and transcription factors containing a Fe(Cys)4 unit have been reported for a number of organisms. No DNA-binding activity has been reported for rubredoxins themselves. Examples of FeS proteins displaying the ability of binding polynucleotides are not limited to rubredoxin-like proteins. Escherichia coli endonuclease III contains a single 4Fe-4S cluster, which apparently only plays a structural role.
To alleviate the toxicity of the byproducts of aerobic respiration, E. coli induces the synthesis of protective enzymes, and this induction is controlled by the regulatory FeS proteins SoxRS, OxyR, and ArcAB. ArcAB, Fnr, SoxRS, and OxyR function in concert, so that E. coli can optimize its energy production and growth rate. Fnr and SoxRS are DNA-binding proteins, and they utilize 4Fe-4S and 2Fe-2S clusters as direct sensors of the redox environment (3).
Perhaps the best-known example of the regulatory function of FeS proteins is represented by the RNA-binding proteins involved in iron homeostasis in mammalian cells. When cells are depleted of iron, iron regulatory proteins IRP1 and IRP2 bind with high affinity to specific RNA stem-loop structures (iron responsive elements, IREs) in messenger RNA transcripts. Binding of IRPs to IRE stabilizes the mRNA coding for the transferrin receptor against degradation, while blocking the translation of the mRNA coding for the iron-storage protein, ferritin. In the presence of iron, IRP1 assembles a 4Fe-4S cluster and loses its IRE-binding properties, whereas IRP2 is rapidly subject to protein degradation. Thus, the presence of iron allows synthesis of ferritin (as well as of d-aminolevulinate synthase, mitochondrial succinate dehydrogenase, and aconitase), while repressing the synthesis of the transferrin receptor and therefore diminishing iron import into the cell (4).
The role of iron in citrate isomerization catalyzed by aconitase was recognized many years ago, and aconitase was found to contain a 4Fe-4S cluster, which can be converted reversibly to an inactive 3Fe-4S form (Fig. 1d). This conversion is made easier by the absence of a coordinating cysteine residue to the "labile" iron atom. Also complex FeS proteins are not limited to redox reactions. A FAD and 4Fe-4S enzyme from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, a reaction involving the cleavage of an unactivated C-H bond; similar activities have been reported for other bacteria.
4. Processing of FeS Proteins and Cluster Assembly
From a chemical standpoint, iron-sulfur clusters are among the easiest cofactors to assemble. Synthetic FeS complexes containing 1, 2, 4, or more iron atoms and an appropriate complement of sulfide readily self-assemble in mixtures of iron, thiol groups, and a suitable source of sulfide (1). This observation, plus the positive charge of the pyrite-like fundamental unit of FeS clusters, and evolutionary considerations, prompted some hypotheses on a role of FeS clusters in the emergence of life in the reducing and acidic environment near submarine hot springs in primordial oceans (5).
A different situation is encountered in the cell, where the toxicity of iron and sulfide must be accounted for, and where protein folding or assembly of multimeric proteins must occur, along with protein targeting of nuclear-encoded proteins to the appropriate compartments. In cells and organelles, sulfide may be derived from cysteine or from thiosulfate through appropriate enzymes, while the actual carriers of iron remain unknown. FeS proteins are imported into organelles as metal-free precursors, and cluster insertion and acquisition of a native structure are very often assisted by more or less complex scaffolding systems. These scaffolding systems are most relevant when assembly of interprotein multimetallic clusters (as in dinitrogenase) or of multiple-component, multiple-cofactor enzymes is required.
