Fe–S clusters are among the most ancient types of prosthetic groups. 33 Two of the nine isc genes, iscS and iscU, are homologous to NifS and NifU. The Isc proteins in general, and IscS and IscU in particular, have been widely conserved, underlining the idea that Fe–S clusters are among the most ancient types of prosthetic groups. IscU is considered to be one of the most conserved amino-acid sequences in nature. 33
Iron and sulfide are necessary for the survival of most, if not all, organisms. To avoid toxic effects of these molecules, the cellular metabolism of Fe and S requires multiple proteins to ensure their storage, trafficking, and delivery 31 The biosynthesis of the MoFe protein is extremely complex. At least six gene products are involved in the biosynthesis of FeMoco: the products of nifQ, nifB, nifV, nifN, nifE, and nifH. 27 FeS cluster assembly is a complex process involving the mobilisation of Fe and S atoms from storage sources, their assembly into [Fe-S] form, their transport to specific cellular locations, and their transfer to recipient apoproteins. 30 Biological Fe-S cluster assembly is tightly regulated within cells. 29
Biosynthesis of FeMo-co. a, Sequence of events during
a, Sequence of events during FeMo-co assembly. The biosynthetic flow of FeMo-co is NifU–NifS → NifB → NifEN → MoFe-protein. The combined action of NifU–NifS generates small Fe–S fragments on NifU (stages 1 and 2), which are used as building blocks for the formation of a large Fe–S core on NifB (stage 3). This Fe–S core is further processed into a molybdenum-free precursor (stage 4), which can be converted to a mature FeMo-co on NifEN on Fe-protein-mediated insertion of molybdenum and homocitrate (stage 5). After the completion of FeMo-co assembly on NifEN, FeMo-co is delivered to its destined location in MoFe-protein (stage 6). The permanent metal centers of the scaffold proteins are colored pink; the transient cluster intermediates are colored yellow. HC, homocitrate.
b, Structures of intermediates during FeMo-co assembly. Shown are the cluster types that have been identified (on NifU, NifEN and MoFe-protein) or proposed (for NifB). Hypothetically, NifB could bridge two [4Fe–4S] clusters by inserting a sulphur atom along with the central atom, X, thereby generating an Fe–S scaffold that could be rearranged into a precursor closely resembling the
core structure of the mature FeMo-co. In the case of the NifEN-associated precursor, only the 8Fe model is shown. The potential presence of X in the intermediates of FeMo-co biosynthesis is indicated by a question mark.
NifS, involved in nitrogenase maturation
The crystal structures of several desulphurases are known and show a dimeric two-domain protein, with one domain harboring the pyridoxal-phosphate-binding site and a smaller domain containing the active-site cysteine that transiently carries the sulfur released from free cysteine as a persulphide. 1
Following we summarize what we know of the biosynthetic processes that lead to the formation of active MoFe protein.
1. Molybdate enters the cell and is processed by NifQ, or possibly just cystine, to form a putative Mo-S containing species.
2. Iron (possibly from NifU) and sulfur (from NifS activity) are combined by NifB to form NifBco.
3. NifBco binds to NifN2E2 .
4. The next events are still obscure, but it is widely assumed that NifN2E2 acts as a scaffold for the combination of NifBco with the putative MoS species to form FeMoco.
5. In the final stage of activation, FeMoco is bound to the ‘‘apo- MoFe protein.’’ The ‘‘apo-MoFe proteins’’ must be bound to NifY or . NifY or dissociate after the activation of the MoFe protein by FeMoco. The role
of (NifY) may be to hold the ‘‘apo-MoFe protein’’ in an open conformation that will allow access of FeMoco to its binding site.
The current model of M-cluster biosynthesis involves three major synthetic platforms: NifU, which interacts with NifS to mobilize Fe and S for the synthesis of small [Fe4S4] units; NifB, where two [Fe4S4] units are coupled and rearranged into a [Fe8S9C] core concomitant with the insertion of an interstitial carbide and a “9th sulfur”; and NifEN, where the [Fe8S9C] core is matured into an M-cluster upon replacement of the “8th Fe” of this core by Mo and homocitrate via the action of NifH. 28 The molecular transformations that take place on these scaffolds are of particular interest, as these reactions involve the influx and efflux of single atoms and simple molecular fragments that are challenging processes in their own right.
Enzymatic degradation of cysteine was hypothesized in the 1940s to take place in bacteria and animals. 6 Forty years later enzymatically catalyzed degradation of cysteine came to focus also on higher plants as a result of the pioneering work of several research groups. In the late 1990s to early 2000s, the nascent field of Fe–S cluster biogenesis expanded as researchers used multiple bacterial and eukaryotic model organisms to characterize the biochemistry, cell biology, and genetics of in vivo cluster biogenesis. These early days of the field led to the important discovery of scaffold proteins where Fe–S clusters are assembled de novo. The complexity of Fe–S biogenesis grew as an array of accessory proteins were identified, many without well-defined biochemical roles. Molecular chaperones, potential iron chaperones, electron transfer proteins, and putative Fe–S cluster carriers were discovered during this time, leading to increasingly intricate models of in vivo Fe–S cluster biogenesis. Throughout the mid- to late-2000s, a consensus began to emerge for several core steps in cluster assembly, including sulfide donation and de novo cluster assembly on scaffold proteins (although the in vivo iron donors for cluster assembly are still being vigorously pursued). Even as some steps in cluster biogenesis were being revealed, new aspects to in vivo biogenesis were emerging. Multiprotein trafficking of Fe–S clusters by an array of “carrier” proteins seems to occur in vivo although the rules that govern target specificity for insertion of clusters into Fe–S metalloproteins are poorly understood. The existence of multiple Fe–S cluster biogenesis pathways in various subcellular compartments (mitochondria, cytosol, nucleus, and chloroplast) has added further complexity to the field, although the key role of mitochondria as the central regulatory organelles for iron and Fe–S metabolism is still uncontested. New model organisms from Archaea have been recently developed to study these questions, owing to their unique physiology, unusual environmental niches, and (in some cases) ancient evolutionary lineages.
The field of Fe–S cluster biogenesis began in earnest in the early 1990s when specific genes required for the maturation of Fe–S clusters in the nitrogenase enzyme were identified and characterized. 5
Two major strategies for metal cofactor biosynthesis can be found in nature. In some cases, the co-factor is assembled while directly attached to its target. The [4Fe–4S] cluster of the scaffold protein a SufU and the nitrogenase [8Fe–7S] P-cluster are examples of in situ cofactor assembly. FeMo-co synthesis is completed outside the target enzyme in a biosynthetic pathway completely independent of the production of the structural polypeptides. Thus, FeMo-co needs to be inserted into the apo-enzyme in order to render the mature, active nitrogenase enzyme. The P-cluster is a [8Fe–7S] cluster in which two [4Fe–4S] cubanes share a sulfide atom in between. The P-clusters are located at the interface between the α and β subunits at around 12Å below the protein surface. In the dithionite-reduced state, amino acid residues α-Cys88 and β-Cys95 provide the thiol groups bridging the two cubanes, whereas residues α-Cys62, α-Cys154, β-Cys70, and β-Cys153 coordinate the remaining Fe sites in the P-cluster.
FeMo-co-assembly occurs outside of the nitrogenase protein holoenzyme (NifDK) in a complex biosynthetic pathway involving a series of biochemical activities that appear to be a common theme in complex metallocluster assembly in nature. FeMo-co biosynthesis requires enzymes, which provide substrates in the appropriate chemical forms and catalyze certain critical reactions such as carbide e insertion, molecular scaffolds to aid in the step-wise assembly of FeMo-co, and metallocluster carrier proteins that escort FeMo-co biosynthetic intermediates in their transit between scaffolds (Table below) Once fully assembled, FeMo-co is transferred from the FeMo-co “biosynthetic factory” into apoprotein ( the protein part of an enzyme without its characteristic prosthetic group ) -NifDK. The insertion of FeMo-co into apo-NifDK generates a mature, functional holoenzyme f -NifDK. The rearrangement of the αIII domain generates an opening for FeMo-co insertion and to provide a positively charged path to drive FeMo-co entrance down to the cofactor binding site.
Considering the toxicity of free iron and sulfide, it is unlikely that protein-bound Fe-S clusters are spontaneously formed in vivo from free iron and sulfide. It is more likely that the iron and sulfur necessary for Fe-S cluster formation are delivered to the cluster assembly site by intermediate carrier proteins. 4
Recruiting Sulfur, Iron, and molybdenum to the assembly site
In the aerobic biosphere, inorganic sulfate is the most abundant source of sulfur that can be utilized by cells. 12 Sulfide is an essential element that is widely required by living organisms because it plays several important roles in cells and the preferred source for the majority of organisms. The synthesis of biologically important sulfur-containing molecules such as cysteine amino acids depends on the transport of sulfate into the cell. Sulfate is taken up from the environment by membrane transporters called Sulfate permeases, which are (ABC)-type transporter transmembrane systems. The uptake process requires a lot of energy in the form of ATP. Once in the cytoplasm, sulfate is further converted to a series of sulfur-containing intermediates in the cysteine synthesis pathway, by an elaborate eight-electron transfer process to hydrogen sulfite and further reduction to sulfide, which is utilized to synthesize the amino acid Serine, and in a further step, Cysteine, amongst other amino acids used in the repertoire of life. Four cytoplasmic enzymes are sufficient for conversion of sulfate to sulfide in an eight electron reduction pathway. Sulfide is a component of the amino acids cysteine amongst a few others, as well as of cellular cofactors and iron-sulfur clusters. The whole uptake process is performed under tight control at the transcriptional level and is additionally modulated by posttranslational modifications.The oxyanions molybdate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC,an (ABC)-type transporter system.
Following are the enzymes required in the pathway:
1. Sulfate permeases
2. ATP sulfurylase
3. APS kinase
4. PAPS reductase
5. Sulfite reductase
6. Cysteine Synthase Complex ( O-acetylserine (thiol)-lyase )
Following the pathway:
Overview of the plant sulfur assimilation pathway
Assimilatory sulfate reduction in plants.
Sulfur assimilatory pathways in plants (Left) and bacteria/cyanobacteria (Right).
Various bacteria and cyanobacteria use APS kinase and PAPS reductase (black arrows) or APS reductase (gray arrow and text marked with an asterisk) for formation of sulfite. 17
Sulfate and thiosulfate transport and assimilation in Salmonella typhimurium and Escherichia coli.
Sulfate is first taken up by the sulfate permease and then is reduced to sulfite and sulfide, which reacts with O-acetylserine to produce cysteine. O-acetylserine is formed by the reaction of serine with acetyl-CoA. Thiosulfate is taken up by the sulfate permease and then reacts directly with O-acetylserine to produce S-sulfocysteine, which is subsequently transformed into cysteine 15
1. Sulfate permeases
Sulfate is taken into the cell by sulfate permeases, which comprise a variety of transporters represented by bacteria by the SulT family. Most of the sulfate and organosulfur transport systems that have been identified in bacteria are members of the ATP binding cassette (ABC) superfamily, where this transport is energized by the hydrolysis of ATP. The canonical form for such transporters consists of three components: a high-affinity, substrate-binding protein located in the periplasm, a membrane-bound permease component, usually present as a dimer, and an ATP-binding component situated peripherally to the membrane on the cytoplasmic face, usually also present as a dimer. The levels of sulfur substrates transported by such systems are comparatively low, since bacteria require relatively little sulfur for growth (sulfur makes up about 1% of the bacterial cell’s dry weight). 12
The SulT group (TC 3.A.1.6.) is the main transporter family responsible for sulfate and thiosulfate uptake in prokaryotes. 13 In Gram-negative bacteria, Escherichia coli and Salmonella typhimurium, the sulfate-thiosulfate permease
is a complex of five types of subunits encoded by: sbp, cysP, cysT (currently named cysU), cysW and cysA genes. Sulfate assimilation is initiated by periplasmic sulfate (Sbp) and thiosulfate binding (CysP) proteins (Fig. 2).
Outline of gram-negative bacterial sulfate/thiosulfate transporters.
The transporter is composed of ABC-type subunits which form a channel. Because of a lack of structural studies of the entire complex, the diagram is based on terse literature descriptions.
The channel for sulfate transport is composed of two transmembrane proteins: CysT(U) and CysW. Homodimer‑forming CysA comprises catalytic and helical domains: a nucleotide-binding (ATP-binding binding) and a regulatory domain (beta sandwich) which changes conformation upon binding of ATP. Interestingly, CysA was also found in a high-throughput screen to bind directly to cysteine synthase CysK, the last enzyme of sulfate assimilation pathway. This five-component system (Sbp, CysA, CysP, CysT, CysW) seems to be similar to that found in cyanobacteria, for instance in Synechococcus sp. Genes encoding these components as well as some other genes involved in sulfur metabolism are tightly regulated at the transcriptional level. This complex response engaging two transcription factors, CysB
Possible topology of a sulfate transporter.
The suggested topology of a sulfate transporter. Consensus positions of 12 trans-membrane helices were determined from an alignment of six plant sulfate transporters. Arrangements of extramembrane loops are arbitrary although the STAS domain is depicted schematically. Within the STAS domain, alpha helices (α1–5) are shaded dark grey and beta sheet structures have no shading. A conserved phosphorylatable threonine in the loop region is indicated by an asterisk. 14
Structure of the ModABC transporter from Archaeoglobus fulgidus.
Front view of the ModAB2C2 complex. The complex in ribbon representation displays the ModA binding protein colored blue, the ModB subunits colored yellow and green, and the ModC subunits colored red and magenta. The oxyanion bound to the center of ModA is shown as green (tungsten) and red (oxygen) spheres. The lipid bilayer is also shown. 15
2. ATP sulfurylase
ATP sulfurylase (ATPS) catalyses the primary step of sulphate activation within cells. The enzyme catalyses the displacement of inorganic pyrophosphate by inorganic sulphate from Pa of ATP by a direct 'in line’ mechanism. The adenosine 5'-phosphosulphate formed is then phosphorylated at the 3' position by APS kinase to give 3'-phosphoadenosine 5'- phosphosulphate, the common sulphating species in biology.
This involves the reaction of inorganic sulphate with ATP (adenosine triphosphate) to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi). It plays a role in the following 3 metabolic pathways: purine metabolism, selenoamino acid metabolism, and sulphur metabolism. ATPS may have a crucial regulatory role in sulfate assimilation 16 Remarkably, algal ATPS proteins show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. According to the occurrence of cysteine residues. ATP sulfurylase activates the sulfate-generating adenosine 5′-phosphosulfate (APS) in preparation for the first two electron reduction. Inorganic pyrophosphate is released which is cleaved by an inorganic pyrophosphatase (DVU1636, ppaC) to “pull” the reaction.
Remarkable, when considering, that it takes these enzymes to make cysteine. So how cysteine was made to make the first ATP sulfurylase is a classic catch22 situation. It takes cysteine to make ATP sulfurylase. But it takes ATP sulfurylase to make cysteine. Which came first ?
Computer model showing the structure of an ATP sulfurylase (ATPS) enzyme.
3. APS kinase
Adenosine-5′-phosphosulfate (APS) kinase (APSK) catalyzes the phosphorylation of APS to 3′-phospho-APS (PAPS). 17 This enzyme is an essential component of primary sulfate assimilation of yeast, fungi, and some bacteria, which require this second activation of sulfate to enable its reduction by a PAPS reductase 19
Adenosine 5-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3-phosphoadenosine 5-phosphosulfate (PAPS) as a sulfur donor in specialized metabolism. The uptake and assimilation of sulfur for a variety of metabolic purposes is a common feature in prokaryotes and eukaryotes alike. In particular, the use of adenosine 5-phosphosulfate (APS), a high energy molecule with twice the energy of the pyrophosphate linkage of ATP, for incorporation of sulfur into compounds of primary and specialized metabolism places constraints on the protein structure required for recognizing this special metabolite. The enzymes responsible for producing APS (ATP sulfurylase) and its conversion to useable forms (APS kinase) are highly conserved in sequence and core structure across a wide range of organisms . 18
4. PAPS reductase
APS dependent pathway is more frequent, being found in plants, algae, and most bacteria, while the PAPS reductase seems to be restricted to fungi, some enteric γ-proteobacteria and many (but not all) cyanobacteria. Sulfite produced by APR or PAPS reductase is then reduced to sulfide by sulfite reductase (SiR). 20 Phosphoadenylyl sulfate (PAPS) reductase catalyzes the reduction of PAPS to sulphite, using thioredoxin as electron donor. This reaction is the essential step in the biosynthesis of cysteine. 21
An overview of the structure of PAPS reductase.
(a) Cα ribbon representation of PAPS reductase drawn. Helices are shown in red and β strands in blue. The N and C termini and the location of the PP motif (P), the DT motif (D) and the flexible loop (F) are indicated. Helices and β strands are labeled with numbers according to their order in the sequence.
(b) Topology diagram of PAPS reductase. Triangles and circles represent β strands and α helices, respectively. Strands that form a β sheet are grouped together. The colour code is the same as in (a).
5. Sulfite reductase
Sulfite reductase catalyzes the six electron reduction of sulfite to sulfide. The electron donor for the reduction is ferredoxin (Fd) in plants or NADPH in bacteria. Sulfite reductases, which belong to the oxidoreductase family, are found in archaea, bacteria, fungi, and plants 22 A vital process in the biogeochemical sulfur cycle is the dissimilatory sulfate reduction pathway in which sulfate (SO4 2-) is converted to hydrogen sulfide (H2S). Dissimilatory sulfite reductase (dSir), its key enzyme, hosts a unique siroheme-[4Fe-4S] cofactor and catalyzes the six-electron reduction of sulfite (SO3 2-) to H2S. 23 Since the early stages of Earth’s biogeochemical emergence, sulfur compounds have been recruited by microbes as electron donors or acceptors for energy conservation and for the biosynthesis of sulfur-containing amino acids and cofactors. Both processes, termed dissimilatory and assimilatory, use the element in oxidation states from SþVI to S-II, with sulfate (SO4 2-), elemental sulfur (ÆS0 æ), and hydrogen sulfide (H2S) being the most abundant sulfur compounds on Earth. The dissimilatory reduction of sulfate to hydrogen sulfide proceeds via three major steps catalyzed by ATP sulfurylase, APS kinase, PAPS reductase, and Sulfite reductase. Sulfite reductases are key enzymes of sulfur metabolism.
Overall architecture of dSir from A. fulgidus using the highly resolved structure of the dSir-SO3 2- complex.
(A) Ribbon diagram of the R2β2 heterotetramer with the R subunits colored red and orange, the β subunits light blue and gray, and the cofactors green. The active site funnel is indicated by a yellow arrow.
(B) Molecular surface representation focusing on the active site funnel. The sulfite ion (yellow for S and red for O) ligated to the catalytic siroheme iron (green for C, blue for N, and brown and Fe) is connected to the bulk solvent via a chain of firmly bound solvent molecules (red spheres).
6. Cysteine Synthase Complex ( O-acetylserine (thiol)-lyase )
Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is
composed of the enzymes serine acetyl transferase (SAT) and O-acetylserine-(thiol)-lyase (OAS-TL). 24 In the first step, serine acetyltransferase generates O-acetylserine by transferring acetate from acetyl-coenzyme A to serine to form O-acetylserine. During the second step, O-acetylserine sulfhydrylase uses pyridoxal phosphate (PLP) as a cofactor to yield cysteine from O-acetylserine and sulfide.
Cysteine biosynthesis is the metabolic link between sulfur assimilation and the myriad of sulfur-containing molecules in the cell. For example, this pathway provides essential metabolites for production of glutathione, a key regulatory agent of intracellular redox environment during abiotic and biotic stresses.
Three-dimensional structure of Cysteine synthase.
C, stereoview of the AtOASS active site. PLP is covalently attached to Lys46 and is shown in the same orientation as A. Side chains of amino acids and the sulfate molecule bound at the active site are shown. Waters are drawn as red spheres. Hydrogen bonds are indicated by the dotted lines.
D, schematic diagram of interactions between PLP and amino acids of AtOASS. W, water. Hydrogen bonds are shown as dashed lines with distances noted in Å.
E, initial SIGMAA-weighted |2Fo - Fc| electron density (1.5 σ) for the Schiff base formed between PLP and Lys46. All structural figures were generated using PyMol. 25
NifU AND NifS ( Scaffold for [Fe–S] cluster biosynthesis. Forms complex with NifS )
Assembly of FeMo-co is probably initiated by NifU and NifS Proteins, which mobilize iron and sulfur for the assembly of small Fe–S fragments 1 The nifU and nifS genes encode the components of a cellular machinery dedicated to the assembly of [2Fe-2S] and [4Fe-4S] clusters required for growth under nitrogen-fixing conditions. The NifU and NifS proteins are involved in the production of active forms of the nitrogenase component proteins, Fe-protein and MoFe-protein subunits. 2 NifS is required for maturation of the Fe– S cofactors in both the Fe and MoFe proteins within the nitrogenase metalloenzyme.
NifS is a cysteine desulphurase. The NifS and NifU proteins are required for the full activation of nitrogenase, and supplies the inorganic sulfide necessary for the formation of the Fe-S clusters contained within the nitrogenase component proteins. 4 It is a pyridoxal phosphate (PLP)-containing homodimer that catalyzes the formation of L-alanine and elemental sulfur by using L-cysteine amino acids b as substrate. Using l-cysteine as a substrate, NifS was shown to catalyze the formation of l-alanine and enzyme-bound sulfane sulfur. To carry out this reaction, NifS and other cysteine desulfurases require pyridoxal 5′-phosphate (PLP) as a tightly bound prosthetic group. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S]2+,+ cluster per subunit, which is referred to as the permanent cluster. NifS activity is extremely sensitive to thiol-specific alkylating reagents, which indicates the participation of a cysteinyl thiolate d at the active site. An enzyme-bound cysteinyl persulfide requires the release of the sulfur from the substrate L-cysteine for its formation ultimately providing the inorganic sulfide required for nitrogenase metallocluster formation. 3
NifU binds one Fe atom at its N-terminal, assembling an FeS cluster that is transferred to nitrogenase apoproteins. NifS transfers sulfane sulfur to Fe–S scaffold proteins, NifU, where the intact cluster is assembled. The mobilization of sulfane sulfur (S0) from l-cysteine by the cysteine desulfurase enzyme NifS was the first well-characterized step in Fe–S cluster biogenesis. 5
Generic scheme for Fe–S cluster biogenesis.
Specific steps known to occur are indicated with solid arrows. Dashed arrows indicate hypothetical steps for electron and iron donation that may or may not occur in vivo
The bridging sulfides (S2−) are a critical component of Fe–S clusters.
Bridging sulfur ligands facilitate spin coupling between iron atoms and the covalency of the metal-ligand bond is greater for S2− than for the thiolate ligands provided directly from Cys residues in the metalloprotein. Thus, the donation of the sulfide ions is an essential step of Fe–S cluster assembly. In the vast majority of characterized Fe–S cluster assembly systems, the bridging sulfides are provided by the decomposition of l-cysteine to release sulfur as an enzyme-bound sulfane sulfur c species (also referred to as a persulfide in many publications).
Iron mobilization and uptake
Iron is a transition metal; its chemical symbol is Fe from the Latin name, ferrum. The melting point of iron is 1536 °C, its boiling point is about 3000 °C and its density is 7.87 g cm− 3 54 Iron can exist in various oxidation states (from − 2 to + 6), the principal forms that occur naturally however are either ferrous or ferric iron (Fe(II) or Fe(III), respectively). Fe(II) is more abundant in anoxic environments whereas, in an oxygen-containing environment, iron is readily oxidized from the Fe(II) to the Fe(III) state. Iron is not found as free metal in nature. It tends to coordinate with organic and inorganic ligands forming a wide number of minerals that play critical roles in environmental chemistry due to their high abiotic reactivity. Iron is known to react with oxygen (O2) in water or air moisture to form various insoluble iron oxide compounds described commonly as rust; there are sixteen known iron oxides and oxyhydroxides. Iron is essential to most life forms. To date, the only organisms that do not depend on iron belong to the Lactobacillus spp.
This metal is an integral part of a number of proteins and enzymes. The two oxidation states of iron (Fe(II) and Fe(III)) make it suitable for numerous biochemical reactions. Iron is an important cofactor in several proteins required for a large range of metabolic processes like (i) the transport, storage and activation of molecular oxygen, (ii) the activation and decomposition of peroxides, (iii) the reduction of ribonucleotides and dinitrogen and (iv) the electron transfer via a variety of electron carriers with a wide range of redox potentials. However, due to its low solubility at neutral pH, iron acquisition poses a problem for neutrophilic organisms. Two distinct molecular mechanisms have been characterized whereby environmental iron is solubilized and transported into the cytosol. Most prokaryotes produce siderophores, that have an extremely high affinity for iron. Siderophores form soluble ferric chelates, that are taken up by the cell via high affinity receptors.
Another example of a key player in iron homeostasis is the globular protein complex ferritin. Each ferritin molecule can hold as many as 4,500 iron atoms inside its spherical structure formed from 24 subunits allowing intracellular iron-storage in Bacteria and Eukarya and keeping iron in a soluble and non-toxic form. Indeed, not only these mechanisms allow the uptake of iron, it also prevents it to be free in the cells where it can generate potent oxidizing hydroxyl radicals that are toxic Therefore, after uptake, the level of cellular iron must be carefully regulated. Within the cells, protein networks involving transporters, metal sensing and metal-storage proteins are required to maintain the proper subcellular concentrations of iron, and, not surprisingly, any perturbation can cause distinct pathological disorders. The positive biological effects of iron are consequently dose-dependent, excessive concentrations of free iron as well as untreated iron deficiency are both actually detrimental to cells.
It is not reasonable to believe that Iron level regulation and mechanisms for proper subcellular iron concentrations could have emerged over large periods of time if cells depend on these mechanisms in order not to be intoxicated.
Iron uptake in Gram-negative bacteria involves four distinct steps:
(i) siderophore synthesis,
(ii) siderophore secretion into the extracellular space,
(iii) iron chelation by the siderophores, and
(iv) siderophore/ iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane. 53
Three major sets of components are involved in iron uptake in Gram-negative bacteria.
First non-ribosomal peptide synthetases. The second component required for proper iron uptake is the export system for siderophores are essential, because they produce the siderophores needed to chelate iron in the extracellular space. Schizokinen, the first identified siderophore secreted by cyanobacteria, was isolated from Anabaena sp. ATCC 27898 about which our knowledge is rather limited.
For the cyanobacterium Anabaena sp. the MFS protein SchE has been recently identified as plasma membrane-localized schizokinen exporter. The transport across the outer membrane occurs via a TolC type protein.
The third set of components is essential for the transfer of the iron-loaded siderophores through the outer membrane and periplasmic space into the cytoplasm. This process requires b-barrel shaped TonB-dependent transporters (TBDTs) in the outer membrane, an Exb/TonB complex localized in the plasma membrane, which regulates the TBDT, and siderophore uptake systems annotated as Fhu, Fec or Fut complexes. These uptake systems beyond TBDT are composed of periplasmic siderophorebinding proteins (e.g. FhuD), membrane-embedded transporters or permeases (e.g. FhuB) and ATP-binding proteins (e.g. FhuC).
Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. 47 While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload. Representatives from all major families of transporters participate in this task, including ion-gradient driven systems and ATP-utilizing pumps. P-type ATPases and ABC transporters both utilize the free energy of ATP hydrolysis to drive transport. Each of these very different families of transport proteins has a distinct role in maintaining transition metal homeostasis: P-type ATPases prevent intracellular overloading of both essential and toxic metals through efflux while ABC transporters import solely the essential ones.
It is estimated that 30–45% of known enzymes are metalloproteins that depend on a metal co-factor for their function. Often, the co-factor is a transition metal such as iron, manganese, zinc, or copper. As a result, many essential physiological processes including respiration, photosynthesis, replication, transcription, translation, signal transduction, and cell division depend on the presence of transition metals. However, transition metals are toxic at elevated intracellular concentrations as they can perturb the cellular redox potential, produce highly reactive hydroxyl radicals k , and displace functionally important metal co-factors from their physiological locations. In both eukaryotes and prokaryotes, a diverse ensemble of membrane-embedded transporters participates in metal translocation across cell membranes. In gram-negative bacteria l, these included ion gradient- and ATP-driven transport systems belonging to the RND, ABC, CDF, and P-type ATPase superfamilies. As depicted in Figure below, each of these superfamilies has a unique architecture and composition: RND transporters are comprised of multiple subunits spanning the inner membrane, periplasm, and the outer membrane. A substrate may enter the translocation pathway either at the cytoplasm or at the periplasm but in both cases the substrate will be expelled to the cell exterior.
For iron transport, three uptake systems are defined: the
- lactoferrin/transferrin binding proteins
- the porphyrin-dependent transporters and
- the siderophore-dependent transporters
ABC transporters are embedded in the inner membrane, and through interactions with their cognate substrate-binding proteins, outer membrane receptors (e.g., BtuB in Figure below), and the ExbB/ExbD/TonB system they participate in metal uptake through the outer membrane, the periplasm, and the inner membrane, delivering transition metals to the cytoplasm.
Outer membrane transporters (TBDT)
ATP-binding cassette (ABC) transporter
Periplasmic binding protein (PBP)
are required for Iron (Fe), Vitamin B12, and other transition metal uptake, while
are used to expell overload of B12 and transition metals.
Despite the low sequence homology between different bacterial species, the available threedimensional structures of homologous proteins are strikingly similar. Examination of the current three-dimensional structures of the outer membrane receptors, PBPs, and ABC transporters provides an overview of the structural biology of iron uptake in bacteria. 51
Filamentous cyanobacteria contain molecular machines for oxygenic photosynthesis under all growth conditions. These machines, as well as those involved in respiration and nitrogen metabolism, depend on non-proteinaceous cofactors such as iron. Even though iron and copper are required for the function of respiratory and photosynthetic complexes, their intracellular level has to be tightly controlled as these ions pose a risk of oxidation 52 Therefore, the uptake of iron is highly regulated in order to avoid intoxication.
" It is hypothesized that iron limitation might have been one of the selective forces in the evolution of cyanobacteria , and one might speculate that those cyanobacteria with the most efficient iron uptake systems might have had an evolutionary advantage ".
Question: Had intracellular Iron homeostasis and regulation , and the regulation mechanisms, import and export membrane proteins, not do emerge all at once, and together ? Homeostasis is only reached, once all interacting parts work together in an orchestrated fashion.
To enhance iron uptake, eubacteria ( also called just "bacteria ")secrete low-molecular-weight iron chelators (siderophores) under iron-limiting conditions. The siderophore-iron complexes are bound by receptor proteins (TonB-dependent transporters, TBDTs) in the outer membrane which are composed of a transmembrane β-barrel domain, a so-called plug domain. The siderophore-iron is subsequently transferred to the cytoplasm by transport proteins in the cytoplasmic membrane. This process is dependent on TonB which provides the energy required for the translocation of siderophore-iron complexes across the outer membrane. In order to facilitate this translocation, the periplasmic domain of TonB interacts with the TonB box of the loaded TBDT. It is proposed that TonB exerts a pulling force on the TonB box and, thereby, partially unfolds the plug domain enabling the translocation of the siderophore into the periplasmic space. The most intensively studied function of TBDTs is the iron uptake in Gram-negative bacteria.
The siderophore TBDTs are sub-classified according to their substrate - that is the chemical nature of the siderophore they bind. Siderophores belong inter alia to hydroxamates, catecholates, phenolates, citrates or combinations thereof. For example, the siderophore transporters FepA, ViuA and IroN recognize catecholates, FhuA, FoxA and FhuE hydroxamate and FecA citrate.
Most prokaryotes produce siderophores, that have an extremely high affinity for iron. Siderophores form soluble ferric chelates h , that are taken up by the cell via high-affinity receptors. The second mechanism for solubilizing iron involves a reductase oriented toward the outside, that reduces Fe(III) into the more soluble Fe(II). After uptake, the level of cellular iron must be carefully regulated. Within the cells, protein networks involving transporters, metal sensing and metal-storage proteins are required to maintain the proper subcellular concentrations of iron. 37
Iron Uptake and Homeostasis in Cells
Iron is an important micronutrient for virtually all living organisms
Only lactic acid bacteria substitute iron through manganese and cobalt 51 Iron can exist in either the reduced ferrous (Fe2+) form or the oxidized ferric (Fe3+) form. The redox potential of Fe2+/Fe3+ makes iron extremely versatile when it is incorporated into proteins as a catalytic center or as an electron carrier. Thus iron is important for numerous biological processes which include photosynthesis, respiration, the tricarboxylic acid cycle, oxygen transport, gene regulation, DNA biosynthesis, etc. Although iron is abundant in nature, it does not normally occur in its biologically relevant ferrous form. Under aerobic conditions, the ferrous ion is unstable. Via the Fenton reaction, ferric ion and reactive oxygen species are created, the latter of which can damage biological macromolecules. The ferric ion aggregates into insoluble ferric hydroxides. Because of iron's reactivity, it is sequestered into host proteins such as transferrin, lactoferrin, and ferritin. Consequently, the cellular concentration of the ferric ion is too low for microorganisms to survive by solely using free iron for survival. Microorganisms overcome this nutritional limitation in the host by procuring iron either extracellularly from transferrin, lactoferrin, and precipitated ferric hydroxides or intracellularly from hemoglobin. This is accomplished by microorganisms via two general mechanisms: iron acquisition by cognate receptors using low molecular weight iron chelators termed siderophores and receptor-mediated iron acquisition from host proteins.
Schematic representation of iron uptake in Gram-negative bacteria.
There are numerous iron uptake pathways in Gram-negative bacteria which include iron uptake from transferrin, siderophores, or heme. All of these uptake pathways require an outer membrane receptor, a PBP, and an inner-membrane ABC transporter. Not all bacteria have all three systems; but some have more than one type. Transport through the outer membrane receptor requires the action of the TonB system (TonB, ExbB, ExbD).
a. Scaffold proteins are crucial regulators of many key signalling pathways. Although scaffolds are not strictly defined in function, they are known to interact and/or bind with multiple members of a signalling pathway, tethering them into complexes. In such pathways, they regulate signal transduction and help localize pathway components (organized in complexes) to specific areas of the cell such as the plasma membrane, the cytoplasm, the nucleus, the Golgi, endosomes, and the mitochondria.
b. Cysteine is a semi-essential proteinogenic amino acid.
c. The unfamiliar term sulfane sulfur sometimes appeared in papers published in the 1970s, and was defined in the review article by Westley in 1983. In the article, sulfane sulfur is described as sulfur atoms that are covalently bound only with sulfur atoms, and as this explanation was somewhat difficult to comprehend, it was not generally accepted. Thus, in the early 1990s, we redefined these sulfur species as "bound sulfur", which easily converts to hydrogen sulfide on reduction with a thiol reducing agent. In other words, bound sulfur refers to a sulfur atom that exists in a zero to divalent form (0 to -2).
d. In organic chemistry, a thiol is an organosulfur compound that contains a carbon-bonded sulfhydryl (R–SH) group (where R represents an alkyl or another organic substituent). Thiols are the sulfur analog of alcohols (that is, sulfur takes the place of oxygen in the hydroxyl group of an alcohol), and the word is a portmanteau of "thion" + "alcohol ". The –SH functional group itself is referred to as either a thiol group or a sulfhydryl group. 8
Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts functional sites with their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). 4 The thiol (or “sulfhydryl”) group of cysteine is ionizable, with a negatively-charged thiolate group being generated after deprotonation, boosting its reactivity (Fig. below).
Structures of cysteinyl residues within proteins.
The aminoacyl groups are shown to the left, with dotted lines representing peptide bonds to the next residue on either side. Both protonated (left) and deprotonated (right) forms of these amino acids are depicted with average pKa values (that can vary in particular protein microenvironments).
This thiol/thiolate group is subject to alkylation by electrophiles and oxidation by reactive oxygen and nitrogen species, leading to posttranslationally modified forms that can exhibit significantly altered functions. 7
e. Carbide, any of a class of chemical compounds in which carbon is combined with a metallic or semimetallic element. 9
f. An apoenzyme together with its cofactor. 10
g. Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia 38
h. Chelate, any of a class of coordination or complex compounds consisting of a central metal atom attached to a large molecule, called a ligand, in a cyclic or ring structure.Chelates are more stable than nonchelated compounds of comparable composition, and the more extensive the chelation—that is, the larger the number of ring closures to a metal atom—the more stable the compound. This phenomenon is called the chelate effect; it is generally attributed to an increase in the thermodynamic quantity called entropy that accompanies chelation. The stability of a chelate is also related to the number of atoms in the chelate ring. In general, chelates containing five- or six-membered rings are more stable than chelates with four-, seven-, or eight-membered rings. 40
Many essential biological chemicals are chelates. Chelates play important roles in oxygen transport and in photosynthesis. Furthermore, many biological catalysts (enzymes) are chelates. A chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand. An example of a simple chelating agent is ethylenediamine. 41
The word chelate (pronounced: “key-late”) is derived from the Greek word “chele” which literally means “claw”, a rather fitting association because chelation is a process somewhat like grasping and holding something with a claw. Chelation occurs when certain large molecules form multiple bonds with a micronutrient, protecting it from reacting with other elements in the nutrient solution and increasing its availability to the plant. Imagine a lobster’s claw made of carbon and hydrogen atoms holding an ion. The more bonds that form between the ion and the carbon atoms, the stronger the ion is held within the chelate. The strength of the chelate’s hold on the ion determines, as pH increases, how long the element will continue to be available to plants. Many micronutrients are unavailable to plants in their basic forms. This is typically due to the fact that these metals, such as iron, are positively charged. The pores or openings on the roots of the plant are negatively charged. As a result, the element can’t enter the plant due to the difference in charges. However, if a chelate is added, it surrounds the metal/mineral ion and changes the charge into a neutral or slightly negative charge, allowing the element to easily pass across the cell membrane and travel into the plant. 42
i In molecular biology, a riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. Thus, a mRNA that contains a riboswitch is directly involved in regulating its own activity, in response to the concentrations of its effector molecule. The discovery that modern organisms use RNA to bind small molecules, and discriminate against closely related analogs, expanded the known natural capabilities of RNA beyond its ability to code for proteins, catalyze reactions, or to bind other RNA or protein macromolecules.
j The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the periplasmic space in gram-negative bacteria. Using cryo-electron microscopy it has been found that a much smaller periplasmic space is also present in gram-positive bacteria. 46
k Hydroxyl Radical (OH•) is the most reactive and the most toxic Reactive Oxygen Species (ROS) known. It is generated at neutral pH by the Fenton reaction between H2O2 and O•−2 catalyzed by transition metals like Fe (Fe2+, Fe3+). It has the capability to damage different cellular components by lipid peroxidation (LPO), protein damage and membrane destruction. Since there is no existing enzymatic system to scavenge this toxic radical, excess accumulation of OH• causes the cellular death (Pinto et al., 2003). 48
l Gram-negative bacteria are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane.
m Carbon monoxide (CO) is a colorless, odorless, and tasteless gas that is slightly less dense than air. It is toxic to hemoglobic animals (both invertebrate and vertebrate, including humans) when encountered in concentrations above about 35 ppm, although it is also produced in normal animal metabolism in low quantities, and is thought to have some normal biological functions. In the atmosphere, it is spatially variable and short lived, having a role in the formation of ground-level ozone. 49
n In chemistry, phosphorylation of a molecule is the attachment of a phosphoryl group. Together with its counterpart, dephosphorylation, it is critical for many cellular processes in biology. Phosphorylation is especially important for protein function, as this modification activates (or deactivates) almost half of the enzymes, thereby regulating their function. Many proteins (between 1/3 to 2/3 of the proteome in eukaryotes) are phosphorylated temporarily, as are many sugars, lipids, and other molecules.
Protein phosphorylation is considered the most abundant post-translational modification in eukaryotes. Phosphorylation can occur on serine, threonine and tyrosine side chains (often called 'residues') through phosphoester bond formation, on histidine, lysine and arginine through phosphoramidate bonds, and on aspartic acid and glutamic acid through mixed anhydride linkages. 50
5. Metals in Cells, page 816
6. Sulfur Metabolism in Phototrophic Organisms, page 72
14. Sulfur Metabolism in Phototrophic Organisms, page 22
20. Sulfur Metabolism in Phototrophic Organisms, page 20
26. NITROGEN FIXATION: FROM MOLECULES TO CROP PRODUCTIVITY, page 56
27. Advances in INORGANIC CHEMISTRY Iron–Sulfur Proteins, page 176
33. Inorganic Biochemistry of Iron Metabolism, page 204
34. The Cell Biology of Cyanobacteria, page 60
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