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Intelligent Design, the best explanation of Origins » Origin of life » Biosynthesis of the Cofactors of Nitrogenase

Biosynthesis of the Cofactors of Nitrogenase

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1 Biosynthesis of the Cofactors of Nitrogenase on Fri Feb 10, 2017 5:26 pm


Biosynthesis of the Cofactors of Nitrogenase  1

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


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

Given the information in the previous two sections, it is now possible to 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 

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.
A, ribbon diagram of the Cysteine synthase (O-Acetylserine sulfhydrylase AtOASS )  dimer. Each monomer is colored either blue or green. PLP is shown as a yellow stick model. The N-terminal and C-terminal residues observed in the electron density of the structure are indicated with residue number in parenthesis. Secondary structure features are labeled.
B, overlay of the Cα traces of AtOASS (green) and StOASS (orange) monomers. The arrows indicate regions of structural variation. 
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.

Cysteine Desulfurases
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 apoproteinsNifS 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 

Sulfur Mobilization
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
Bacterial ATP-driven transporters of transition metals: physiological roles, mechanisms of action 47
Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. 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)
ExbB/ExbD/TonB system
ATP-binding cassette (ABC) transporter
Periplasmic binding protein (PBP)

are required for Iron (Fe), Vitamin B12, and other transition metal uptake, while

P-type ATPase

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 [6], 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 is an important micronutrient for virtually all living organisms except lactic acid bacteria where manganese and cobalt are used in place of iron 51 Under physiological conditions, 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).

The main difference between these two mechanisms is that siderophores and heme can be taken up by the bacterial cell as intact molecules whereas iron must be extracted from host carrier proteins such as transferrin or lactoferrin prior to being transported into the bacterial cell. The uptake of iron from transferrin, lactoferrin, hemoglobin, and siderophores has been identified in both Gram-negative and Gram-positive bacteria. In Gram-negative bacteria, the outer membrane is a permeability barrier, protecting the bacterium from toxins, degradative enzymes, and detergents. The presence of trimeric β-barrel proteins termed porins in the outer membrane allows for passive diffusion of small solutes that have a molecular weight less than 600 Da. Transferrin, lactoferrin, hemoglobin, and most ferric-siderophore complexes exceed the molecular weight cut off of porins and thus require specific outer membrane receptors for uptake into the periplasmic space. All of these iron uptake pathways involve an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. The Gram-negative outer membrane lacks an established ion gradient or ATP to  provide the energy for transport. This energy requirement is accomplished through the coupling of the proton motive force of the cytoplasmic membrane to the outer membrane via three proteins: TonB, ExbB, and ExbD. Unlike Gram-negative bacteria, Gram-positive bacteria have no outer membrane. A cell wall composed of murein, polysaccharides, teichoic acids, and cell wall proteins is all that separates the bacterial cytoplasm from its environment. Uptake of an iron source involves a membrane-anchored binding protein, which resembles the PBP in Gram-negative organisms, as well as an ABC transporter (Fig. 2). Recent papers describe the molecular basis of iron uptake in Gram-positive bacteria. However, when compared to Gramnegative bacteria, there is still relatively little information on iron transport in Gram-positive bacteria. The growing availability of genome sequences of Gram-positive bacteria allow for the identification of genes that encode iron transporters that are related to those found in Gram-negative bacteria. This review is limited to a discussion of the structural biology of ferric ion uptake systems. 

P-type ATPases and CDF transporters catalyze metal translocation across the inner membrane, and in both systems the substrate is transported from the cytoplasm to the periplasm.

The different transporter types participating in transition metal translocation in gram-negative bacteria. 
Arrows indicate the directionality of transport of protons and transition metals, and proteins are colored according to chains. From left to right: RND transporters have a trimeric organization and traverse both the inner and outer membranes. They utilize the energetically downhill movement of protons across the inner membrane (DmH+) to drive metal efflux from the cytosol and the periplasm to the cell exterior. ABC transporters have a dimeric organization and are embedded in the inner membrane. They use the energy of ATP hydrolysis and require a cognate substrate binding protein for their function (e.g., BtuF). ABC transporters partner with the energy-transducing ExbB/ExbD/TonB complex  and high affinity outer membrane transporters (e.g., BtuB) to deliver essential trace elements from the environment to the cytosol. P-type ATPases are monomers , and like ABC transporters, are powered by ATP to catalyze metal efflux from the cytosol, across the inner membrane, to the periplasm. A similar task is performed by the dimeric CDF transporters that utilize the energy of DmH+ to drive metal efflux across the inner membrane.

Physiological roles of bacterial transition metal transporters
In many enzymatic reactions, the limited chemical reactivity of the side chains of the 20 amino acids is supplemented by the heightened reactivity of transition metal co-factors . It is however the same heightened reactivity that makes transition metals potentially toxic. As such, a delicate intracellular balance is required to maintain the essential supply of these metals while preventing toxic overload. One mechanism by which bacterial (and mammalian) cells maintain this balance is through a combination of sequestration and controlled release. Since the toxic form of transition metals is their unliganded ionic form, the metals may be stored as bound moieties by specialized sequestering proteins. Examples of such storage proteins include the iron-storage protein ferritin and the zinc and copper-storing metallothioneins. Metal coordination by the sequestering protein neutralizes the metals’ reactivity. The sequestered metals are then released in a controlled manner as the need arises, thereby providing the mechanisms of both protection and storage. An additional mechanism of bacteria to control their intracellular transition metal concentrations is by active uptake and extrusion. Given the low passive membrane permeation of transition metals, the net change in intracellular metal concentrations is determined by the rates and directionality of active transport. In this respect, the roles of P-type ATPases and ABC transporters are readily distinguished: transition metal P-type ATPases function as detoxifiers through their efflux activity while their ABC counterparts function exclusively as high-affinity importers. 

P-type ATPases export both essential transition metals (e.g., Zinc, Copper, and Carbon monoxyde m ) and metals that are exclusively toxic (e.g., silver (Ag), cadmium (Cd), lead (Pb), and mercury (Hg). Such a recognition spectrum is beneficial since all transition metals (including the essential ones) are toxic at elevated intra-cellular concentrations. In contrast, ABC transporters of transition metals import only the essential ones (e.g., Iron (Fe), zinc (Zn), manganese (Mn), nickel (Ni), and copper (Co) ). Clearly, it is disadvantageous to invest energy in the import of non-essential toxic compounds. Several reports have implicated P-type ATPases as having roles in influx, rather than efflux, of transition metals. However, P-type ATPases that function as importers are uncommon, and the great majority of P-type ATPases characterized to date operate as efflux pumps. In addition to their role in maintaining the balance between influx and efflux, prokaryotic transition metal transporters appear to be involved in other cellular processes. Many bacterial genomes contain more than one gene encoding a Cu+-transporting P-type ATPase. Of these, usually only the copA1 gene is essential for copper tolerance. In Rhizobia, copA2 (fixI) has been proposed to be important for nitrogen fixation, through its role in supplying copper to coppercontaining enzymes such as cytochrome c oxidase and nitrous oxide. In the photosynthetic bacteria Synechocystis, the complementary action of two P-type ATPases has been shown to be important to respiration and photosynthesis through the supply of copper to plastocyanin.

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.
Cysteine biosynthesis

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.[1][2][3][4] 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.

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

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.[1][2][3] Many proteins (between 1/3 to 2/3 of the proteome in eukaryotes[4][5]) 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
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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ABC transporters
Molecular organization of ATP-driven transporters
The molecular organization and structural fold of prokaryotic P-type ATPases and ABC transporters are very different (Figure below).

Comparison of the structural folds of an ABC transporter (BtuCD–F) and a P-type ATPase (CopA Cu+-ATPase 1).
Proteins are colored by chain, and co-factors are shown as ball and stick models. 
Left: BtuCD  displays a dimeric organization of the NBDs and TMs. One molecule of vitamin B12 is bound by the monomeric BtuF. Two cyclo-tetrametavanadate (blue spheres) molecules are bound at the interface of the NBDs, indicating the location of the binding site for two ATP molecules. 
Right: P-type ATPases of transition metals are monomers, as exemplified by the class IB Cu+-ATPase, CopA.

The functional unit of prokaryotic transition metal transporting P-type ATPases (also referred to as heavy metal or type P1B ATPases) is a monomer, composed of a single polypeptide chain, encoded by a single gene. They contain a membrane-spanning domain and three cytoplasmic domains: A (actuator), P (phosphorylation) and N (nucleotide) domains (Figure above). The N-domain binds the nucleotide and phosphorylates n the conserved aspartic residue in the canonical DKTGT sequence of the P-domain. The role of the A domain is to dephosphorylate the P-domain, thus completing the ATPase cycle. The membrane-spanning domain serves two functions: it creates the permeation pathway through the membrane and is responsible for substrate binding and selectivity. ABC transporters of transition metals are minimally comprised of three subunits, each encoded by a different gene: a nucleotide binding domain (NBD), a trans-membrane permease (TM), and a soluble, periplasmic Substrate-Binding Protein (SBP). The three genes are transcribed as an operon, sometimes with overlapping open reading frames. At the protein level, both the NBD’s and TM’s function as homodimers, resulting in a functional unit that is composed of two copies each of the ATPase and permease, and one copy of the substrate-binding protein (Fig. above). Unlike P-type ATPases, the membrane translocation pathway, formed by the homodimeric permease, offers little in terms of substrate selectivity, which is almost entirely determined by the periplasmic substrate binding protein.

Abstract | ATP-binding cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins that are responsible for the ATP-powered translocation of many substrates across membranes. The highly conserved ABC domains of ABC transporters provide the nucleotide-dependent engine that drives transport. By contrast, the transmembrane domains that create the translocation pathway are more variable. 28

Any object that enters or leaves a cell, whether a nutrient, a virus or a waste product, must penetrate one or more enclosing membranes. The magnitude of this phenomenon might be estimated, for example, by the need of an actively growing Escherichia coli cell to take up ~106 glucose molecules per second to support its requisite metabolic demands. Cells must not only be able to import preferred substrates, but they also often have the capability to use a wide range of alternative nutrients when available. With a few exceptions (such as oxygen and nitrogen), the movement of small molecules, ions and even some macromolecules across membranes is mediated by specialized membrane proteins that are known as transporters. To accommodate the diversity of molecules that a cell might need to acquire from the environment, many different transporters are encoded in the genomes of organisms. In E.coli, for example, ~10% of the genome has been classified as participating in transport processes and, overall, more than 550 different types of transporters have been identified . The importance of transport activity can be appreciated from the non-trivial metabolic costs of pumping molecules across cell membranes, which are estimated to consume ~10–60% of the ATP requirements of bacteria and humans , depending on conditions.

One of the largest classes of transporters is the ATPbinding cassette (ABC) transporter superfamily. These transporters use the binding and hydrolysis of ATP to power the translocation of a diverse assortment of substrates across membranes, ranging from ions to macromolecules. ABC transporters function as either importers, which bring nutrients and other molecules into cells, or as exporters, which pump toxins, drugs and lipids across membranes . Members of the ABC transporter family are present in organisms from all kingdoms of life; whereas exporters are found in both eukaryotes and prokaryotes, importers seem to be present exclusively in prokaryotic organisms.  At the sequence level, the superfamily of ABC transporters is identified by a characteristic set of highly conserved motifs that are present in the ABCs. For prokaryotic ABC transporters that function as importers, substrate translocation is also dependent on another protein component, a high-affinity binding protein that specifically associates with the ligand in the periplasm for delivery to the appropriate ABC transporter (FIG. a below ). Originally recognized by Heppel16, these binding proteins are released following osmotic shock. Therefore, the associated transport systems, which are now recognized as ABC transporters, were initially identified as ‘shock-sensitive’.

Molecular architecture of aBc transporters. 
a | A cartoon of the modular organization of ATP-binding cassette (ABC) transporters, which are composed of two transmembrane domains (TMDs) and two ABC domains (or nucleotide-binding domains). The binding protein component that is required by importers is also shown. Two conformational states of the ABC transporter — outward facing and inward facing, with the substrate-binding site orientated towards the periplasmic (extracellular) and cytoplasmic (intracellular) regions, respectively — are depicted to show the alternating access mechanism of transport. 
b | The Escherichia coli vitamin B12 importer BtuCDF22. The core transporter consists of four subunits: the two TMD BtuC subunits (purple and red) and the two ABC BtuD subunits (green and blue). This complex also contains one copy of BtuF, the periplasmic binding protein (cyan).

Kinetic mechanism of ABC transporters 
The similarities in ABC structures support a common mechanism by which ABC transporters, both importers and exporters, orchestrate a series of nucleotide- and substrate-dependent conformational changes that result in substrate translocation across the membrane. The ‘alternating access’ model, in which the substratebinding site alternates between outward- and inwardfacing conformations, provides a productive framework for this mechanistic analysis. The successful operation of transporters that move molecules across membranes against a concentration gradient requires the elimination of short-circuiting. This is achieved by preventing the uncoupled processes from occurring in their energetically favourable directions — that is, leakage of the accumulated substrate across the membrane or the futile cycling of ATP hydrolysis.

Mechanism of Iron transport
The first step of active uptake of iron (and iron complexes) occurs across the outer membrane, and is mediated by specific, high affinity transporters ( BtuB ). Regardless of their substrate specificity, these outer-membrane transporters share a common architecture, composed of a 22-stranded b-barrel plugged by a hatch domain. A common mechanistic feature is their high substrate affinity, dictated by the scarcity (or insolubility in the
case of iron) of the target substrate. For example, the outermembrane transporters BtuB  and FepA (Fe-enterobactin) have a KD of 0.2–0.3 nM towards their substrates. Since there is no ATP in the periplasm and no ion gradients across the outer membrane, conventional bacterial energy currencies cannot be used to drive active transport across the outer membrane. Therefore, the energy utilized by outermembrane transporters is transduced from the inner membrane by the ExbB/ExbD/TonB complex , by a mechanism that is poorly understood. Once in the periplasm, the substrate is captured by the system’s substrate binding protein. To complete the uptake of iron into the cytoplasm, BtuF must associate with its inner-membrane partner and deliver its bound cargo. Formation of the BtuCD–F transport complex occurs spontaneously, and does not require an energy input. Upon formation of the ultra-stable BtuCD–F complex, iron is released into the cytoplasm, again in an energy-independent manner (Fig.below).

Proposed catalytic cycle of Iron (Fe) transport by BtuCD–F. 
The pie chart in the background indicates which part of the cycle is ATP dependent. As iron is transported into the periplasm by the combined action of BtuB and the ExbB/ExbD/TonB complex, it is captured by BtuF. BtuF has the highest affinity towards the nucleotide-free BtuCD, and once the BtuCD–F complex is formed, iron is released from BtuF and is not retained by the complex. The now substrate- and nucleotide-free complex is extremely stable (KD E 0.1 pM) and will only dissociate in the presence of ATP and free iron, with both the nucleotide and the substrate contributing towards complex dissociation. ATP binding, hydrolysis, and release of ADP and phosphate reset the system for a subsequent cycle of transport. In the absence of periplasmic iron, BtuF will remain bound to BtuCD, essentially plugging the permeation pathway.

In the maltose ABC import system these two steps, complex formation and substrate release, are not spontaneous and do require energy. In the vitamin B12 system the sub-nM affinity of the initial step of translocation across the outer membrane, followed by the spontaneity of the two down-stream events (complex formation and intracellular substrate release), provide a ‘de-facto’ unidirectional inwardly directed substrate flux. Only after the substrate is released to the cytoplasm does the system require energy (Figure above). Perhaps non-intuitively, the role of energy input (ATP binding) in BtuCD–F is to break the complex, rather than to stabilize it. Whether this phenomenon is specific to the iron system or is a general mechanism of high-affinity uptake of trace metals has yet to be determined.
Another unique (and counter-intuitive) feature of Iron (Fe) import is that the substrate reduces the equilibrium affinity (KD) between the transporter and the binding protein 10^5-fold. The seeming paradox of this substrate effect is better understood by examining the effects of iron on the non-equilibrium kinetics of complex formation: Fe accelerates both complex formation and complex dissociation rates, by 15- and B10^7-fold respectively (resulting in the observed decreased equilibrium affinity). Such a substrate effect may prove detrimental to a receptor or an enzyme, but not for a transporter. In order for the transport reaction to proceed in a cyclic manner, the binding protein and the transporter must continuously associate and dissociate in a cyclic manner. Slow association rates are unfavorable as are slow dissociation rates. In a sense, Fe exerts a positive feedback on its own transport by increasing both the kon and koff. This effect is so pronounced that in the absence of a substrate, the dissociation rate (koff) of the BtuCD–F complex is reduced to 108 s1, an infinity in terms of bacterial life cycle. 

The hypothetical LUCA is predicted to possess all classes of ABC systems.  33
That raises the question, of course, how these life-essential transmembrane proteins emerged, since they cannot be explained by darwinian mechanisms, since they are a pre-requirement for life to start, and so - evolution. 

Outer membrane transporters (TBDT)
Given iron’s limited bioavailability, there are several Fe acquisition strategies. Among these is siderophore mediated iron uptake, which involves the synthesis and secretion of low molecular weight chelators that tightly bind Fe(III). The ferrisiderophore complex is transported as a whole into the cell where iron is released from the complex. This strategy has been well characterized in plants, yeast and bacteria.  In many environments, dissolved iron is predominantly bound by strong organic chelators that maintain Fe(III) in solution yet buffer extremely low concentrations of free iron.  Hereafter, unchelated iron, present as free hydrated species will be referred to as Fe′.

As ferri-siderophores are too large to passively diffuse through the OM porins, they must be actively transported across the membrane by specific receptor proteins 43 TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates called siderophores, as well as vitamin B12, nickel complexes, and carbohydrates. 44 The transport process requires energy in the form of protonmotive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins like serum transferrin and haemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch.  45 In recent years many new structures of TBDTs have been solved in various states, resulting in a more complete picture of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with TonB-ExbB-ExbD.

Transport into Gram-negative organisms is initiated by passage of the transported species across the outer membrane and into the periplasmic space j prior to inner membrane translocation. The uptake of iron is particularly important for bacterial growth and synthesis of outer membrane iron transporters (called TonB-dependent transporters, TBDTs) is therefore regulated in a variety of ways. While iron complexes constitute the majority of substrates for TBDTs, vitamin B12, nickel chelates, and carbohydrates are also transported by this mechanism. These transporters show high affinity and specificity for metal chelates called siderophores and require energy derived from the protonmotive force across the inner membrane to transport them. To tap this energy source, TBDTs must interact with an inner membrane protein complex consisting of TonB, ExbB, and ExbD. The first crystal structures of two Escherichia coli TonB-dependent transporters, ferrichrome transporter (FhuA) and ferric enterobactin transporter (FepA), showed that TBDTs use a 22-stranded β-barrel to span the outer membrane with an unanticipated ‘plug’ domain folded into the barrel interior. The plug domain functions to bind a specific metal chelate at the extracellular side of the membrane and to interact with TonB-ExbB-ExbD at the periplasmic side of the outer membrane. In these ‘ground state’ structures, the plug domain completely occludes the barrel pore, revealing an unexpected complexity for siderophore transport.

Synthesis of TonB-dependent transporters TBDTs is Regulated at Multiple Levels
Genes encoding the seven TBDTs in E. coli are scattered throughout the chromosome. Several genes encode ABC transporters that transport the siderophores across the cytoplasmic membrane. Downstream of fepA is the gene entD, involved in the synthesis of the enterobactin siderophore. Expression of all of these genes is highly regulated both at the transcriptional and post-transcriptional levels.

Fur repressor regulates transcription of TBDTs for ferric siderophores
Although iron is essential for most living organisms, iron accumulation can be toxic because it may lead to production of reactive radicals and it is therefore crucial to keep cellular iron levels under tight control. In E. coli, the Fur (Ferric Uptake Regulator) transcriptional repressor plays a key role in this process by regulating expression of genes involved in iron homeostasis as a function of cellular iron concentration. When iron is limiting, Fur cannot bind DNA, leading to derepression of genes that encode iron transporters and proteins involved in siderophore biosynthesis and iron metabolism, but also other cellular functions. Consistent with their role in iron transport, all TBDTs for ferric siderophores are controlled by Fur and their expression is therefore repressed when iron reaches a certain level.  Fur binds in vitro to the promoter regions of fepA-entD, fecABCDE, fhuACDB and cirA. In addition, Fur boxes were identified not only in these promoter regions, but also upstream of fhuE and fiugenes. Interestingly, Fur also directly represses transcription of the tonB gene as well as the exbB-exbD operon by binding upstream of exbB.

Transport and regulation of siderophores
Transport of ferric siderophores across the outer membrane derives energy from the inner membrane protonmotive force. This requires an energy-transducing TonB complex in the inner membrane (blue), consisting of TonB, ExbB and ExbD proteins. TonB interacts with outer membrane transporters (TBDT) at the TonB-box motif. Transport of ferric siderophores across the inner membrane requires a periplasmic binding protein and an ABC transporter. Once the ferric siderophore enters the cytoplasm, ferric ion (Fe3+) is reduced to ferrous ion (Fe2+), which is destined for storage or incorporation into enzymes. Excess Fe2+ (which could induce the formation of radicals harmful to the cell) binds to the repressor protein Fur, which in turn binds target promoters (Pfur) and inhibits transcription of siderophore transport genes. Some TBDTs, such as E. coli FecA are additionally regulated by σ/anti-σ factor systems. In addition to transporting diferric dicitrate, FecA regulates the expression of fecABCDE transport genes initiated by the binding of ferric citrate to FecA. This involves the N-terminal extension of FecA (green), the inner membrane regulator FecR (σ regulator, pink), and the cytoplasmic sigma factor FecI (ECF σ factor, pink). Both transport and induction require energy transduction from the TonB-ExbB-ExbD complex in the inner membrane.

Structure and Function of TonB-dependent transporters (TBDTs)
An analysis of the original four TBDTs shows that all of them have the same domain architecture: a 22-stranded transmembrane β-barrel encloses a globular plug domain (Figure below).

The structure of the (prototype) TBDT FhuA
TBDTs have an N-terminal plug domain that sits inside a C-terminal 22-stranded β-barrel domain. The conserved TonB box is found near the N-terminus of the plug domain facing the periplasm and is generally thought to remain sequestered inside the β-barrel domain in the absence of ligand. Upon binding ligand, a conformational change leads to exposure of the TonB box and subsequent interaction with TonB and siderophore transport. 
Panel a represents the FhuA-ferrichrome crystal structure (1BY5) with FhuA shown in ribbon and ferrichrome in spacefill model, 
panel b represents only the beta-barrel domain, 
panel c represents only the plug domain, and 
panel d shows the FhuA apo structure (1BY3) with those residues with at least 50% conservation highlighted in blue. Top view represents the extracellular view, side view represents the membrane view, and bottom represents the periplasmic view. The TonB box was found disordered in both structures and is represented by dashed lines.

Ligand binding sites are formed from residues on the extracellular side of the plug domain, as well as from residues on the walls and extracellular loops of the β-barrel. The TonB box is found at the N-terminus of the plug domain, and in some structures protrudes into the periplasm. In others, the TonB box is tucked up into the plug domain within the barrel or is disordered and not visible in the structures. A structure-based sequence alignment revealed conserved motifs in the plug and barrel which are close to one another and interact. Finally, an analysis of water molecules located at the plug-barrel interface revealed that the plug is highly solvated, resembling a transient protein complex and suggesting conformational change and/or movement of the plug within the barrel during transport. In the following sections, we will outline some of the significant structural and functional studies done with TBDTs in recent years.

Siderophore binding transduces a signal across the outer membrane
The binding of a siderophore to its TonB-dependent transporters (TBDTs) transduces a signal across the outer membrane that results in a disordering (also called unfolding or undocking) of the TonB box. The nature of the transduced signal is not completely clear, but for some TBDTs it appears to involve large conformational changes in extracellular loops which fold in over the top of the TBDT when siderophore binds, sequestering the ligand and contributing new residues to the binding site. This type of induced fit mechanism has been observed for FecA , ShuA , and FyuA. Ligand binding also induces smaller conformational changes in the plug domain (observed in many TBDT structures) but exactly how binding of a small molecule at the extracellular surface results in disordering of the TonB box is not completely clear.

TBDTs associate with TonB through β-strand pairing
When a TBDT has bound its siderophore and signalled to TonB-ExbB-ExbD, the next step appears to be a physical association between the TonB box of the TBDT and the C-terminal (periplasmic) domain of TonB. . In both structures TonB assumes an alpha-beta fold containing a 3-stranded β-sheet. The TonB box of either transporter adopts a β-strand conformation that pairs with the existing β-sheet of TonB. Association through strand pairing has been observed for many protein complexes and although details differ for the two complexes described here, we can conclude that the binding interface is relatively small. In both structures the plug domain still resides inside the β-barrel just like in all the other ground state structures of TBDTs. 

Membrane proteins play vital roles in inside-out and outside-in signal transduction by responding to inputs that include mechanical stimuli. 1  We confirm the feasibility of protein-only mediated mechanical gating by demonstrating that the interaction between TonB and BtuB (a TBDT) is sufficiently strong under force to create a channel through the TBDT. In addition, by comparing the dimensions of the force-induced channel in BtuB and a second TBDT (FhuA), we show that the mechanical properties of the interaction are perfectly tuned to their function by inducing formation of a channel whose dimensions are tailored to the ligand.

Perfect tuning, and tailored dimensions to the ligand are clear evidence of design. How, otherwise, could its perfect arrangement be explained ?  

Belying its name, the outer-membrane (OM) envelope of Gram-negative bacteria is relatively devoid of lipid and instead is packed with β-barrel OM proteins (OMPs), which function as enzymes, foldases, assembly platforms and both specific and non-specific transporters. Many of these functions, such as transport against a concentration gradient, are energy dependent and, as the periplasmic space is devoid of adenosine triphosphate (ATP), it is necessary to couple OMP transporters to machinery from the energized inner-membrane (IM) to facilitate these processes. A well-studied example is the interaction of OM transporters with TonB—a periplasmic protein. The TonB-dependent transporter family (TBDTs) bind and then transport scarce but vital nutrients such as  metallo-organic compounds including vitamin B12 (BtuB), haem (HasR and HemR) and siderophores (FepA, FhuA, FecA and FhuE). Their importance is vital to  cell viability.

Structurally, TBDTs are characterized by 22-stranded β-barrels whose lumens are occluded by an N-terminal globular ‘plug’ domain (Figure below).

(a) Schematic of TonB-dependent vitamin B12 transport in E. coli. PG, peptidoglycan. 
(i) The lumen of BtuB, a 22-stranded β-barrel OM protein, is occluded by an N-terminal plug domain (arrow) preventing transit of vitamin B12 (red space filling model)
(ii) the binding of vitamin B12 induces an allosteric rearrangement of the plug domain, releasing the Ton box into the periplasmic space, where it forms a 1:1 complex with the C-terminal domain of TonB (TonBCTD, residues 153–233 (PDB: 1XX3). The transmembrane helix at the N-terminus of TonB forms a complex with ExbB and ExbD (grey cylinders), which is necessary, together with an energized IM, for TonB-dependent transport
(iii) linkage of the OM and IM via this non-covalent complex is thought to trigger the full or partial unfolding of the plug domain, allowing the passage of vitamin B12. While the precise mechanisms vary, most models for TonB-dependent transport suggest that remodelling is induced by application of force (red arrow).

The plug domain contains a conserved binding motif known as the Ton box9 on the periplasmic side, which upon binding of extracellular substrate becomes disordered. This transition allows the formation of a non-covalent complex with the C-terminal globular periplasmic domain of TonB (TonBCTD) (Fig. 1b). TonB is anchored to the IM by a single N-terminal transmembrane helix and these domains are linked by a central proline-rich periplasmic spanning domain. This linker adopts an extended polyproline type II helical rod conformation, allowing TonBCTD to reach the OM. In vivo, TonB forms a complex with the IM proteins ExbB and ExbD. These proteins are thought to function as a scaffold (ExbB) and to harness the proton motive force (ExbD), a pre-requisite for TonB-dependent activity.

While it is known that TonB-mediated transport requires TonB to be tethered to an energized inner membrane (IM), the mechanism by which TonB remodels the plug domains of TBDTs remains unclear. Currently favoured models, such as the pulling hypothesis or the rotational surveillance and energy transfer model, suggest that TonB applies a mechanical remodelling force driven by its interaction with the ExbBD complex in the energized IM. This induces partial or full-plug domain unfolding via the non-covalent interaction of the Ton box with TonBCTD3. For these models to be viable, the Ton box–TonB inter-protein interaction must be sufficiently stable under tension to allow the unfolding of the plug domain before its dissociation. Intriguingly, the crystal structure of BtuB (the vitamin B12 TBDT receptor of E. coli) in complex with TonB shows that the Ton box binds to the β-sheet of TonBCTD in a parallel orientation and that this augmented β-sheet is rotated ∼90° with respect to the β-strands of the plug domain. If the plug domain of BtuB is extended by TonB in vivo, the relative geometry of the β-strands involved in these inter- and intra-protein interactions are ideally oriented to engender these mechanical phenotypes as shown using molecular dynamics (MD).

Alternative iron uptake pathway
Many cyanobacterial species do not produce siderophores, and alternative Fe acquisition mechanisms exist. Here we present a study of the iron uptake pathways in the unicellular, planktonic, nonsiderophore producing strain Synechocystis sp. PCC 6803.  We found that Synechocystis 6803 is capable of acquiring iron from exogenous ferrisiderophores (Ferrioxamine-B, FeAerobactin) and that unchelated, inorganic Fe is a highly available source of iron. Inhibition of iron uptake by the Fe(II)-specific ligand, ferrozine, indicated that reduction of both inorganic iron and ferrisiderophore complexes occurs before transport through the plasma membrane. The reduction-based uptake strategy is well suited for acquiring iron from multiple complexes in dilute aquatic environments and may play an important role in other cyanobacterial strains. . Iron has two environmentally relevant oxidation states: Fe(II) and Fe(III). . In oxygenated waters at circumneutral pH, Fe(II) is soluble but thermodynamically unstable while dissolved Fe(III) concentrations are limited by the low solubility of Fe(III) hydroxide species. In many environments, dissolved iron is predominantly bound by strong organic chelators that maintain Fe(III) in solution yet buffer extremely low concentrations of free iron.39 

The TonB, ExbB and ExbD complex
TonB is a key protein in active transport of essential nutrients like vitamin B12 and metal sources through the outer membrane transporters of Gram-negative bacteria. This inner membrane protein spans the periplasm, contacts the outer membrane receptor by its periplasmic domain and transduces energy from the cytoplasmic membrane pmf to the receptor allowing nutrient internalization. Whereas generally a single TonB protein allows the acquisition of several nutrients through their cognate receptor, in some species one particular TonB is dedicated to a specific system.   29

The TonB–TBBtuB interaction is sufficiently strong under extension to allow the mechanical unfolding of a defined region of the plug domain of BtuB to create a substrate channel through the receptor before its dissociation. This mechanism is similar to that originally proposed a decade ago, which was supported by an in silico study which showed that extension of TBBtuB by ∼20 nm leads to the creation of a channel that would allow substrate passage. All the suggested models involve the formation of a force-transduction pathway, which apply mechanical force to the plug domain. In these models, the vertical displacement of TBBtuB that is required for channel opening may be a result of ‘passive' processes (for example, variations in the width of the periplasmic space and/or the differential diffusion of the IM and OM proteins. Alternatively, active processes dependent on the proton motive force may play a role. This includes a conformational change of the TonB linker resulting in a shortening in the end-to-end length of some part of the linker region by a transition of residues from an extended polyproline type II to a shorter polyproline type I helix, or by the ExbBD-dependent rotary motion of TonB itself.

Iron in the form of ferric siderophore complexes and vitamin B12 are transported through the outer membrane of Gram-negative bacteria by a mechanism which consumes energy. There is no known energy source in the outer membrane or in the adjacent periplasmic space so that energy is provided by the electrochemical potential across the cytoplasmic membrane. Energy flows from the cytoplasmic into the outer membrane via a complex consisting of the TonB, ExbB and ExbD proteins which are anchored in the cytoplasmic membrane. It is proposed that the TonB-ExbB-ExbD complex opens — via an energized conformation of the TonB protein—channels in the outer membrane, formed by proteins which serve as highly specific binding sites for the various ferric siderophores and vitamin B12. 34

Energy-coupled transport through the outer membrane is a bioenergetic paradox since there is no energy source within this membrane, or within the adjacent periplasmic space. The outer membrane certainly cannot be energized by an electrochemical potential because it contains open protein channels, and ATP, PEP or other high energy metabolites, have not been found in the periplasm. Nevertheless, compelling evidence exists that the uptake of certain compounds through the outer membrane requires energy. In Escherichia coli these compounds include ferric siderophores (ferrichrome, ferric enterochelin, also designated ferric enterobactin, ferric citrate, ferric 2,3' dihydroxybenzoylserine, ferric aerobactin), vitamin B12, and group B colicins. Ferric siderophores, vitamin B12 and the colicins bind to highly specific receptor proteins in the outer membrane, even in energy-depleted cells, but require energy for uptake. Outer membrane transporters, or "receptors",   are essential for the uptake of ferric siderophores, vitamin B12 and colicins. These substrates are usually too large for the channels of the type I and II porins, and they occur at very low concentration, necessitating the use of specific cell surface receptors to govern uptake. 

Nutrients that are either poorly permeable through porins (those greater than 600 Da) or present at very low concentration, are transported via outer membrane specific transporters by an energized process.  The following substrates are internalized by this active transport process: vitamin B12, heme, iron-siderophore and nickel-nickelophore complexes and some carbohydrates. No energy source is present at the OM, the energy for this process is provided by an inner membrane complex composed of TonB, ExbB and ExbD proteins. which couples the inner membrane proton-motive force (pmf) to the TonB-dependent outer membrane transporter (TBDT). These TBDTs share common structural features and are composed of a C-terminal β-barrel and a N-terminal globular domain, folding back into and closing, like a “plug”, the channel formed by the barrel

Iron uptake systems in Cyanobacteria
Iron is considered the critical micronutrient for marine diazotrophs g due to their use of the iron-nitrogenase protein complex containing a homodimeric iron protein with a 4Fe∶4S metallocluster (NifH) and a heterotetrameric molybdenum-iron protein with a P cluster and a  MoFe cofactor Field experiments and models both predict the distribution of oceanic nitrogen fixation to be primarily constrained by the availability of iron. Despite this importance of iron on marine nitrogen fixation, there is a limited understanding of how marine diazotrophs have adapted to this low iron environment. 35 

There are three Fe-substrates:

dissolved inorganic iron
(Fe') and the Fe-siderophores Ferrioxamine B (FOB)
FeAerobactin (FeAB)

All strains were found to employ a reductive step in the uptake of Fe' and FOB. Our data support the existence of a common reductive Fe uptake pathway amongst cyanobacteria, functioning alone or in addition to siderophore-mediated uptake. Cyanobacteria combining both uptake strategies benefit from increased flexibility in accessing different Fe-substrates. Iron is characterized by its exceedingly low solubility in oxic, circum-neutral pH waters, and, as such, Fe rapidly precipitates out of solution as ferric oxyhydroxide species.  The more accessible soluble iron pool (<0.02 μm) is typically found at sub-nanomolar levels and can be roughly divided into two fractions: free inorganic iron (Fe') and organically complexed Fe. While Fe' has proven to be a highly bioavailable Fe substrate to eukaryotic phytoplankton and some cyanobacteria, while, it found at pM levels, accounting for less than 1% of dissolved iron in surface waters. The overwhelming majority (>99%) of dissolved Fe is bound to organic ligands. However, the chemistry and structures of complex organic ligands are obscure, and thus the bioavailability of this heterogeneous fraction remains poorly defined. 36

The bioavailability of any particular Fe-substrate depends not only on its chemistry but also on the Fe-uptake mechanisms available to an organism. Different cyanobacterial species may possess different iron uptake strategies. Therefore, iron bioavailability is not only a question of “what?” but also of “how?” As prokaryotes, cyanobacteria are often associated with the siderophore-mediated iron uptake pathways implemented by many heterotrophic bacteria a. Siderophores are low molecular weight compounds secreted by iron-limited microorganisms with the purpose of scavenging iron from the environment. These compounds have very high Fe(III) affinities. Once bound to iron, the ferric-siderophore complexes are transported back into the cell via siderophore specific transporters. Decomplexation of the ferric siderophores usually occurs in the cytoplasm. In recent years, the paradigm of siderophore-mediated uptake amongst cyanobacteria has been re-evaluated in the face of experimental and genetic studies showing that 

(a) not all cyanobacteria possess the components of a siderophore based uptake system and 
(b) alternative Fe-uptake pathways exist in cyanobacteria. 

Genetic studies demonstrated that siderophore biosynthesis and transporter genes are absent from open ocean cyanobacteria and several freshwater strains e.g. Moreover, even in species that are known to produce siderophores, experimental work points to the operation of a siderophore independent Fe-uptake pathway. Reductive iron uptake directly mediated by the cell may be important in natural populations as well as in open ocean Synechococcus species. This reductive strategy is well studied in eukaryotic phytoplankton and involves the reduction of free or complexed ferric iron into its ferrous form prior to its transport across the plasma membrane (either in a re-oxidized ferric or ferrous form) 

The TonB-dependent transport systems
Cyanobacteria require large amounts of iron, generally 10 times more than non-photosynthetic prokaryotes. Iron is taken up bound to siderophores, which are Fe(III)-chelators secreted by microorganisms. Siderophore production and secretion occurs under iron starvation to foster iron uptake, but it also occurs when the cells are exposed to high concentrations of other metal ions, such as copper, to protect the organism from such toxic compounds. The molecular mode of siderophore secretion into the cellular surrounding has been studied in Anabaena sp. PCC 7120, which produces the siderophore schizokenin.

The marine cyanobacterium Trichodesmium sp. accounts for approximately half of the annual ‘new’ nitrogen introduced to the global ocean but its biogeography and activity is often limited by the availability of iron (Fe). 35 Fe is an absolute requirement in the catalysts of both photosynthesis and dinitrogen (N2) fixation.  Cyanobacteria display a number of distinct Fe acquisition pathways including Fe3+ and Fe2+ transporters, the latter often coupled to biological reduction of Fe3+ to Fe2+, alongside production of siderophores which are released from the cell, bind Fe and are subsequently taken up through dedicated transporters.  Homologs to Fe2+ (FeoAB), Fe3+ (FutABC) and siderophore (FhuD) transporter components are encoded in the Trichodesmium genome; however, the proteins involved in Fe reduction are not well characterized.  Inorganic Fe reduction and uptake is possibly facilitated by reactive oxygen species (ROS) produced intracellularly. The mechanisms and physiological impacts of cell-to-substrate contact for acquisition of Fe from dust are yet to be fully determined.

NifS and NifU as essential components for full activation and cluster assembly in both nitrogenase component proteins. 33 IscU corresponds to the N-terminal domain of NifU, and recent studies have indicated that IscU functions as a scaffold for the IscS-mediated assembly of both [2Fe–2S] and [4Fe–4S] clusters that are subsequently used for the maturation of apo Fe–S proteins. 

NifS is a cysteine desulfurase that probably supplies the inorganic sulfur necessary for nitrogenase metallocluster assembly. It has now been shown that NifU binds iron that is destined for iron-sulfur core formation and that it provides a site for the assembly of a 2Fe-2S unit that serves as a precursor for the nitrogenase metallocluster cores. Although a specific function for NifU in nitrogenase [Fe-S] cluster formation is not known, the available evidence points to a role either as the iron source necessary for [Fe-S] cluster formation or as an intermediate site for [Fe-S] cluster assembly. For NifU to serve either of these functions it must have the ability to transiently bind iron destined for [Fe-S] cluster formation or as an intermediate site for [Fe-S] cluster assembly. 26 For NifU to serve either of these functions it must have the ability to transiently bind iron destined for [Fe-S] cluster formation. Previous work has shown that isolated NifU is a homodimer that contains two identical [2Fe-2S] clusters.

NifU is a homodimer of 33-kDa subunits with 2 Fe atoms per subunit. Spectroscopic studies showed the presence of [Fe2S2]2, clusters with Em 254 mV and only cysteinyl coordination, but with properties unlike other [Fe2S2] containing ferredoxins. The exact role of NifU in the full activation of nitrogenase is still unclear. 27 

In the NIF system, NifS and NifU are required for the formation of metalloclusters of nitrogenase in Azotobacter vinelandii, and other organisms, as well as in the maturation of other FeS proteins. Nitrogenase catalyses the fixation of nitrogen. It contains a complex cluster, the FeMo cofactor, which contains molybdenum, Fe and S. NifS is a cysteine desulphurase. NifU binds one Fe atom at its N-terminal, assembling an FeS cluster that is transferred to nitrogenase apoproteins 30

The [Fe-S] biosynthetic systems has a cysteine desulfurase and a molecular scaffold. The cysteine desulfurase enzyme NifS catalyzes the removal of atomic sulfur from L-cysteine. Labile [Fe-S] clusters are built on the molecular scaffolding proteins NifU presumably from S provided by a cysteine desulfurase and Fe provided by a Fe trafficking molecule such as the frataxin homolog CyaY 31

The biogenesis of iron–sulphur clusters requires the co-ordinated delivery of both iron and sulphur. 32 It is now clear that sulphur in iron–sulphur clusters is derived from l-cysteine by cysteine desulphurases. However, the iron donor for the iron–sulphur cluster assembly still remains elusive.

Molybdenum trafficking to FeMo-co can be divided into in five processes: 
(1) molybdate harvesting, 
(2) molybdate transport and discrimination against tungstate, 
(3) molybdenum accumulation and homeostasis, 
(4) molybdenum sorting to the appropriate pathway, and 
(5) molybdenum insertion into the cofactor.

a A heterotroph  is an organism that ingests or absorbs organic carbon (rather than fix carbon from inorganic sources such as carbon dioxide) in order to be able to produce energy and synthesize compounds to maintain its life.



Further readings:
Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site
Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism
Stepwise formation of P-cluster in nitrogenase MoFe protein
Formation and Insertion of the Nitrogenase Iron−Molybdenum Cofactor

Last edited by Admin on Wed Feb 14, 2018 3:34 pm; edited 34 times in total

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In the aerobic biosphere, inorganic sulfate is the most abundant source of sulfur that can be utilized by cells. 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. 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. So, the process is

Sulfate uptake by Sulfate permeases

Afterwards, transformation from sulfate to sulfide is transformed by three enzymes :

Phosphoglycerate dehydrogenase
Phosphoserine transaminase
phosphoserine phosphatase

to get Serine amino acids, which serve as a substrate to get cysteine amino acids, by going through another elaborate synthesis process, requiring following three enzymes:

Serine O-acetyltransferase
Cysteine synthase


In Behe's book "Black Box", just a few irreducibly complex systems were mentioned, and some, like the flagellum, have been debated for years. It's remarkable, that during two decades, not many other irreducibly complex systems have been mentioned or proposed. Fact is, however, that such structures are found in basically ALL living beings, starting with biological cells. What I want to bring to attention, is the fact, that in many cases, it is tiny things, like a single protein, or even co-factor, that are LIFE -ESSENTIAL. And removing a single one will make ALL life on earth impossible.

In a car, there are many parts that do not make the cars main function impossible. if you remove the Rearview Mirror, not much will change. If you remove however the electrical wire that connects the ignition lock to the power starter, the car will not turn on.

But things get acuter. The electrical wire of the ignition lock has to be manufactured.  Let's suppose there are a factory and a robotic production line that makes that electrical wire. Several complex machines or robots are required in that production line. if one tiny part of one of the robots has a malfunction, the production line will cease to make electrical wires, the factory will not produce the wire, and in the end,  a car cannot function.    

In life, there are literally MILLIONS of such tiny parts, that MUST exist, and exist only to make proteins, cofactors, vitamins, and all kind of molecules, that are required for a higher end, a higher function.

An example. i wrote recently about Nitrogenase enzymes, which fix nitrogen, essential as building block of amino acids, DNA, RNA etc.

Nitrogenase is one of the most unique enzymes in nature.    It performs one of the most biologically difficult reactions there is:  as a molecular sledgehammer, it reduces and break the triple bond of dinitrogen. The only comparable mechanism is the electricity of lightning. That gives an idea about the force required for the split. One author summed up the situation well by remarking, ‘Nature is really good at it (nitrogen-splitting), so good in fact that we've had difficulty in copying chemically the essence of what bacteria do so well.’   It so happens, that this reaction is absolutely vital for every living organism.

Nitrogenase carries the largest iron-sulfur cluster in Nature, clearly a masterpiece in transition metal catalysis.

Not only the enzyme and its function per se is remarkable, but as well, how it is biosynthesized or made. Over 18 genes are required to instruct bacterias how to make the Nitrogenase enzyme complex.  

Nitrogenase contains 3 Iron-sulfur metal clusters. In order to make them Complex import, transport, and transformation machinery of sulfur and Iron are required.  

The cluster assembly occurs outside of the nitrogenase protein holoenzyme  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 cluster biosynthesis requires enzymes, which provide substrates in the appropriate chemical forms and catalyze certain critical reactions such as carbide insertion, molecular scaffolds to aid in the step-wise assembly of FeMo-clusters, and metallocluster carrier proteins that escort FeMo-co biosynthetic intermediates in their transit between scaffolds. 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 ) . 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.

So, there has to be a fully operational import and transport machinery of sulfur, iron, molybdenum. To give you an idea, to get sulfur, six extremely complex protein complexes are required:

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 )

If Inorganic pyrophosphate ( ppaC) is missing to “pull” the reaction, ATP sulfurylase, just one of the six proteins, cannot do its job, and guess what - nothing done. Nitrogenase metal clusters cannot be biosynthesized without sulfur, and - no life. A tiny part - but life essential.

Now you have just the sulfur substrate, to begin the assembly of the Iron-Sulfur cluster.

Biosynthesis of the Cofactors of Nitrogenase

Once, all basic materials are in place, the complex synthesis can begin, going through six complex synthesis steps. And then you have just one of the 3 metal clusters. And all instructions must be there to insert it in the right way into the active site of the enzyme.

Natural selection would not select for components of a complex system that would be useful only in the completion of that much larger system.

Sorry, Mr.Darwin..... intelligence, much of it, is required, to create life. Natural, unguided, random events are too limited for the job.

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Intracellular homeostasis of metals, and origin of life scenarios


Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. 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, 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. 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 through the cytoplasm or at the periplasm but in both cases, the substrate will be expelled to the cell exterior.

My comment: Think about this: replication, transcription, translation, signal transduction, and cell division depend on the presence of transition metals. Life depends on all these intracellular proceedings, that means, the uptake and homeostasis of these metals had to be fully existent and in operation when life and cell self-replication began. That means a cell membrane, and BOTH, P-type ATPase pumps, and ABC transporters - membrane transport proteins, mechanisms to recognize either overload or too little metals inside the cell had to be fully operational, besides the biosynthesis of Iron/Sulfur clusters, essential co-factors in life-essential enzymes and protein complexes.

I am tracking down the mechanisms of Iron and Sulfate import, and metal-cluster biosynthesis. It is a staggeringly complex process, which is fully regulated, depending on scaffolding proteins and a battery of finely tuned processes of import, transformation of substrates into usable form, then handing over to sophisticated production lines, until the finished clusters are made, and ready to be joined in an incredibly complex manner with the apo-protein. These apo-proteins have the precise shape, size, and place where to insert the co-factors, and specific binding sites to annex it. The insertion process also requires the precise conformational change of the protein, in order that the co-factor can enter and fit into its binding pocket.

You cannot invoke evolution to explain these processes. Neither physical necessity. There was no need for physical affinity, necessity or any other physical requirement, to produce the blueprint for all these procedures. Now, all you have left is either unguided, lucky accidents or a super-intelligent creator had the intention to create life, and knew what was required, and implemented it. No gradual, stepwise, gradual slow evolutionary processes will do. This is an either all or nothing situation. Either life was fully set up at once, in the beginning, or nothing.

The choice what explanation makes the most sense is yours. Cheers.

Biosynthesis of the Cofactors of Nitrogenase

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