Molybdenum, essential for life

Molybdenum, essential for life

https://reasonandscience.catsboard.com/t2430-molybdenum-essential-for-life

Molybdenum is bioavailable as molybdate (MoO42−) 20 Once molybdate enters the cell, it is subsequently incorporated by complex biosynthetic machineries into metal cofactors.  Molybdenum cofactor Molybdenum utilization is very likely an ancient trait present in LUCA because (i) it is utilized by almost all phyla of Archaea and Bacteria and (ii) a number of molybdo-enzymes, including the arsenite oxidase, the formate dehydrogenase, the nitrate reductase and the polysulfide reductase, have been predicted to have existed before the Archaea/Bacteria divergence The synthesis of the molybdo-pterin cofactor is a conserved multi-step pathway that is present in molybdenum-utilizing organisms in all three domains of life (see Ref. [52] for review and references therein) and therefore might have been present in LUCA. 19  Molybdenum cofactor (Moco) is a metal-containing prosthetic group common to nearly all molybdoenzymes and is ubiquitous to all kingdoms of life. Moco-dependent enzymes play central roles in many biologically important processes such as purine and sulfur catabolism in mammals, anaerobic respiration in bacteria, and nitrate assimilation in plants. 16 Molybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. 17 Metals play unique and critical roles in biology, promoting structures and chemistries that would not otherwise be available to proteins alone. 8 Molybdenum is a naturally occurring element which is found in soil, water and in our bodies. In humans, molybdenum is needed to produce enzymes which play a vital role in maintaining our bodily functions. In fact, it’s essential for all human, animal and plant life. 14 The transition element molybdenum (Mo) is of essential importance for (nearly) all biological systems as it is required by enzymes catalyzing diverse key reactions in the global carbon, sulfur and nitrogen metabolism. 15 Molybdenum metabolism is strictly dependent on iron metabolism at different levels. FeMo-co biosynthesis and nitrogenase maturation are based on the synthesis of complex Fe–S clusters. 10

At the book A priviledged Planet, page 201,  Gonzales writes:
The strong nuclear force  is responsible for holding protons and neutrons together in the nuclei of atoms. In such close quarters, it is strong enough to overcome the electromagnetic force and bind the otherwise repulsive, positively charged protons together. It is as short-range as it is strong, extending no farther than atomic nuclei. But despite its short range, changing the strong nuclear force would have many wide-ranging consequences, most of them detrimental to life.  The periodic table of the elements would look different with a changed strong nuclear force. If it were weaker, there would be fewer stable chemical elements. The more complex organisms require about twenty-seven chemical elements, iodine being the heaviest (with an atomic number of 53). Instead of ninety-two naturally occurring elements, a universe with a strong force weaker by 50 percent would have contained only about twenty to thirty. This would eliminate the life-essential elements iron and molybdenum.

Minerals containing the elements boron and molybdenum are key in assembling atoms into life-forming molecules. It is thought that the boron minerals needed to form RNA from pre-biotic soups were not available on early Earth in sufficient quantity, and the molybdenum minerals were not available in the correct chemical form. 11 The researcher points out that boron minerals help carbohydrate rings to form from pre-biotic chemicals, and then molybdenum takes that intermediate molecule and rearranges it to form ribose, and hence RNA. This raises problems for how life began on Earth, since the early Earth is thought to have been unsuitable for the formation of the necessary boron and molybdenum minerals. It is thought that the boron minerals needed to form RNA from pre-biotic soups were not available on early Earth in sufficient quantity, and the molybdenum minerals were not available in the correct chemical form. "It’s only when molybdenum becomes highly oxidised that it is able to influence how early life formed. "This form of molybdenum couldn’t have been available on Earth at the time life first began, because three billion years ago, the surface of the Earth had very little oxygen.

Hundreds of other minerals that incorporate relatively rare elements such as lithium, beryllium, and molybdenum appear to have taken a billion years or more to first appear because it is difficult to concentrate these elements sufficiently to form new minerals. So those slow-forming minerals are also excluded from the time of life’s origins. 12

That made Leslie Orgel propose the esoteric proposal of directed panspermia, as he wrote:
The chemical composition of living organisms must reflect to some extent the composition of the environment in which they evolved. Thus the presence in living organisms of elements that are extremely rare on the Earth might indicate that life is extraterrestrial in origin. Molybdenum is an essential trace element that plays an important role in many enzymatic reactions, while chromium and nickel are relatively unimportant in biochemistry. The abundance of chromium, nickel, and molybdenum on the Earth are 0.20, 3.16, and 0.02%, respectively. We cannot conclude anything from this single example, since molybdenum may be irreplaceable in some essential reaction nitrogen fixation, for example. However, if it could be shown that the elements represented in terrestrial living organisms correlate closely with those that are abundant in some class of star molybdenum stars, for example we might look more sympathetically at "infective” theories. 18

Chromium, molybdenum, selenium, and vanadium, for example, are essential for building proteins, and proteins serve as life’s molecular “factories.” 13

The atmosphere is the primary source of nitrogen, and the continents are the primary source of several mineral nutrients, including molybdenum. This suggests that planetary environments lacking a nitrogen-rich atmosphere and continents may not be able to support a robust biosphere.

Metals are essential in microbial cells. 4 Cells require a number of  elements that could potentially provide biosignatures, including bioactive trace metals. Biological use of copper and molybdenum has developed along with bioavailability.

The majority of the presented subfamilies and, as a consequence, the Molybdo-enzyme superfamily as a whole, appear to have existed in LUCA. 5

Molybdoenzymes have been classified into three families:

1.xanthine oxidase (XO) family
2.sulfite oxidase (SO) family
3.the dimethyl sulfoxide (DMSO) reductase family

Molybdenum in Biology - An Essential Trace Element 1
Molybdenum is an essential trace element for several enzymes important to animal and plant metabolism, in special nitrate reductase and nitrogenase. Molybdenum functions as an electron carrier in those enzymes that catalyse the reduction of nitrogen and nitrate. Molybdenum is essential to plants and  humans. Molybdenum is needed for at least three enzymes. Sulfite oxidase catalyses the oxidation of sulfite to sulfate, necessary for metabolism of sulfur amino acids. Sulfite oxidase deficiency or absence leads to neurological symptoms and early death. Xanthine oxidase catalyses oxidative hydroxylation of purines and pyridines including conversion of hypoxanthine to xanthine and xanthine to uric acid. Aldehyde oxidase oxidises purines, pyrimidines, pteridines and is involved in nicotinic acid metabolism. Low dietary molybdenum leads to low urinary and serum uric acid concentrations and excessive xanthine excretion.

Molybdenum (Mo)  are trace elements that catalyze, upon binding to the appropriate cofactors, diverse and important redox reactions in the global carbon, nitrogen, and sulfur cycles. 7  Mo is found in two forms of oxygen-labile metal cofactors, a pterin-based and a Fe – S-cluster-based scaffold. Both oxyanions enter the cell via an ABC-type high affinity uptake system and are subsequently processed by a multistep biosynthetic machinery forming either Mo and W-pterin cofactors (Moco or Wco) in a large variety of Mo- and W-containing enzymes or the FeMo cofactor (FeMo-co) in nitrogenase-catalyzed nitrogen fixation. The functional diversity of pterin-based Mo and W cofactors is reflected by a large number of enzymes such as nitrate reductase, dimethyl sulfoxide reductase, formate dehydrogenase, aldehyde oxidoreductase and CO dehydrogenase. In these enzymes Mo and W are bound via thiolates to one or two unique tricyclic pterin moieties, commonly referred to as molybdopterin but the term “metal binding pterin” (MPT) is more appropriate due to its association with both, Mo and W. It is commonly believed, but still not demonstrated, that Moco and Wco are synthesized by a similar and highly conserved pathway. Synthesis of the Moco can be divided into four major steps, according to the biosynthetic intermediates cyclic pyranopterin monophosphate, MPT, and adenylated MPT. In contrast, FeMo-co biosynthesis is less understood in terms of reaction intermediate and mechanisms of different reactions catalyzed by the involved proteins. It starts with the formation of Fe – S cluster core structures that are assembled and arranged to a topology similar to mature FeMo-co. In the next steps, Mo and homocitrate are transferred before the mature cofactor is inserted into nitrogenase. Finally, a brief overview about Mo- and W-pterin enzymes as well as FeMo- and FeW-nitrogenases is given.

Molybdoenzymes emerged as a superfamily of respiratory oxidoreductases that require a catalytic molybdenum/tungsten-based cofactor to catalyze redox reactions. 5 These enzymes are further classified into three families based on the active site structure that coordinates the molybdenum atom. A key feature that separates the dimethyl sulfoxide (DMSO) reductase family members from xanthine oxidase and sulfite oxidase families is that it has two pyranopterin groups coordinating the Mo atom, whereas the others have only one. Members of each family have similar structural folds around the catalytic cofactor, and a recent study demonstrated that the protein fold is directly correlated to the pyranopterin conformation



The complex iron sulfur containing molybdoenzyme DMSO reductase. (a) Structure of the molybdo-bis(pyranopterin guanine dinucleotide) (MobisPGD) catalytic cofactor and [4Fe-4S] clusters that makeup the electron transfer chain within DMSO reductase. (b) Overall architecture and composition of DMSO reductase from Escherichia coli, demonstrating the catalytic DmsA, electron conduit DmsB, and membrane anchor DmsC subunits

The distribution of life on earth is constrained also by the distribution of 20 bio-essential nutrients such as Calcium (Ca), Chloride (Cl−), Chromium (Cr), Cobalt (Co), Copper (Cu), Magnesium (Mg), Manganese (Mn), Nickel (Ni), Iodine (I), Iron (Fe), Molybdenum (Mo), Phosphorus (P), Potassium (K), Selenium (Se), Sodium (Na), Sulfur (S), Tugsten (W), Vanadium (V), and Zinc (Zn), which are relatively rare, but that are key components of DNA, RNA, enzymes, and other biomolecules. 2

Molybdenum in pre-biotic chemistry-the nitrogen cycle
The nitrogen cycle provides essential nutrients to the biosphere, but its antiquity in modern form is unclear. In a drill core though homogeneous organic- rich shale in the 2.5- billion- year- old Mount McRae Shale, Australia, nitrogen isotope values vary from +1.0 to +7.5 per mil (parts per thousand) and back to +2.5 parts per thousand over similar to 30 meters. These changes evidently record a transient departure from a largely anaerobic to an aerobic nitrogen cycle complete with nitrification and denitrification. Complementary molybdenum abundance and sulfur isotopic values suggest that nitrification occurred in response to a small increase in surface- ocean oxygenation. These data imply that nitrifying and denitrifying microbes had already evolved by the late Archean and were present before oxygen first began to accumulate in the atmosphere

Molybdenum and the origin of life
An evolutionary tree of key enzymes from the Complex-Iron-Sulfur-Molybdoenzyme (CISM) superfamily distinguishes "ancient" members, i.e. enzymes in the last universal common ancestor (LUCA) of prokaryotes, from more recently evolved subfamilies. The molybdo-enzyme superfamily existed in LUCA. The results are discussed with respect to the nature of bioenergetic substrates available to early life and to problems arising from the low solubility of molybdenum under conditions of the primordial Earth.

Whereas the details of iron's and copper's involvement in numerous biological reactions have been studied for more than a century, the precise role of other metals, although recognized as vital trace elements in enzyme catalysis, became elucidated only recently. Molybdenum (Mo) has during the last 2 decades been shown to constitute an essential cofactor in at least 3 distinct enzyme superfamilies, the most widespread of which is the so-called Complex Iron-Sulfur Molybdoenzyme (CISM) superfamily of molybdo-pterin containing enzymes. Incidentally, this denomination ignores the fact that a few members of the family use tungsten (W) instead of molybdenum in their active sites. In the periodic table of elements, tungsten lies directly below molybdenum in the d-block and is thus expected to feature chemical properties related to those of Mo.

The ineluctable requirement for the trans-iron elements molybdenum and/or tungsten in the origin of life 3
The majority of the presented subfamilies and, as a consequence, the Molybdo-enzyme superfamily as a whole, appear to have existed in LUCA.
A vast number of enzymes rely on metal cofactors for catalysis and/or redox conversions. The structural unit of the CISM protein thus appears to have served multiple purposes for life, especially in energy harvesting, right from its very beginnings. CISM enzymes in LUCA likely performed energy conversion through the reduction of carbon dioxide, polysulfide or nitrate as well as from the oxidation of arsenite. Reduction of CO2 and sulfur with H2 as electron donor would be viable bioenergetic pathways in the geochemical setting of the early Archaean and have indeed been put forward as ancestral bioenergetic mechanisms. The presence of the CISM superfamily in LUCA implies a vital role of its metal cofactors in early life.

What then of the availability of these two transition metals?
W occurs in both acid and alkaline solutions and was thus available to emerging life, whereas Mo is relatively insoluble in reduced and neutral waters, but does occur in mixed valence sulfide and selenide and/or oxide complexes in alkaline solutions. Mo's insolubility at neutral pH values, exacerbated by an anoxic atmosphere, suggested a low bioavailability of this element for early life. Mo-isotope analyses on samples from the Archaean era indeed show substantially lower levels than during Phanerozoic times. Two scenarios can reconcile the results of molecular phylogeny and paleogeochemistry. (i) The ancestral CISM enzyme exclusively used W (tungsten) which was later replaced by Mo. (ii) CISM-catalyzed reactions in early life used Mo supplied by alkaline hydrothermal vents, proposed as cradles for life.

What we see here, are the usual "ad-hoc" explanations. 

Certainly tungsten and most likely molybdenum ought to be added to the list of metals vital already to earliest life on Earth

The biosynthesis of the molybdenum cofactor 17

In E. coli, nine proteins with known function are directly involved in Moco biosynthesis (MoaA, MoaC, MobA, MocA, MoaD, MoaE, MoeA, MoeB, MogA) 17 In all prokaryotes, Moco is synthesized by a conserved pathway which can be divided into four general steps:

(i) the synthesis of cyclic pyranopterin monophosphate (cPMP) from 5′GTP ,
(ii) insertion of two sulfur atoms into cPMP and formation of MPT,
(iii) formation of Moco by insertion of molybdate to the sulfur atoms of MPT,
(iv) further modification of Moco by the attachment of GMP or CMP to the phosphate group of MPT, forming the MGD cofactor  or MCD cofactor  (Fig. 1). The four steps of Moco biosynthesis are described below:

First step: The biosynthesis of Moco starts from 5′-GTP. The first stable intermediate of Moco was isolated in 1993 and later identified to be a 6-alkyl pterin with a cyclic phosphate group at the C2′ and C4′ atoms, named cPMP . The reaction of cPMP formation from 5′GTP is catalyzed by the two proteins MoaA and MoaC in bacteria. MoaA belongs to the superfamily of S-adenosyl methionine (SAM)-dependent radical enzymes. The protein contains two [4Fe4S] clusters, with the N-terminal [4Fe4S] cluster binding SAM and generating the 5′-deoxyadenosyl radical, and the C-terminal [4Fe4S] cluster binding the 5′GTP. While the individual catalytic functions of MoaA and MoaC had long been unknown, recent studies showed that MoaA catalyzes the conversion of 5′GTP to (8S)-3′,8-cyclo-7,8-dihydroguanosine 5′triphosphate (3′,8-cH2GTP), and MoaC catalyzes the conversion of 3′,8-cH2GTP to cPMP . In this reaction, the C8 of GTP is inserted between the C2′ and C3′ carbons of the ribose. MoaC further converts 3′,8-cH2GTP to cPMP, a reaction which involves pyrophosphate cleavage in addition to the formation of the cyclic phosphate group.

Second step: In the next step of Moco biosynthesis, cPMP is converted to MPT by the insertion of two sulfur atoms to the C1′ and C2′ positions of cPMP. This reaction is catalyzed by MPT synthase, a (αβ)2 heterotetrameric complex composed of two MoaD and two MoaE subunits. The sulfur atoms required for this reaction are present at the C-terminus of MoaD in form of a thiocarboxylate group. Studies on the reaction mechanism lead to a model in which two MoaD molecules are required for the sulfur insertion and cPMP is bound to MoaE during the reaction. Both subunits of the MoaE dimer core act independently, so that the two MoaD proteins are exchanged on each side of the dimer during the reaction. The first sulfur is added to the C2′ position cPMP by one MoaD-SH molecule, resulting in a hemisulfurated intermediate. This reaction is coupled to the hydrolysis of the cPMP cyclic phosphate group. In the hemisulfurated intermediate, the MoaD C-terminus is covalently linked via a thioester linkage. In the next reaction, the thioester is hydrolyzed by a water molecule, the first MoaD subunit dissociates from the MPT synthase complex  and a new MoaD-SH molecule associates with the complex. After the opening of the cyclic phosphate in the first sulfur transfer step to the C2′ position, the hemisulfurated intermediate is proposed to shift its location within MoaE protein, so that the C1′ position becomes more accessible to the attack by the second MoaD-SH. In the second sulfur transfer step, a covalent intermediate is formed with the new MoaD-SH protein. Further, MPT is formed by hydrolysis of this MoaD-thioester intermediate and release of MoaD.

For a new round of catalysis, a new thiocarboxylate group needs to be formed on MoaD. The activation of MoaD is catalyzed by the MoeB protein, which forms a (MoaD-MoeB)2 complex with MoaD. During the reaction, the C-terminus of MoaD is activated by the formation of an acyl-adenylate group at its terminal glycine. In this complex, MoaD-AMP receives the sulfur from a sulfurtransferase and MoaD-SH is formed. The sulfur is directly transferred to MoaD in the (MoaD-MoeB)2 complex, additionally releasing (MoeB)2 and AMP. MoaD-SH dissociates from the complex, and reassociates with MoaE to form the active MPT synthase heterotetramer (Fig. 1) . It was shown that in E. coli, L-cysteine serves as the origin of the MPT dithiolene sulfurs. In the sulfurtransfer reaction to MoaD, the proteins IscS and TusA are involved in E. coli, forming a sulfur relay system in which a persulfide sulfur is transferred from one protein to another. It is believed that under specific conditions TusA can be replaced by other sulfurtransferases in the cell, like the rhodanese-like protein YnjE or the L-cysteine desulfurase SufS. It has been proposed that the sulfur of one of these persulfide-containing proteins interacts with the (MoaD-MoeB)2 complex and attacks the MoaD-AMP bond, releasing AMP and creating a transient MoaD perthiocarboxylate intermediate with the sulfurtransferase, which is further reductively cleaved, finally releasing MoaD-SH.



The biosynthesis of the molybdenum cofactors. 
Shown is a scheme of the biosynthetic pathway for Moco biosynthesis. The central part shows the three conserved steps of Moco biosynthesis present in all organisms, the formation of cPMP, MPT and Mo-MPT. Unstable intermediates formed during the reactions are shown in brackets: 3,8-cH2GTP, the hemisulfurated MPT intermediate, MPT-AMP and bis-Mo-MPT. Bacteria contain a fourth step of Moco modification in which Mo-MPT is further modified by the addition of nucleotides, GMP or CMP. Additionally, Moco can be further modified by the replacement of one oxo ligand by a sulfido ligand, forming the mono-oxo Moco present in the xanthine oxidase family of molybdoenzymes. The SO family contains the Mo-MPT cofactor with a proteinogenic cysteine ligand. The DMSO reductase family of molybdoenzymes present only in bacteria binds the bis-MGD cofactor in which the molybdenum atom contains an additional ligand, which can be a cysteine, a selenocysteine, a serine, an aspartate or a hydroxo-ligand. Here, also a Moco sulfuration step exists, in which an oxo-ligand at the bis-MGD cofactor is replaced by a sulfur ligand. The proteins involved in the reactions are colored in red, and additional cosubstrates required for the reactions are colored in blue.

Third step: For the formation of Moco, molybdate is bound to the dithiolene sulfurs of MPT. The specific insertion of molybdenum into MPT was shown to be catalyzed by the joined action of the MoeA and MogA proteins. During the reaction, MogA thereby forms an MPT-AMP intermediate under ATP consumption, and this intermediate is further transferred to MoeA, which mediates molybdenum ligation at low concentrations of MoO42-. The end product of the MoeA and MogA reaction is Mo-MPT in a tri-oxo form, the basic form of the molybdenum cofactor which can be further modified by nucleotide addition in the next step. Alternatively, the Mo-MPT cofactor can be directly inserted into enzymes of the SO family, where Moco is coordinated by a cysteine ligand which is provided by the polypeptide chain of the protein, forming an MPT-MoVIO2 core in its oxidized state (Fig. 2).



Figure 2. The families of molybdoenzymes.
The basic form of Moco is a 5,6,7,8-tetrahydropyranopterin, named Mo-MPT, which coordinates the molybdenum atom by the characteristic dithiolene group at the C1′ and C2′ positions of the pyranopterin ring. Mo-MPT (shown in the tri-oxo structure; Reschke et al.2013) can be further modified and three different molybdenum-containing enzyme families are classified according to their coordination at the molybdenum atom: the XO, SO and DMSO reductase families. The SO family is characterized by a MPT-MoVIO2Cys ligand sphere. The XO family contains a MPT-MoVIOS(OH) core. Here, the MPT core can be modified by an additional CMP nucleotide at the phosphate group, forming MCD. The DMSO reductase family contains a MGD2-MoVIXY core with X being either a sulfur or an oxygen ligand and Y either being a hydroxo or amino acid ligand (Ser, Cys, Sec and Asp ligands were identified so far). Shown are structures of enzymes representative enzymes from each family: chicken sulfite oxidase (pdb 1SOX), bovine xanthine dehydrogenase (pdb 1FIQ) and S. massilia TMAO reductase (pdb 1TMO). The surface representations show that the Moco in each enzyme is deeply buried at the end of a funnel-like passage, giving access only to the substrate molecules (entrance site is shown by the arrow).

Fourth step: In the fourth step of Moco biosynthesis in bacteria, Mo-MPT can be further modified by the addition of a GMP or CMP to the terminal phosphate group. (A) The proteins of the DMSO reductase family in bacteria contain the bis-MGD cofactor (Fig. 2). The synthesis of the bis-MGD was shown to occur in a two-step reaction which requires Mo-MPT, MobA and Mg-GTP (Reschke et al.2013). In the first reaction, the bis-Mo-MPT intermediate is formed on MobA with Mo-MPT as substrate. For this reaction, the ligation of molybdenum to MPT is essential but no further cofactors or molecules are required (Temple and Rajagopalan 2000). In the second reaction, two GMP moieties from GTP are added to the C4′ phosphate of bis-Mo-MPT, forming the bis-MGD cofactor (Palmer et al.1996; Lake et al.2000) (Fig. 1). After the attachment of two GMP molecules to the bis-Mo-MPT intermediate, the bis-MGD cofactor is formed and released from MobA. Since bis-MGD is not stable in its free form, it is immediately bound by Moco-binding chaperones, which insert the cofactor specifically into target enzymes of the DMSO reductase family (see below).

(B) Enzymes of the XO family in some bacteria like E. coli contain the MCD form of Moco (Fig. 2). MCD formation is catalyzed by MocA, a protein that shares a high amino acid sequence identities to MobA. The overall catalytic reaction of MocA is similar to the second part of the reaction of MobA, in that it acts as a MPT CTP transferase and covalently links MPT and CMP with the concomitant release of the α- and γ-phosphates of CTP as pyrophosphate (Fig. 1). However, in this reaction MCD is the end product and the bis-Mo-MPT form is not formed. Instead, the MCD cofactor for all enzymes of the XO family is further modified and contains an equatorial sulfido ligand at its active site, which is essential for enzyme activity.

Molybdenum cofactor synthesis is linked to copper: a copper ion is bound to molybdopterin dithiolate sulfurs as an intermediate in the biosynthetic pathway (Kuper et al. 2004). 7 Its synthesis involves the insertion of molybdenum into molybdopterin by the Cnx1 G-domain. The identification of copper bound to the molybdopterin dithiolate sulphurs in Cnx1G, coupled with the observation that copper inhibited Cnx1G activity, suggests a link between molybdenum and copper metabolism, which would require cytoplasmic copper (Schwarz and Mendel 2006).

MoCo-binding Chaperones
The crystal structures of several molybdoenzymes revealed that Moco is deeply buried inside the proteins, at the end of a funnel-shaped passage giving access only to the substrate (Fig. 2). These structures suggested that chaperones might be required to facilitate the insertion of Moco into the target enzyme. Additionally, it was suggested that only after the insertion of Moco, the apo-enzymes adopt their final structure. It was also shown that Moco insertion is usually the final step of molybdoenzyme maturation, which occurs after protein folding, subunit assembly and the insertion of additional redox cofactors such as cytochromes, FeS clusters or flavin mono/dinucleotides. The Moco insertion step is catalyzed by Moco-binding molecular chaperones, which bind the respective Moco variant and insert it into the specific target molybdoenzyme (Fig. 3). It is suggested that most molybdoenzymes in bacteria, especially enzymes of the DMSO reductase family, have a specific chaperone for Moco insertion. Deletion of the chaperone gene generally leads to the loss of activity of the molybdoenzyme due to the lack of Moco insertion into the enzyme.


Figure 3. Chaperone-assisted Moco insertion into molybdoenzymes.
On the left side, the TorD/TorA system for bis-MGD insertion is shown: TorD binds bis-MGD and inserts the cofactor into apo-TorA. Shown are the structures of dimeric TorD from S. massilia (pdb 1N1C) and monomeric TorA from S. massilia (1TMO). 
In the middle, a model of the FdsC/FdsA system for insertion of sulfurated bis-MGD from R. capsulatus is shown. Rhodobacter capsulatus FdsC binds bis-MGD and further transfers it to the FdsA subunit of R. capsulatus FDH, which is composed of the (FdsGBA)2 heterotrimer. It is proposed that bis-MGD is further modified by sulfuration. For the homologous E. coli system, it was shown that IscS is involved in sulfurtransfer to bis-MGD. In R. capsulatus, the NifS4 protein performs a similar role for the XdhC/XdhB system. Here it is suggested that FdsC binds bis-MGD and an L-cysteine desulfurase (IscS/NifS4) transfers the sulfur to the Mo atom by exchanging an oxo-group and adding the sulfur ligand. Afterwards, sulfurated bis-MGD is inserted into FdsA, which is already assembled as a (FdsGBA)2 heterotrimer containing various FeS clusters and FMN. The crystal structure for the FdhD-homologous protein from Desulfotalea psychrophila is shown (pdb 2PW9). 
On the right-hand side, the XdhC/XdhB system for insertion of sulfurated Mo-MPT from R. capsulatus is shown. It was shown that XdhC binds Mo-MPT. The equatorial Mo=S ligand of Mo-MPT is inserted into Moco while bound to XdhC by the sulfurtransferase function of the NifS4 protein. After the formation of sulfurated Mo-MPT, XdhC interacts with XDH (here the XdhB subunit of R. capsulatus XDH is shown, pdb 1JRO) for final Moco insertion. The crystal structure for the XdhC-homologous protein from Bacillus halodurans is depicted (pdb 3ON5).

One well-studied example for the action of a molecular chaperone with its apo-enzyme is the TorD/TorA system for TMAO reduction in E. coli. TorD was shown to be the specific chaperone of TorA  and plays a direct role in the insertion of Moco into apoTorA. In E. coli, specific chaperones were also identified for nitrate reductase, DMSO reductase and FDH and their specific function was analyzed in detail: NarJ is the chaperone for nitrate reductase A NarGHI, NarW is the chaperone for nitrate reductase Z NarZYV, DmsD is the chaperone for DmsABC and YnfE/F , NapD is the chaperone for periplasmic nitrate reductase NapA and FdhD is the chaperone for FdhF . However, in the same host, no defined specific chaperone has been identified so far for the cytoplasmic molybdoenzyme BisC or the periplasmic molybdoenzyme TorZ. Crystal structures of several molybdoenzyme-specific chaperones were solved, and TorD, DmsD, NarJ and NapD chaperones were fully described. These dedicated chaperones are sometimes also called REMPs for redox enzyme maturation proteins.

Molecular chaperones were also identified for the XO family of molybdoenzymes. Here, the best characterized chaperone from this family is the Rhodobacter capsulatus XdhC protein which is essential for the maturation of R. capsulatus XDH (Neumann and Leimkühler 2011) (Fig. 3, Table 1).

The TorD family of Moco-binding chaperones
The TorD family of molecular chaperones was shown to contain hundreds of members that are mainly bacterial but also a few archaeal cytoplasmic proteins. Except YcdY, which in E. coli is linked to a zinc protein, these chaperones carry out essential roles in the biogenesis of both periplasmic and cytoplasmic molybdoenzymes from the DMSO reductase family. Thus, it was shown that for all studied chaperone–molybdoenzyme couple, the absence of the chaperone affects the stability or the activity of their target protein. In general, these Moco-binding chaperones are highly specific for their target molybdoenzyme. 

While the TorD-like proteins in general present a low level of sequence identity (20% or less), the members of this family are characterized by a common fold. Their 3D structures showed that they generally are organized in 10–12 α-helices that account for 60–70% of the protein residues. They contain in addition a long loop region separating the N- and C-terminal domains of the proteins. At first, a motif, ‘E(Q)PxDH’, held by the loop region was proposed to characterize members of the TorD family, but with the increase of available sequences it became clear that the sequence motif is not present in all species. It was established that these chaperones bind at least to one region of their target corresponding to the twin-arginine signal sequences of exported molybdoenzymes to the periplasm or N-terminal sequences of cytoplasmic ones. The maturation process of various bis-MGD containing enzymes has been reported. A common framework in the maturation mechanism has been suggested according to the complexity or the location of the molybdoenzymes; however, differences also appear.

Maturation of TorA, a simple periplasmic molybdoenzyme
The mechanism of TorA maturation by TorD has been studied in detail in the past. TorA is a bacterial respiratory enzyme catalyzing the reduction of TMAO into the volatile compound trimethylamine (TMA). It is a monomeric protein located in the periplasm and contains solely the bis-MGD cofactor as prosthetic group. Therefore, it is a well-defined model system to study the insertion of Moco without interferences linked to the assembly of subunits or the incorporation of other cofactors, e.g. iron–sulfur centers. TorA maturation is a cytoplasmic event which occurs before its translocation across the membrane. It is now established that TorD is involved in the stabilization and maturation of TorA  (Fig. 4).



Figure 4. Model for TorA and NarGHI maturation in E. coli.
TorD-dependent TorA maturation: by interacting with the leader peptide (LP) and the core of apoTorA, TorD protects it against proteolytic attack of the Lon protease and maintain the apoenzyme in a competent conformation for Moco insertion. TorD recruits the components involved in the final step of bis-MGD synthesis and transfers the mature cofactor to the catalytic site of TorA. Translocation of TorA is dependent on the TAT machinery, role of TorD in TorA targeting to the TAT components is not defined. NarJ-dependent NarGHI maturation: NarJ binds the N-terminal part and the core of NarG. NarJ is required to insert the iron–sulfur cluster and Moco into the NarG subunit. NarJ interacts with enzymes involved in Moco synthesis but a direct binding to bis-MGD was not shown yet. When NarG maturation is complete, the NarGHI complex is anchored to the membrane.
 
Maturation of membrane-associated complex molybdoenzymes
NarGHI is a membranous nitrate reductase responsible for anaerobic respiration when nitrate is abundant. The catalytic subunit NarG is facing the cytoplasm and contains the bis-MGD cofactor and also an iron–sulfur cluster named FS0 , NarH is an electron transfer unit containing four iron–sulfur centers and NarI contains a b-type cytochrome and permits the anchoring of the complex to the cytoplasmic membrane (Fig. 4). The molecular chaperone for NarGHI is the soluble protein NarJ. NarJ is required for the stability and the location of the complex and the insertion of Moco. Like TorD, the role of NarJ is mediated by its interaction with two distinct regions on NarG, the N terminal part and the core of the protein . The 1–15 N-terminal amino acids of NarG adopt an α-helical conformation in solution, which is recognized by NarJ via hydrophobic interactions. Moreover, the NarJ conformation is modified when interacting with NarG. The absence of NarJ leads to a lack of the cofactor in NarG, a defect in the assembly of the NarGHI complex which results in a soluble NarGH complex and an alteration of the b-type heme content of the membranous NarI cytochrome. Finally, it was also demonstrated that the maturation of the FS0 center must precede bis-MGD insertion into NarG. Thus, NarJ ensures complete maturation and anchoring of the cytoplasmic NarGH complex to NarI.

TorD interacts with two distinct regions of the Moco-free apoform of TorA, which encompasses the signal sequence at the N-terminal part of the protein and a binding site in the core of the apoprotein. This was confirmed by analysis of the TorA–TorD complex by SAXS analyses which showed a 1:1 binding stoichiometry of the two proteins (Dow et al.2013). Moreover, TorD is able to bind to both sites simultaneously. Recently, it was demonstrated that TorD binding to the core of apoTorA prevents proteolytic attack of the Lon protease and also the proteolysis of the N-terminal extremity by an additional, still unknown protease (Genest et al.2006a,b; Redelberger et al.2013). While the region of TorD involved in the recognition of the core of apoTorA was defined and corresponds to the fifth alpha helix in TorD structure, the region of TorD responsible for the protection of the apoTorA signal sequence is still controversial (Jack et al.2004; Genest et al.2008; Genest, Mejean and Iobbi-Nivol 2009). However, two residues (D124 and H125, E. coli numbering) located in the loop region were predicted to be involved in the binding of TorA leader sequence (Jack et al.2004; Hatzixanthis et al.2005). The TorD chaperone is also required for the insertion of the Moco into the catalytic site of TorA (Pommier et al.1998; Ilbert, Mejean and Iobbi-Nivol 2004) (Fig. 3). By binding to the core of the apoprotein, TorD induces a conformational change of apoTorA that becomes thus competent for Moco insertion. Further, TorD plays a role also in the last step of the Moco maturation by interacting with the MobA protein involved in bis-MGD formation (Genest et al.2008). Thus, the chaperone could act as a facilitator to insert the synthesized bis-MGD cofactor into the binding site of apoTorA and thereby render the mature enzyme protease resistant (Redelberger et al.2013). After bis-MGD insertion into apoTorA, mature TorA and release of TorD from the complex, TorA has to be targeted to the TAT machinery (Fig. 4). This step is facilitated by the fact that insertion of Moco into the catalytic site of TorA modifies the affinity between the interacting region of TorA and TorD allowing the release of TorD (Pommier et al.1998). This event can be considered as a proofreading mechanism directed by TorD or a passive competition between proteins of the TAT machinery and TorD, whose affinity for the signal sequence is thereby decreased (Jack et al.2004; Genest, Mejean and Iobbi-Nivol 2009). Consequently, the TAT leader peptide is exposed after TorD release and bis-MGD containing TorA can be targeted to the TAT machinery. Alternatively, a competition can occur between TorD and the TAT components leading to the release of TorD. These models remain to be proven.

Conclusions
Although their biosynthesis is a complex multistep process, molybdoproteins are ancient enzymes which are present in all kingdoms of living organisms. It has been shown that their maturation requires a precise pattern drawn almost specifically for each of them. This characteristic can surely be linked to both the variety of forms of the Moco that can be inserted and the diversity in the catalytic sites and localizations of these enzymes. The specific chaperones are a control point for the discrimination of the correct Moco to be inserted. Moreover, the way they handle the protection or the maturation process appears adapted to their proper target.

Importance of molybdoenzymes was already depicted in humans where the absence of Moco leads to a death in early childhood. In bacteria, it appears that the wide range of enzymatic activities catalyzed by these enzymes can be a great advantage for survival in many niches including polluted sites and an efficient adaptive mechanism. In an era submitted to ecological pressure to counteract anthropogenic discharges, bacterial molybdoenzymes can be surely adapted tools.

Parts in the cell required for the biosynthesis of Molybdenum cofactor synthesis
High-affinity ATP-binding cassette (ABC) transporter for molybdenum uptake
Copper
pterin
iron
ATP  
Moco-binding proteins
MoaA and MoaC
MPT synthase, a (αβ)2 heterotetrameric complex
MoaD
MoeA  
MogA 
bis-MGD cofactor
TorD/TorA system
periplasmic molybdate-binding protein (ModA)
transmembrane channel (ModB)
cytoplasmic protein (ModC)
Transmembrane protein, ModB
Molybdenum metabolism is strictly dependent on iron metabolism at different levels. FeMo-co biosynthesis and nitrogenase maturation are based on the synthesis of complex Fe–S clusters

1. http://www.imoa.info/HSE/environmental_data/molybdenum_trace_element.php
2. Molybdenum Cofactors and Their role in the Evolution of Metabolic Pathways , page 10
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278043/
4. http://www.ajsonline.org/content/305/6-8/467.abstract
5. http://www.nature.com/articles/srep00263
6. Prokaryotic Systems Biology page 215
7. Molecular microbiology of heavy metals, page 271
8. http://www.cell.com/current-biology/fulltext/S0960-9822(11)01079-7
9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650355/
10. http://www.nature.com/nature/journal/v460/n7257/full/nature08302.html
11. http://www.bbc.com/news/science-environment-23872765
12. https://www.sciencedaily.com/releases/2013/11/131125164814.htm
13. http://www.reasons.org/articles/vital-poisons
14. http://www.imoa.info/essentiality/molybdenum-essential-for-life.php
15. http://www.sciencedirect.com/science/article/pii/S0167488906001017
16. https://en.wikipedia.org/wiki/Molybdenum_cofactor
17. https://academic.oup.com/femsre/article-lookup/doi/10.1093/femsre/fuv043
18.http://www.checktheevidence.co.uk/Disclosure/PDF%20Documents/Directed%20Panspermia%20F.%20H.%20C.%20CRICK%20AND%20L.%20E.%20Orgel.pdf
19. http://www.sciencedirect.com.https.sci-hub.hk/science/article/pii/S0005272812010407
20. http://sci-hub.tw/https://www.nature.com/articles/nature08302

Further readings:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028170

Molybdenum cofactors, enzymes and pathways 1

The trace element molybdenum is essential for nearly all organisms and forms the catalytic centre of a large variety of enzymes such as nitrogenase, nitrate reductases, sulphite oxidase and xanthine oxidoreductases. There are two scaffolds holding molybdenum in place, the iron–molybdenum cofactor and pterinbased molybdenum cofactors. Despite the different structures and functions of molybdenum-dependent enzymes, there are important similarities, which we highlight here. The biosynthetic pathways leading to both types of cofactor have common mechanistic aspects relating to scaffold formation, metal activation and cofactor insertion into apoenzymes, and are ‘toolboxes’ to mediate additional cellular functions in eukaryotic metabolism.

Molybdenum is bioavailable as molybdate (MoO4 2−). Once molybdate enters the cell, it is subsequently incorporated by complex biosynthetic machineries into metal cofactors. One type of molybdenum cofactor is the iron–sulphur-cluster-based iron–molybdenum cofactor (FeMo-co) that is unique to the molybdenum nitrogenase6, with one [4Fe–3S] and one [Mo–3Fe–3S] partial cubane bridged by three sulphides and one μ6 central atom, X (which may be carbon, oxygen or nitrogen). The molybdenum of FeMo-co is further coordinated by homocitrate (Fig. 1a). The core structure of the other type of molybdenum cofactor (Moco) is a pterin-based cofactor (molybdopterin or metal-binding pterin (MPT)), with a C6-substituted pyrano ring, a terminal phosphate and a unique dithiolate group binding molybdenum8. The metal can be attached to one or two pterin moieties with additional terminal oxygen and sulphur ligands (Fig. 1c, d). Both cofactors are oxygen sensitive and very unstable outside their respective apoenzymes.

Molybdenum-dependent enzymes
On the basis of cofactor composition and catalytic function, molybdenum- dependent enzymes can be grouped into two categories: 

1.bacterial nitrogenases containing an FeMo-co in the active site, and 
2.pterin-based molybdenum enzymes. 

The second category is divided into three families, exemplified by 

sulphite oxidase 
xanthine oxidase and 
dimethyl sulphoxide reductase (DMSOR)

which each have a distinct activesite structure(Fig. 1). Tungsten-dependent formate dehydrogenase is classified as part of the DMSOR family, whereas aldehyde:ferredoxin oxidoreductases form a separate family of tungsten-cofactor (W-co) containing enzymes found only in archaea. 

Molybdenum nitrogenase
Nitrogenases provide the biochemical machinery for nucleotide-dependent reduction of dinitrogen (N2) to ammonia (NH3). The overall reaction catalysed by nitrogenases is usually depicted as N2 + 8H+ + 16MgATP + 8e− → 2NH3 + H2 + 16MgADP + 16Pi, where Pi denotes an inorganic phosphate. This reaction not only represents a major entry point of reduced nitrogen into the global nitrogen cycle, but also embodies the complex chemistry of breaking the triple bond of N2 under ambient conditions. Three homologous nitrogenase systems have been identified so far. The best-characterized molybdenum nitrogenase is a binary enzyme system comprising two redox-active metalloproteins (Fig. 2a).



Synthesis and Maturation of Molybdenum enzymes
The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity.  9 Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.

The transition element molybdenum is an essential micronutrient for microorganisms, plants, and animals. Surprisingly, molybdenum itself is catalytically inactive in biological systems until it is complexed by a special scaffold (2). One type of scaffold is the ubiquitous pterin-based molybdenum cofactor (Moco),2 which, in different variants, forms part of the active centers of all molybdenum enzymes in living organisms, except one. This exception is bacterial nitrogenase, which harbors the other type of cofactor, namely the Fe-S cluster-based FeMo cofactor, which is found only once in nature (for details, compare the minireview of Hu and Ribbe (62) in this thematic series). Molybdenum belongs to the group of trace elements, i.e. the organism needs it only in minute amounts; however, unavailability of molybdenum is lethal for the organism. Molybdenum is very abundant in the oceans in the form of the molybdate anion (3). In soils, the molybdate anion is the only form of molybdenum that is available for bacteria, plants, and fungi. More than 50 enzymes are known to be molybdenum-dependent. The vast majority of them are found in bacteria, whereas only seven have been identified in eukaryotes (4, 5). It is somewhat surprising that not all organisms need molybdenum. The commonly used eukaryotic model organism yeast plays no role in molybdenum research, as Saccharomyces cerevisiae does not contain either molybdenum enzymes or the Moco biosynthesis pathway. Also Schizosaccharomyces pombe does not use molybdenum, whereas Pichia pastoris needs molybdenum. Genome-wide database analyses revealed a significant number of bacteria and unicellular eukaryotes that do not need molybdenum, whereas all multicellular eukaryotes are dependent on molybdenum (6). In addition, mainly anaerobic archaea and some bacteria are molybdenum-independent, but they require tungsten for their growth (7). In the periodic table of elements, tungsten lies directly below molybdenum and features chemical properties similar to molybdenum.








Biosynthesis of FeMo-co 10
Assembly of nitrogenase FeMo-co is a considerable chemical feat because of its complexity and intricacy. Recent progress in the chemical synthesis of FeMo-co analogues has provided significant insights into this process. Elucidation of the biosynthesis of FeMo-co, on the other hand, is further complicated by the large ensemble of participating gene products. The exact functions of these gene products and the precise sequence of events in FeMo-co assembly have remained unclear until recently, when the characterization of a number of assembly-related intermediates afforded a better understanding of this biosynthetic ‘black box’ (Fig. 3).




Formation of the Fe–S core of FeMo-co
Assembly of FeMo-co is probably initiated by NifU and NifS, which mobilize iron and sulphur for the assembly of small Fe–S fragments.  NifS is a pyridoxal phosphate-dependent cysteine desulphurase and is responsible for the formation of a protein-bound cysteine persulphide that is subsequently donated to NifU for the sequential formation of [2Fe–2S] and [4Fe–4S] clusters  (Fig. 3). These small Fe–S clusters are then transferred to NifB and further processed into a large Fe–S core that possibly contains all the iron and sulphur necessary for the generation of a mature cofactor. The exact function of NifB in this process is unclear. Nevertheless, NifB is an indispensable constituent of FeMo-co biosynthesis, as deletion of nifB results in the generation of a cofactor-deficient MoFe-protein. Sequence analysis indicates that NifB contains a CXXXCXXC (where X is any amino acid) signature motif at the amino terminus, which is typical for a family of radical S-adenosyll- methionine (SAM)-dependent enzymes1,. In addition, there is an abundance of potential ligands in the NifB sequence that are available to coordinate the entire complement of iron atoms of FeMo-co. Thus, formation of the Fe–S core on NifB may represent a new synthetic route to bridged metal clusters that relies on radical chemistry at the SAM domain of NifB. For example, NifB could link two [4Fe–4S] subcubanes by inserting a sulphur atom along with the central atom, X, thereby building a fully complemented Fe–S core that could be rearranged later into the core structure of FeMo-co (Fig. 3).


Insertion of molybdenum into the Fe–S core on NifEN
The function of NifEN (NifE–NifN) as a scaffold protein for FeMo-co maturation was initially proposed on the basis of a significant degree of sequence homology between NifEN and the MoFe-protein, which has led to the hypothesis that NifEN contains a ‘P-cluster site’ that houses a P-cluster homologue and an ‘FeMo-co site’ that hosts the conversion of FeMo-co precursor to a mature cofactor1,. Whereas the P-cluster homologue in NifEN was identified earlier as a [4Fe–4S] cluster, a molybdenum-free precursor of FeMo-co was captured on NifEN only recently. Iron K-edge X-ray absorption spectroscopy reveals that this precursor closely resembles the Fe–S core of the mature FeMo-co despite slightly elongated interatomic distances40 (Fig. 3). This finding implies that, instead of being assembled by the previously postulated mechanism that involves the coupling of [4Fe–3S] and [Mo–3Fe–3S] subclusters, the FeMo-co is assembled by having the complete Fe–S core structure in place before the insertion of molybdenum. The precursor on NifEN can be converted, in vitro, to a fully complemented FeMo-co on incubation with Fe-protein, MgATP, molybdate and homocitrate41. Iron and molybdenum K-edge X-ray absorption spectroscopy reveals that the FeMo-co on NifEN is nearly identical in structure to the native cofactor in MoFe-protein, except for an asymmetric coordination of molybdenum that is probably due to the presence of a different ligand environment at the molybdenum end of the cofactor in NifEN41. Homocitrate is supplied by NifV (that is, homocitrate synthase) in vivo, but molybdenum mobilization within the cell that occurs before the intervention of Fe-protein remains a topic of debate.

Nevertheless, the fact that the cluster is completely converted before its exit from NifEN points to Fe-protein having a significant role in FeMoco maturation. Fe-protein re-isolated after incubation with molybdate, homocitrate
and MgATP is ‘loaded’ with molybdenum and homocitrate that can be subsequently inserted into the precursor on NifEN45. The molybdenum K-edge X-ray absorption spectrum of the loaded Fe-protein is consistent with a decreased number of Mo=O bonds (two or three instead of the four found in molybdate) as well as a decrease in the effective oxidation state of molybdenum due to either a change in the formal oxidation state of molybdenum or a change in molybdenum ligation. Interestingly, the electron paramagnetic resonance spectrum of loaded Fe-protein assumes a line shape intermediate between those of the MgADP- and MgATP-bound states of the Fe-protein. This observation is consistent with that from the initial crystallographic analysis of an ADP-bound form of Fe-protein, in which molybdenum is attached at a position that corresponds to the γ-phosphate of ATP. Such an ADP/ molybdenum-binding mode (Fig. 4a) may reflect the initial attachment of molybdenum to Fe-protein, particularly when the structural analogy between phosphate and molybdate is considered. Remarkably, similar nucleotide-assisted processes are proposed for the molybdenum insertion in pterin-based cofactors (see below; Fig. 4b).



Insertion of FeMo-co into apo-MoFe-protein
The completion of FeMo-co assembly on NifEN signals the delivery of FeMo-co to its destined location in MoFe-protein. The absolute requirement of intermediary FeMo-co carrier(s) between NifEN and MoFe-protein was precluded by the observations of unaffected nitrogen-fixing activity of the host after deletions of proposed carrierencoding gene(s) and direct FeMo-co transfer between NifEN and MoFe-protein on protein–protein interactions. Sequence comparison between NifEN and MoFe-protein reveals that certain residues that either provide a covalent ligand or tightly pack FeMo-co within the polypeptide matrix of MoFe-protein are not duplicated in the corresponding NifEN sequence. It is possible, therefore, that the respective cluster sites in NifEN and MoFe-protein are brought into close proximity, allowing the subsequent diffusion of FeMo-co from its biosynthetic site in NifEN (low-affinity site) to its binding site in MoFe-protein (high-affinity site). On its delivery to MoFe-protein, FeMo-co interacts with a number of MoFe-protein residues en route to its target location within the protein. Identification of these residues was assisted by the crystallographic analysis of a P-cluster-intact yet FeMo-co-deficient form of MoFe-protein, which contains a positively charged funnel in the α-subunit that is of sufficient size to accommodate the insertion of the negatively charged FeMo-co.

Biosynthesis of pterin-based molybdenum cofactors
Although widespread in all kingdoms, Moco is synthesized by a conserved biosynthetic pathway divided into four steps according to the biosynthetic intermediates: 

1.cyclic pyranopterin monophosphate (cPMP)
2.MPT 
3.MPT–AMP.

The biosynthetic pathway has been summarized in detail with particular focus on plants, bacteria and humans, and is believed to be very similar to W-co synthesis. In prokaryotes a final modification by a nucleotide can occur, whereas in MPT-type enzymes Moco maturation either involves a terminal sulphuration (xanthine oxidase family) or cysteine ligation to the apoenzyme (sulphite oxidase family).

Synthesis of the metal-binding pterin
Biosynthesis starts with the conversion of GTP into cPMP (previously identified as precursor Z) catalysed by two proteins: a radical SAM enzyme (for example MoaA in bacteria) harbouring two oxygen- sensitive [4Fe–4S] clusters, and an accessory hexameric protein involved in pyrophosphate release (for example MoaC in bacteria). MoaA harbours an N-terminal Fe–S cluster involved in radical SAM generation and a MoaA-specific C-terminal Fe–S cluster crucial for substrate binding. Although the reaction mechanism of cPMP synthesis is not yet fully understood, it is well established that each carbon of the ribose and purine is incorporated into cPMP. Furthermore, the structure of cPMP as a fully reduced pyranopterin with a terminal cyclic phosphate and geminal diol (Fig. 5) supports its physicochemical properties. With respect to the observed geminal diol, it remains to be determined at which point interconversion into a keto function takes place. The functions of MoaA and MoaC are conserved, as eukaryotic orthologues are able to restore Moco biosynthesis in bacteria.




To form the MPT dithiolate, two sulphur atoms are incorporated into cPMP by MPT synthase, a heterotetrameric complex of two small (MoaD in E. coli) and two large (MoaE in E. coli) subunits. MoaD carries a sulphur atom as thiocarboxylate at the conserved C-terminal double-glycine motif60, which is deeply buried in the large subunit to form the active site61. As one sulphur atom is bound per small subunit, a two-step mechanism for MPT dithiolate synthesis with the formation of a singly sulphurated intermediate has been demonstrated62. MPT synthase homologues in higher eukaryotes have been identified and characterized2. The expression of human MPT synthase is unusual, as both subunits are encoded by a bicistronic messenger RNA63. In a separate reaction, sulphur is transferred to the small subunit of MPT synthase (Fig. 5). For this, in E. coli MoeB catalyses the adenylylation of the C-terminal glycine residue of MoaD64 in a process that is notably similar to the action of the ubiquitin-activating enzyme Uba1. Together with MoaD, which has a ubiquitin-like fold, MPT synthase provides an  origin for ubiquitin-like protein conjugation. AMP-activated MoaD becomes sulphurated by sulphide transfer, which is catalysed by a cysteine desulfurase and a rhodanese; the latter is fused in eukaryotes, as the C-terminal domain, to an MoeB-homologous domain.


Metal insertion and nucleotide attachment
On completion of MPT synthesis, the metal is transferred by a multistep reaction. Whereas E. coli encodes two separate proteins involved in this step, eukaryotes catalyse metal transfer by homologous two-domain proteins, such as Cnx1 (plants) and gephyrin (human) (Fig. 5), pointing to a functional cooperation between their domains.

The physiological functions of their domains were discovered by determining the crystal structure of the N-terminal G domain of Cnx1 in complex with substrate and product69. The latter was found to be MPT–AMP, a common intermediate in bacterial and eukaryotic Moco synthesis synthesized by G domains and homologous proteins (MogA in bacteria). Subsequently, a transfer of MPT–AMP to the E domain in Cnx1 was demonstrated. In the presence of divalent cations and molybdate, bound MPT–AMP is hydrolysed and molybdenum is transferred to the MPT dithiolate, resulting in Moco release. This Moco most probably carries two oxo ligands and one OH group depicted (Figs 4a and 5) in a deprotonated form, as supported by preliminary spectroscopic data derived from a storage-protein-bound Moco (see below; G.S., unpublished observation). There is no experimental evidence for a reduction of molybdenum at this state. W-co biosynthesis is believed to be conserved up to MPT formation, with differences in metal transfer. The tungsten-dependent archaeon Pyrococcus furiosus and related thermophiles lack mogA; instead, they all express genes encoding an MoaB-like protein, which also catalyses MPT adenylylation, confirming MPT–AMP as an essential and general prerequisite before metal insertion. Furthermore, P. furiosus expresses two different MoeA-like proteins, suggesting metal-selective activities. Finally, enzymes of the DMSOR family need to be further modified by the attachment of a nucleotide molecule (Fig. 5), a reaction dependent on the preceding metal insertion72. In E. coli, MobA catalyses the conversion of MPT and GTP to Mo–bis-MGD74. Interaction studies with proteins catalysing metal insertion and Mo–bis-MGD formation identified a transient Moco-synthesizing machinery comprising MogA, MoeA, MobA and molybdenum-enzyme-specific chaperones.

Cofactor maturation, storage, and transfer
Molybdenum hydroxylases such as aldehyde oxidase and xanthine oxidase require a final step of maturation to gain enzymatic activity, namely the addition of a terminal sulphido ligand to the molybdenum centre, which is catalysed by a Moco sulphurase (that is, Aba3 in plants or HMCS (also known as MOCOS) in humans), a two-domain protein76 acting as a homodimer (Fig. 6). In a pyridoxal phosphatedependent manner, the N-terminal NifS-like domain abstracts sulphur from l-cysteine and forms a persulphide intermediate on a conserved cysteine residue77. Subsequently this sulphur is transferred via a second cysteine persulphide intermediate to bound Moco. Both of these steps are catalysed by the C-terminal Moco-binding domain of Aba3 (ref. 78), which selectively stabilizes sulphurated Moco. The same mechanism operates in HMCS (R.R.M., unpublished observations). Among prokaryotes, no homologues to eukaryotic Moco sulphurases have been found. However, for xanthine dehydrogenase from R. capsulatus, its enzyme-specific chaperone XdhC was found to fulfil Moco sulphuration. By contrast with enzymes of the xanthine oxidase family, sulphite oxidase and nitrate reductase incorporate Moco without further modification. The proposed tri-oxo coordination of molybdenum in mature Moco (Figs 4b and 5) suggests a simple mechanism of cysteine ligation to the molybdenum accompanied by loss of one of the oxygens as water. As Moco is highly unstable once liberated from proteins, it was assumed that Moco does not occur in a ‘free state’; rather, Moco might be bound to a carrier protein that protects and stores it until further use. Whereas some bacteria have molybdate-binding proteins such as Mop, the alga Chlamydomonas reinhardtii produces a homotetrameric protein that holds four Moco molecules in a surface-exposed binding pocket. In higher plants, gene families with 8–12 homologous Moco-binding proteins have been discovered recently (R.R.M., unpublished observations). It is still unclear whether these proteins represent a buffer in which to store Moco or whether they are part of the default pathway for Moco allocation and insertion into molybdenum enzymes, a mechanism poorly understood in eukaryotes. Because Moco is deeply buried within the holoenzymes, it needs to be incorporated before the completion of folding and oligomerization of enzyme subunits/ domains; for this, many bacterial molybdenum enzymes require the presence of chaperones, such as NarJ for E. coli nitrate reductase, TorD for trimethylamine N-oxide reductase and DmsD for DMSOR, which bind and protect the apoenzymes, assist in cofactor insertion and control transmembrane targeting.

Molybdenum homeostasis and disorders
Cellular uptake
Bacterial molybdenum uptake requires specific systems to scavenge molybdate in the presence of competing anions. This involves a high-affinity ATP-binding cassette (ABC) transporter: molybdate is captured by one component, a periplasmic molybdate-binding protein (ModA), and transferred to another, the transmembrane channel (ModB). The crystal structure of an ABC transporter from Archaeoglobus fulgidus suggests a conserved two-state mechanism by which ATP hydrolysis and the release of ADP plus Pi at the cytoplasmic protein (ModC) controls conformation of the transmembrane protein, ModB. For tungstate, two ABC-type transporters, TupA– TupB–TupC and WtpA–WtpB–WtpC, have been identified, the latter being highly selective for tungstate over molybdate owing to a unique octahedral substrate coordination. Algae and multicellular plants are the only eukaryotes for which the molybdate-uptake mechanisms have been recently determined. Two proteins belonging to the large sulphatecarrier family have been shown to transport molybdate with high affinity. Unexpectedly, none of them was found to reside in the plasma membrane. Contradictory reports localized them to the endomembrane system or the mitochondrial envelope. It is likely that additional transporters, not only in autotrophs but also in animals, will be discovered soon.

Molybdenum–iron and –copper crosstalk
Molybdenum metabolism is strictly dependent on iron metabolism at different levels. FeMo-co biosynthesis and nitrogenase maturation are based on the synthesis of complex Fe–S clusters, and enzymes participating in the first step of Moco biosynthesis contain two [4Fe–4S] clusters. Furthermore, all molybdenum hydroxylases and several members of the DMSOR family use Fe–S clusters for intramolecular electron transfer.  Recently, another link between the metabolic pathways of molybdenum and iron was discovered. In plants (and most probably also in animals), enzymes catalysing cPMP synthesis, such as Cnx2 and Cnx3, were localized within the mitochondrial matrix, which necessitates the export of cPMP from mitochondria into the cytosol. Here, the mitochondrial ABC-type transporter Atm3 (also known as Sta1) from A. thaliana seems to fulfil a dual function: it not only exports Fe–S-cluster precursors to the cytosol, but it is somehow also involved in cPMP translocation. Atm3-deficient plants showed defects in Fe–S-dependent cytosolic enzymes and accumulated large amounts of cPMP in mitochondria; consequently, activities of all molybdenum enzymes were strongly reduced . Only a few cases and conditions of limited molybdate availability have been reported so far. Among these, the shortage of molybdenum in Australian farmland triggered excessive fertilization, resulting in molybdenum overload of the soil that caused pathological symptoms of molybdenosis in animals; this, in particular in ruminants, triggered secondary copper deficiency. Later, these molybdenum-induced conditions of copper deficiency revealed the pathology of two copper-homeostasis disorders: Menkes disease (copper deficiency) and Wilson’s disease (copper overload). Consequently, potent copper chelators such as tetrathiomolybdates were used to treat Wilson’s disease and a number of other disorders that are linked to copper homeostasis, such as neurodegeneration, cancer and inflammation. Another antagonism between molybdenum and copper has been found recently. The crystal structure of Cnx1G, which catalyses MPT adenylylation, revealed the presence of a covalently bound copper ion (most probably Cu1+) at the MPT dithiolate in both the substrate- and product-bound states. The function of copper during Moco biosynthesis is still unknown. It may participate in the sulphur-transfer reaction enabled by MPT synthase, act as a protecting group for MPT and/ or function within molybdenum insertion. In vitro studies suggested a competition between copper and molybdenum during Moco formation, ultimately raising the question of whether Moco biosynthesis might be affected under conditions of copper overload or deficiency.


1. http://www.nature.com/nature/journal/v460/n7257/full/nature08302.html
2. Engineering Novel Metalloproteins: Design of Metal-Binding Sites into Native Protein Scaffolds

Engineering Novel Metalloproteins: Design of Metal-Binding Sites into Native Protein Scaffolds 2

Two Steps of Protein Design
Proteins play an essential role in biology. Whether it is in catalysis or molecular recognition, proteins set a golden standard of efficiency and selectivity that few other natural or artificial molecules can match. For this reason, the study of protein structure and function has been the focus of many years of research. Given the amount of time and effort devoted to this endeavor and the accomplishments achieved, it is fair to ask how much we have learned from these studies. The best way to test our knowledge is to design a protein with a desired structure and function. The protein design process can be divided into two steps: the design of overall scaffold and the design of active sites (Figure 1).



All enzymes that depend on molybdenum catalyse redox reactions by taking advantage of the versatile redox chemistry of the metal, which is controlled by the cofactor itself and the enzyme environment3. Within the enzyme, molybdenum shuttles between three oxidation states (+4, +5 and +6), thereby catalysing two-electron reduction–oxidation (redox) reactions. In most cases, regeneration of the active site involves single-electron steps, resulting in a paramagnetic molybdenum intermediate. Molybdenum enzymes are found in nearly all organisms. In nature, two very different systems exist to control the redox state and catalytic power of molybdenum, which functions as an efficient catalyst in oxygen-transfer reactions. In either case, at least three sulphur and two oxygen atoms form ligands to molybdenum (Fig. 1).

Molybdenum Uptake in Bacteria

In Bacteria, molybdenum is transported into the cytoplasm in the form of the oxyanion molybdate (MoO42−), which is taken up through the membrane by highaffinity ABC-type transporters.

To get life, one thing you need, and is life essential, are Molybdenum Co-factors. The get them, you need 1. a fine tuned strong atomic force 2. Molybden 3. Iron 4. Sulfur. The elements to make the cell membrane, that is to say:phospholipids, glycolipids, and sterols. 5. Molybden co-factors 6. FE/S clusters for maturation of Molybdenum cofactors (Moco) 7. The cell membrane and all functions and biosynthesis pathways to make it 8. sulfur import proteins and 9. the assembly line to make them 10. siderophore import proteins and 11. the assembly lines to make them 12. Siderophores, and the 13. Assembly lines for siderophore biosynthesis and all proteins involved 14 the genetic information to make all of it. With that, you have only ONE of several other co-factors required for the beginning of life. If one of these is missing, no life.

Transport of molybdates into the cell

Many bacteria and Archaea actively take up molybdate when its concentration in the environment is low. 8 An ATP-binding cassette (ABC) transporter performs this high-affinity uptake. ABC transporters are arguably the most important molecular pumps that transport small solute molecules into and out of prokaryotic and eukaryotic cells. They consist of functionally distinct protein components that are usually separate proteins in prokaryotes. Specific substrate-binding proteins capture their cognate solute molecules and transfer them to a protein channel in the cell membrane. Transport through the membrane involves conformational changes, which are driven by ATP hydrolysis in an ATPase that is associated with the cytoplasmic side of a membrane-spanning protein. The names ModA, ModB and ModC are usually given, respectively, to the substrate-binding protein, the transmembrane protein, and the ATP-binding protein of the molybdate-transport system.

Acquisition of molybdenum by prokaryotes involves transporting molybdate into the cell from the surrounding medium. It is considered that a low-affinity transport system with a Km of 300 μM functions at molybdate concentrations greater than 10μM  In several bacteria, it has been established that there is a high-affinity transport system for molybdate. The periplasmic binding protein, ModA, does not bind arsenate, sulfate, phosphate, nitrate, selenate, or chlorate. On the inner side of the plasma membrane is a peripheral membrane protein that binds ATP. With ATP hydrolysis, molybdate is transported into the cytoplasm where, in excess, it binds to repressor proteins that are soluble in the cytoplasm. 5

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. 6 ABC2 importers bring vital substrates across the cellular membrane into the cytoplasm. These transporters play a critical role in nutrient and metabolite uptake, transporting a variety of nutrients including peptides, metals, ions, and amino acids necessary for viability. All ABC importers have similar architecture: the transmembrane domain that forms the translocation pathway and the nucleotide binding domain that binds and hydrolyzes ATP, driving the active transport of substrate through the transmembrane domains.  For ABC importers, substrate specificity is facilitated by the substrate binding protein that binds to and delivers substrate to its cognate transporter

A structural classification of substrate-binding proteins 7
20 April 2010
Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The pro-teins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven trans-porters, DNA-binding proteins, as well as channels and receptors from both pro- and eukaryotes. Substrate-binding proteins (SBPs), and substrate-binding do-mains (SBDs), form a class of proteins (and protein domains) thatare often associated with membrane protein complexes for trans-port or signal transduction. SBPs were originally found to be asso-ciated with prokaryotic ATP-binding cassette (ABC)-transporters but have more recently been shown to be part of othermembrane protein complexes as well.

The protein ModA of the molybdate transporter is the first protein ligand for molybdate. It is present in the periplasm of Gram-negative bacteria. In Grampositive bacteria, such as Staphylococcus carnosa, which have only a single
membrane and no periplasmic region, ModA is attached to the outer side of the membrane by an N-terminal lipid anchor. ModA enables the cell to select molybdate in the presence of competing anions. Only molybdate
and tungstate bind to E. coli ModA. The anions vanadate, sulfate, phosphate, arsenate, nitrate, chlorate, selenate, perchlorate, and permanganate do not bind. Tungstate, but neither vanadate nor sulfate, competes for radioactive molybdate transported into A. vinelandii and An. variabilis. 

In addition to the binding protein, ModA, the molybdate transporter consists of an integral membrane protein or permease, and its associated ATP-binding protein. ModC has a domain of about 200-240 amino acids that is highly conserved from bacteria to man. This domain belongs to the large AAA protein-domain family (ATPases Associated with diverse cellular Activities) that is involved in effecting molecular movements. 

Molybdate must be able to enter cells by at least two pathways; a high-affinity system that functions at low molybdate concentrations, and a system or systems that operate at high concentrations. Wildtype cells use the low-affinity route, when high molybdate concentrations repress expression of the high-affinity transport system.

The iron-molybdenum cofactor (the M-cluster) serves as the active site of molybdenum nitrogenase.  1 Molybdate enters the bacterium via a high‐affinity transporter system which comprises three proteins (ModA, ModB and ModC) 3 The transport of molybdate has been characterized best in Escherichia coli, which has a high-affinity ABC transport system for molybdate. ModA is the periplasmic Mo-binding protein, ModB is the membrane-spanning component, and ModC provides the energizer function on the cytoplasmic side of the membrane. The diazotrophic bacteria, Rhodobacter capsulatus and Azotobacter vinelandii, have a similar high-affinity molybdate-transport system with homologues of the genes found in E. coli. Little is known about the mechanism of molybdate transport in cyanobacteria. However, in A. variabilis molybdate at very low concentrations represses V-nitrogenase, suggesting that there is a high-affinity molybdate-transport system. We demonstrate here that A. variabilis has a molybdate-transport system that appears similar to those of E. coli and A. vinelandii. 2


ABC transporter Overall structure. 
Front view of the ModB2C2A complex in ribbon representation, with the ModB subunits coloured yellow and blue, the ModC subunits coloured green and magenta, the binding protein ModA coloured red, and with bound tungstate in van der Waals representation (yellow and blue spheres). The grey box depicts the probable location of the lipid bilayer on the basis of the hydrophobicity of the protein surface. N, amino terminus; C, carboxy terminus. Note that there is a vertical two-fold molecular and non-crystallographic symmetry axis for ModB2C2. 4

ATP-binding cassette (ABC) transporter proteins carry diverse substrates across cell membranes.  Bacterial ABC importers are essential for the uptake of nutrients , including rare elements such as molybdenum. A detailed understanding of their mechanisms requires direct visualization at high resolution and in distinct conformations. Our recent structure of the multidrug ABC exporter Sav1866 has revealed an outward-facing conformation of the transmembrane domains coupled to a closed conformation of the nucleotide-binding domains, reflecting the ATP-bound state . Here we present the 3.1 A˚ crystal structure of a putative molybdate transporter (ModB2C2) from Archaeoglobus fulgidus in complex with its binding protein (ModA). Twelve transmembrane helices of the ModB subunits provide an inward-facing conformation, with a closed gate near the external membrane boundary. The ATP-hydrolysing ModC subunits reveal a nucleotide-free, open conformation, whereas the attached binding protein aligns the substrate-binding cleft with the entrance to the presumed translocation pathway. Structural comparison of ModB2C2A with Sav1866 suggests a common alternating access and release mechanism, with binding of ATP promoting an outward-facing conformation and dissociation of the hydrolysis products promoting an inward-facing conformation.

ABC transporters consist of two transmembrane domains (TMDs) that provide a translocation pathway, and two cytoplasmic nucleotide-binding domains (NBDs) that hydrolyse ATP and drive the transport reaction. The NBDs contain highly conserved motifs6 , whereas the TMDs have distinct primary sequences and even vary in the number of transmembrane helices. Although exporters feature a conserved core of 12 transmembrane helices, ABC importers contain between 10 and 20 transmembrane helices, depending on the mass and the chemical nature of the substrates they translocate. Also, the substrates of ABC exporters enter the translocation pathway from the cytoplasm or from the lipid bilayer, whereas ABC importers require a binding protein that captures the solutes and feeds them to the external face of the transporter.

With the structures of four ABC transporters available, a mechanistic scheme emerges that rationalizes ATP-driven transport. The key to unidirectional transport seems to be the ATP-driven conversion of the inward-facing state.  . Binding of two ATP molecules at the interface of the NBDs closes the gap between the conserved ATP-binding motifs. As this gap closes, so does the distance between the attached coupling helices, causing the TMDs to flip from the inward-facing to the outward-facing conformation.


Nucleotide-binding domains (NBD's) conformations and interfaces. 
a, Schematic summarizing the conformational changes observed by superimposing the NBDs of Sav1866 and A. fulgidus ModC. The light grey and dark grey areas represent individual NBDs, and the helical and the RecA-like subdomains are indicated. The upper panels represent a bottom view and the lower panels a side view of the two NBDs. The ATP-free state is depicted on the left, whereas the ATP-bound state is shown on the right. Red and yellow lines indicate the conserved P-loops (consensus sequence GxxGxGKST) and LSGGQ motifs. The hook-like features represent the C termini of the NBDs as observed in ModC, suggesting permanent contact of the NBDs throughout the reaction cycle. 
b, The architecturally conserved coupling helix of ABC transporters. A single ModC subunit in ribbon representation is shown with the RecA-like subdomains and helical subdomains coloured green and cyan, respectively. Segments corresponding to the P-loop and the LSGGQ motif are coloured red and light yellow, respectively. The ‘coupling helix’ (helix 4a) of ModB is indicated in dark yellow ribbon representation, with the Ca position of the conserved Gly 166 depicted as an orange sphere. After superposition of the NBDs, the analogous coupling helices of Sav1866, BtuCD and HI1470/71 superimpose well with that of ModB, but for clarity only that of Sav1866 is depicted in black. N and C reflect the amino and carboxy termini of the polypeptide stretches that form the coupling helices.


1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650356/
2. http://sci-hub.tw/https://link.springer.com/article/10.1007/s00203-002-0499-y
3. http://emboj.embopress.org/content/18/6/1435.figures-only
4. http://sci-hub.tw/https://www.nature.com/articles/nature05626
5. Structural and Functional Relationships in Prokaryotes, page 506
6. http://www.jbc.org/content/289/21/15005.full
7. https://febs.onlinelibrary.wiley.com/doi/epdf/10.1016/j.febslet.2010.04.043
8. Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria, page 242