Proteins with molybdenum clusters, essential for life

Proteins with molybdenum clusters, essential for life

https://reasonandscience.catsboard.com/t2910-proteins-with-molybdenum-clusters-essential-for-life

Why, of all obscure elements known to man, would molybdenum be necessary for life? The biological versatility of Mo and W result not only from their redox-activity, ranging through oxidation states VI to IV, but because the
intermediate V valence state is also accessible, they can act as interfaces between one- and two-electron redox systems, which allows them to catalyze hydroxylation of carbon atoms using water as the ultimate source of oxygen.

Another factor which characterizes Mo (and W) enzymes is that, with the exception of bacterial nitrogenase, instead of having the metal itself directly coordinated to amino acid side chains of the protein, they contain a molybdenum pyranopterindithiolate cofactor (MoCo), which is the active component of their catalytic site. This cofactor coordinates the metal ion via a dithiolate side chain. The MoCo cofactor can exist in the fully oxidised (Mo(VI)) and fully reduced (Mo(IV)) forms, with some enzymes generating the (Mo(V)) form as a catalytic intermediate 17

The answer lies with the electrons that whirl around the molybdenum core. Like many other metals, molybdenum is eager to take an extra electron under its wings or give a spare one away. Life has eagerly exploited this ability to juggle electrons around. Cells not only incorporate molybdenum ions into their enzymes, but also zinc, copper, iron and nickel. Many of these metal containing proteins shuttle electrons between molecules, as if they are playing a massive game of hot potato, changing, breaking and building molecules along the way. Electrons truly are what makes life go round. Or, as the Hungarian Nobel prize winner Albert Szent-Györgyi put it: "Life is nothing but an electron looking for a place to rest". 15

Molybdenum is needed for at least three enzymes.
Sulfite oxidase catalyses the oxidation of sulfite to sulfate, necessary for metabolism of sulfur amino acids. 16
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.

LUCA was supposedly an anaerobe, as long predicted by microbiologists. Its metabolism was replete with O2-sensitive enzymes. These include proteins rich in O2-sensitive iron-sulfur (FeS) clusters and enzymes that entail the generation of radicals (unpaired electrons) via S-adenosyl methionine (SAM) in their reaction mechanisms. That fits well with the 50-year-old but still modern view that FeS clusters represent very ancient cofactors in metabolism. 13

The Molybdo-enzyme superfamily as a whole, appear to have existed in LUCA. 14 A vast number of enzymes rely on metal cofactors for catalysis and/or redox conversions. 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.

Numerous forms of life (including man) do not use proteins/enzymes with Tungsten metal atoms (W), and at least some species (Pyrococcales) do not use Molybdenum ( Mo) , but a cell that does not use either one is yet to be found. 9 Molybdenum (Mo) is a transition metal that plays an essential role in metabolism in the three domains of life. It is a trace element; living beings require it in small doses. The trace element molybdenum (Mo) is the catalytic component of important enzymes involved in global nitrogen, sulfur, and carbon metabolism in both prokaryotes and eukaryotes. 4 The trace element molybdenum (Mo) plays a critical role in several metabolic pathways and functions as a catalytic component of certain metalloenzymes that are essential for nearly all living organisms, including animals, plants, fungi and bacteria. In spite of that scarcity, molybdenum is essential to most organisms, from archaea and bacteria to higher plants and mammals, being found in the active site of enzymes that catalyze oxidation–reduction reactions involving carbon, nitrogen and sulfur atoms of key metabolites.  Some of the molybdenum-dependent reactions constitute key steps in the global biogeochemical cycles of carbon, nitrogen, sulfur and oxygen, with particular emphasis on the atmospheric dinitrogen fixation (reduction) into organic ammonium (nitrogen cycle/nitrogenase enzyme).

While bacteria contain enzymes of all three enzyme families, enzymes of the DMSO reductase family are not present in eukaryotes. A significant number of bacteria and unicellular eukaryotes that do not require molybdenum, while all multi-cellular eukaryotes contain molybdoenzymes, which are essential for their viability. 

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. They are required for respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. In the first known biochemical reactions on earth, molybdenum and iron-sulfur (Fe-S) clusters enabled electron transfers turning inorganic molecules into hydrogenated carbon molecules. 1 The majority of the  Molybdo-enzyme superfamily as a whole, appear to have existed in LUCA. 2  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.

The presence of the CISM superfamily in LUCA implies a vital role of its metal cofactors in early life. Mo's insolubility at neutral pH values, exacerbated by an anoxic atmosphere, suggested a low bioavailability of this element for early life. Certainly tungsten and most likely molybdenum ought to be added to the list of metals vital already to earliest life on Earth.

The only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. 3  

Involvement of molybdenum in purine metabolism is common to virtually all forms of life and only a small number of organisms use other mechanisms to oxidize xanthine (e.g. some yeasts), thus confirming the essential role of molybdenum for life on Earth. Molybdoenzymes are widespread in all domains of life and they catalyze key steps in carbon, sulfur and nitrogen metabolism.6 The physiological role of molybdoenzymes is fundamental since they are essential for most organisms and play a crucial role in fundamental biogeochemical cycles.

Mo-containing enzymes hold key positions both in the biogeochemical redox cycles of carbon, nitrogen and sulfur on Earth and in the metabolism of every the individual organism. 11

Molybdenum and Tungsten Enzymes Carola Schulzke, Martin L. Kirk, Russ Hille, page 6:
Both molybdo- and tungstoenzymes probably existed in the last universal common ancestor (LUCA).

There is a discrepancy of information, since in the coming text below, the authors claim that Molybden enzymes did substitute Tungsten enzymes at the great oxygenation event. What is it ?

The two cofactors that hold the metals in the enzymes active site would also have to have been present. This is particularly remarkable when we realize how elaborated the two cofactors are (particularly the nitrogenase one) and how “limited” their utilization compared to, for instance, porphyrin-related structures. Why do living organisms expend so much effort to use these metals in a (comparatively) small number of reactions? This effort (including synthesizing the protein machinery to scavenge the metals from the environment, producing and inserting the specialized cofactors and regulating the whole process) underscores how important both metals would have been, and still are to extant organisms, particularly in the case of molybdenum.

Another important aspect of molybdenum in biology can be seen in sulfite- oxidizing enzymes, which are used by almost all forms of life in the catabolism of sulfur-containing amino acids and other sulfur-containing compounds, oxidizing sulfite to sulfate.

More than 50 molybdenum-containing enzymes are known. The great majority are prokaryotic, with eukaryotes holding only restricted number of molybdoenzymes. When we think about the elements that are essential for life on Earth, we hardly ever consider molybdenum. The biological role of molybdenum can only be appreciated when put in perspective. Nitrogen is the fourth most abundant element in living organisms (only behind hydrogen, oxygen and carbon) and life on Earth depends on the nitrogen biogeochemical cycle to keep this element in forms that can be used by the organisms.Noteworthy, the “closing” of the nitrogen cycle, with the atmospheric dinitrogen fixation into ammonium depends virtually entirely on the trace element molybdenum : nitrogenase, a prokaryotic enzyme responsible for dinitrogen reduction to ammonium, requires one molybdenum atom in its active site. 



The biologically available form of Mo is the oxyanion molybdate (MoO42−) from which organisms take up Mo along their daily life. Although only a minor constituent of the Earth’s crust, Mo is readily available due to its presence as a trace element in aquatic environments (as molybdate, MoO24−).   The metal itself is biologically inactive unless it is complexed by a special cofactor. Mo is bound to a pterin, thus forming the molybdenum cofactor (Moco) which is the active compound at the catalytic site of all other Mo-enzymes.

The choice of molybdenum versus tungsten
Mo's insolubility at neutral pH values, exacerbated by an anoxic atmosphere1, suggested a low bioavailability of this element for early life1,3. Mo-isotope analyses on samples from the Archaean era indeed show substantially lower levels than during Phanerozoic times 22

Two scenarios can reconcile the results of molecular phylogeny and paleogeochemistry. 
1. The ancestral CISM enzyme exclusively used W which was later replaced by Mo.
2. CISM-catalyzed reactions in early life used Mo supplied by alkaline hydrothermal vents, proposed as cradles for life23. The exclusiveness for Mo of many CISM-members as well as findings that primary productivity involving Mo has been comparable to the present since the geological record began at 3.8 Ga 24 lead us to favor the second scenario.

In oxic aqueous systems, these metals exist in the form of their tetrahedral MoO42− or WO42− oxyanions, and are therefore easily mobilizable into enzyme systems with the important caveat that they must be distinguished during enzyme maturation to ensure proper redox function and therefore catalysis. It is claimed that prior to approximately 2.3 billion years ago the earth’s biosphere was essentially anoxic, high in sulfur, and highly reducing.Mo and W have broadly equivalent natural abundance in the earth’s crust, but sulfides of W are soluble in water whereas those of Mo are not. 

Most microbes cannot distinguish tungstate from molybdate, and substitution of one for the other usually affects enzymic activity. Tungstate similarly inactivates many enzymes with molybdopterin cofactors. In a few cases, molybdenum replaces tungsten functionally and vice versa. For example, tungsten can replace molybdenum in the catalytic center of Rhodobacter capsulatus dimethylsufoxide reductase to give an active enzyme that accesses the same range of oxidation states as the molybdenum enzyme 10 Tungstoenzymes predominantly occur in thermophilic and hyperthermophilic organisms in the specialised niche of oceanic hydrothermal vents

Moco is very labile and sensitive to air oxidation. 

As a result,W would have been bioavailable in the primordial earth’s biosphere whereas Mo would not. Following the emergence of biological water oxidation, photosynthetic organisms supposedly caused a dramatic increase in ambient redox potential that paralleled the increase in atmospheric oxygen, and this resulted in the appearance and bioavailability of Mo in the form of MoO42−. As a consequence, the bioavailability of the two elements were reversed, with Mo being present at a concentration in sea water at 100 times that of W. It is claimed that this idea is supported by the significant correlation between archaeal life and W-biochemistry. The name archaeon (previously: archaebacterium) is of course intended to transmit the notion that these forms of life are thought to be the most similar to ‘primitive’ life as it must have existed in early geological times not long after the appearance of the first living cell. Most archaea are anaerobes (or perhaps microaerophiles) and the link may simply be one of mutual exclusion of molecular oxygen and tungsten biochemistry.

Both elements are able to assemble into mononuclear molybdoenzymes in an essentially identical manner. This presents a critical biological problem, because redox reactions catalyzed by W typically occur at much lower potentials than those catalyzed by Mo, and the active sites of the enzymes have to modulate the redox properties of their cognate metal ion. Thus, assembly of W in place of Mo in a bona fide molybdoenzyme would elicit dramatic changes in catalytic efficacy.

The toxicity of W towards molybdoenzymes and the inferred toxicity of Mo towards tungstoenzymes present cells with a serious problem: toxicity of the antagonist oxyanion via incorrect metal insertion. Mo and W have the same atomic radii (1.75 ˚A and 1.78A˚ , respectively), the same electronegativity, the same free energy of solvation (−226.8 kcal mol−1 and −230.1 kcal mol−1, respectively) and the same covalent solution radii (2.75 ˚A and 2.83A˚ , respectively). In order to distinguish them, exquisitely discriminating systems are necessary at the levels of metal uptake into bacterial cells.

Molybdo and tungsto-enzymes coexist in some bacteria, which must be able to discriminate between the two anions. 

In some microorganisms (mostly thermophilic archaea), tungsten (W) is also coordinated by pyranopterin (Wco). W can still today be selectively transported into prokaryotic cells by certain transporters. Some organisms can distinguish between tungstate and molybdate. In the thermophilic methanogenic Archaea Methanobacterium wolfei and M. thermoautotrophicum, the enzyme formylmethanofuran dehydrogenase catalyzes the first step in methane formation from carbon dioxide. Both organisms synthesise distinct tungsten- and molybdenum-containing enzymes. Expression of the tungstoenzyme is constitutive. Molybdate induces expression of the molybdoenzyme, whereas tungstate does not. Some organisms have a high-affinity ABC transport system that specifically transports tungstate in the presence of molybdate. In the Gram-positive anaerobe Eubacterium acidaminophilum, which contains at least two tungstoenzymes, an equimolar amount of molybdate does not affect the transport of tungstate.

The basis for this extraordinary discrimination between tungstate and molybdate is not yet known, but it could depend on differences in pK between molybdate and tungstate, as the latter is more basic.

In order to understand, how the transition from Tungsten to Molybdenum could have ocurred, it has to be elucidated how the two metals are recognized and imported into the Cell.

The bioinorganic chemistry of tungsten 5
Tungsten is the bioelement with the highest atomic number,74. Tungsten is widely distributed in biology, however, it is not a universal bioelement. For some species tungsten is essential: their life depends on the presence of the element.  Molybdenum is in many ways the twin element of tungsten. Also in biology the coordination chemistries of W and Mo are similar in structural and functional aspects. It has been suggested that in an evolutionary sense tungsten is an ‘old’ element on its way to be replaced by ‘modern’ molybdenum. 



The structure of tungsten-containing pterin b cofactors and intermediates: tungsten-containing metal binding pterin (W-MPT) (I), tungsto-bispterin (W-bis- MPT) (II), tungsto-bispterin guanine dinucleotide (W-bis-MGD) (III)

In general, the biochemistry of a metal in a monocellular organism encompasses several processes: sequestering and transport over the cytoplasmic membrane, storage and release, metal-cofactor biosynthesis, metalloenzyme catalysis, and metal-controlled regulation of transcription and/or translation.


A schematic overview of the stages in cellular metabolism of tungsten (in various chemical forms): uptake, storage, regulation, cofactor biosynthesis, and incorporation in enzymes. Dashed arrows correspond to hypothetical processes based on cellular processes known for molybdate.


Biochemical cycle of nitrogen. 
Dinitrogen fixation, blue arrow; “organic nitrogen pool”, green arrows; assimilatory ammonification, pink arrow; dissimilatory nitrate reduction to ammonium, violet arrow; nitrification, yellow arrows; denitrification, red arrows; anaerobic ammonium oxidation (AnAmmOx), orange arrows. The steps catalyzed by molybdenum-containing enzymes are highlighted with thick arrows, nitrogenase (blue), nitrate reductase and nitrite oxidoreductase (grey).

With this wide perspective in mind, the molybdenum biological role certainly assumes another dimension. In fact the lack of molybdenum, while hampering the existence of an efficient nitrogenase, would have been one of the limiting factors for life to emerge.  The involvement of molybdenum in the nitrogen cycle is not restricted to the dinitrogen fixation, as the element is also essential for the reduction of nitrate to nitrite and for the oxidation of nitrite to nitrate,  both processes being exclusively dependent (as far as is presently known) on the molybdenum- containing enzymes nitrate reductases (from both prokaryotic and eukaryotic sources) and nitrite oxidoreductases (from prokaryotes only).

Noteworthy, molybdenum is also essential for nitrite reduction to nitric oxide for biological signalling purposes. Nitric oxide is a signalling molecule involved in several physiological processes, in both prokaryotes and eukaryotes, and nitrite is presently recognized as a nitric oxide source particularly relevant to cell signalling and survival under challenging conditions. 

The primitive carbon cycle would have also been dependent on molybdenum, as the metal (together with tungsten)would have been essential for the earliest, strictly anaerobic, organisms to handle aldehydes and carboxylic acids, catalyzing their interconversion.

 With the exception of nitrogenase containing the iron–molybdenum cofactor, all other molybdoenzymes possess in their active site a molybdenum atom coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT) .  74% of bacteria representing almost all phyla utilize the molybdenum cofactor.

The different Moco-containing enzymes

One type of cofactor is the iron–sulfur-cluster-based iron-Mo cofactor, which is found only once in nature, namely in bacterial nitrogenase. The other type of cofactor is the pterin-based Mo-cofactor (Moco) that, in different variants, forms part of the active centers of all Mo-enyzmes in living organisms. Mo has a versatile redox chemistry that is used by the enzymes to catalyze diverse redox reactions. This redox chemistry is controlled both by the different ligands at the Mo atom and by the enzyme environment. Mo is very abundant in the oceans in the form of the molybdate anion. 


four families based on the nature of the cofactor and other ligands bound in their active centres in the oxidized Mo(VI) forms



Molybdenum- and tungsten-containing cofactors and enzymes
(A) Chemical structures of molybdenum cofactor (Moco), Mo/W-bis pyranopterin guanosine dinucleotide (PGD) and W-bis pyranopterin cofactor. 
(B) Domain structures of eukaryotic Mo enzymes of the xanthine oxidase (XO) and sulfite oxidase (SO) families. D, dimerization domain.


The first group is termed the xanthine oxidase (XO) family and features a single pterin cofactor and terminal sulfido and oxido-ligands. 
The second group comprises the sulfite oxidase (SO) family and carries a single cofactor, a cysteine ligand and two oxido-ligands.
The third group is the dimethyl sulfoxide (DMSO) reductase family and harbours a bis-pyranopterin guanine dinucleotide cofactor and two oxido-ligands plus, and depending on the particular enzyme, an extra cysteine, seleno-cysteine, serine or aspartate ligand.
The fourth group comprises the archaeal aldehyde oxidoreductase family that harbours a bis-pyranopterin cofactor preferentially bound to a W atom.

Five molybdo-enzymes are found in eukaryotes and belong to only two of the families (Figure B): 

nitrate reductase (NO), SO and the amidoximereducing component (mARC) are members of the SO family, while 
XO/xanthine dehydrogenase (XDH) and aldehyde oxidase (AO) form part of the XO family.

Molybdo-enzymes (mainly bacterial) are involved in key processes in the global carbon, nitrogen and sulfur cycles, such as nitrate reduction, sulfite detoxification and purine catabolism.

Nitrate Reductase
Eukaryotic NR (EC 1.6.6.1) is a cytoplasmic, water-soluble enzyme involved in the reduction of nitrate to nitrite as the first step of assimilation of nitrogen in plants, algae and fungi.

Sulfite Oxidase
SO (EC 1.8.3.1) is essential in sulfur catabolism. In vertebrates, it catalyses the two-electron oxidation of sulfite to sulfate coupled to the reduction of two molecules of cytochrome c.37 Sulfite oxidation is the terminal step in the
oxidative degradation of cysteine.

Xanthine Dehydrogenase and Oxidase
Eukaryotic XDH/XO (EC 1.17.1.4) systems participate in the degradation of purines by oxidation of hypoxanthine to xanthine and xanthine to uric acid. The enzyme can function either as a dehydrogenase using NAD1 as electron
acceptor or, upon reversible cysteine oxidation, as an oxidase using dioxygen as terminal electron acceptor.

Aldehyde Oxidase
AO enzymes (EC 1.2.3.1) originate from a duplication of the xdh gene in eukaryotes before the origin of multicellularity.49 Consequently, both enzymes contain the same cofactor-binding domains (Fe-S clusters, FAD and
Moco) as well as a dimerization domain (Figure B).





The families of molybdoenzymes.
The cofactor is not located on the surface of the protein, but is buried deeply within the interior of the enzyme and a tunnel-like structure makes it accessible to the cognate substrates.
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 ) 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).

In eukaryotes, the most prominent Mo-enzymes are

(1) sulfite oxidase, which catalyzes the final step in the degradation of sulfur-containing amino acids and is involved in detoxifying excess sulfite,
(2) xanthine dehydrogenase, which is involved in purine catabolism and reactive oxygen production,
(3) aldehyde oxidase, which oxidizes a variety of aldehydes and is essential for the biosynthesis of the phytohormone abscisic acid, and in autotrophic organisms also
(4) nitrate reductase, which catalyzes the key step in inorganic nitrogen assimilation.

All Mo-enzymes, except plant sulfite oxidase, need at least one more redox active center, many of them involving iron in electron transfer. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also includes iron as well as copper in an indespensable way. Moco as released after synthesis is likely to be distributed to the apoproteins of Mo-enzymes by putative Moco-carrier proteins. Xanthine dehydrogenase and aldehyde oxidase, but not sulfite oxidase and nitrate reductase, require the postranslational sulfuration of their Mo-site for becoming active. This final maturation step is catalyzed by a Moco-sulfurase enzyme, which mobilizes sulfur from l-cysteine in a pyridoxal phosphate-dependent manner as typical for cysteine desulfurases. 11 

How does it work, what does it do
Mo-enzymes generally catalyze the transfer of an oxygen atom, ultimately derived from or incorporated into water, to or from a substrate. Each reaction, either reduction or oxidation, involves the transfer of two electrons, thereby causing a change in the oxidation state of the Mo atom in the substrate-binding site from IV to VI or vice versa.

The task of the cofactor is to position the catalytic metal Mo correctly within the active center, to control its redox behaviour and to participate with its pterin ring system in the electron transfer to or from the Mo atom. 11 The pterin with its several possible reduction states as well as different structural conformations might also be important in channeling electrons from or to other prosthetic groups

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

The path goes from molybdenum uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes 12  In soils, the molybdate anion is the only form of Mo that is available for plants and bacteria.  Moco is a tricyclic pterin that co-ordinates the metal via a dithiolene group at the third (pyrano) ring.



The task of the cofactor is to position the catalytic metal Mo correctly within the active centre, to control its redox behavior, and to participate with its pterin ring system in the electron transfer to or from the Mo atom. The pterin, with its several possible reduction states as well as different structural conformations, might also be important in channeling electrons from or to other prosthetic groups

Once entered the cell, molybdate has to be complexed by a unique scaffold in order to gain biological activity. This compound is a unique tricyclic pterin called molybdopterin or metalcontaining pterin (MPT)


The molybdenum cofactor as found in eukaryotic molybdenum enzymes. In enzymes of the sulfite oxidase family, X is represented by a single-bonded sulfur provided by a cysteine residue of the respective protein, while Y corresponds to a double-bonded oxygen. In enzymes of the xanthine oxidase family, X is represented by a double-bonded inorganic sulfur and Y by a hydroxyl group.

Protein folding and assembly into macromolecule complexes within the living cell are complex processes requiring intimate coordination. The biogenesis of complex iron sulfur molybdoenzymes (CISM) requires use of a system specific chaperone – a redox enzyme maturation protein (REMP) – to help mediate final folding and assembly. The CISM dimethyl sulfoxide (DMSO) reductase is a bacterial oxidoreductase that utilizes DMSO as a final electron acceptor for anaerobic respiration. The REMP DmsD strongly interacts with DMSO reductase to facilitate folding, cofactor-insertion, subunit assembly and targeting of the multi-subunit enzyme prior to membrane translocation and final assembly and maturation into a bioenergetic catalytic unit. 

There must be correct folding and targeting coordinated with cofactor insertion.  Proteins must be targeted to their correct subcellular locations, and there has to be precise control of the assembly of large multimeric complexes.
 
Targeting, coordination, correct insertion, precise control of assembly are usually actions either directly performed by intelligence, or preprogrammed by intelligence to be exsercised in a robot-like manner. 

Maturation of a CISM into a functional holoenzyme requires numerous stages that involve initial translational ribosome integrated folding, cofactor insertion and coordination, subsequent folding and assembly with other subunits that may have had similarly complex folding pathway. The many steps comprising the cytoplasmic biogenesis processes are highly complex and must be intricately coordinated by numerous assistant proteins to produce a functional CISM.

Of special interest to CISM maturation are system specific chaperones that help mediate the complete final folding and assembly. Such chaperones were termed redox enzyme maturation proteins (REMPs) and are essential for the proper assembly of CISMs albeit absent in the final assembled holoenzyme.

REMPs ‘escort’ their CISM substrates though the entire maturation process. Accordingly, many potential roles for REMPs have been proposed, including functioning as: 

1. foldases to ensure correct secondary and tertiary structure; 
2. unfoldases to correct folding mistakes; 
3. avoidance chaperones to prevent incorrect membrane targeting during folding and assembly; 
4. cofactor-assembly chaperones to maintain apoenzymes in a cofactor-binding competent conformation; 
5. cofactor-binding proteins, which bind the cofactor prior to its transfer to the apoenzyme; 
6. targeting proteins directing substrates to specific cellular locations; 
7. escort chaperones to promote transmembrane transport of enzyme complexes; 
8. proofreading chaperones to suppress transport until essential prior steps in the assembly process are complete;
9. protease protection chaperones to prevent degradation during assembly

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. Chaperones are required to facilitate the insertion of Moco into the target enzyme. Only after the insertion of Moco, the apo-enzymes adopt their final structure. 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. Most molybdoenzymes in bacteria, especially enzymes of the DMSO reductase family, have a specific chaperone for Moco insertion.



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  and monomeric TorA from S. massilia .
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. 
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).

Chaperones in maturation of molybdoenzymes: Why specific is better than general? 25 Apr 2018
An interesting feature is that molybdoenzymes and their cognate chaperones, which are usually genetically related, seem to have coevolved. This latter point could be an explanation concerning the high level of specificity between the molybdoenzyme and the associated chaperone  7

This is a remarkable claim. Since these enzymes had to become functional at LUCA, they had to emerge without evolution as a causal mechanism. Secondly, chaperones have the only function when used in the maturation process of the enzyme. The high specificity on top of that seems far better to infer that a designer projected these enzymes and the requirement of "helper" proteins for maturation and synthesis of the protein complex. 

a Hydroxylation is a chemical process that introduces a hydroxyl group (-OH) into an organic compound. In biochemistry, hydroxylation reactions are often facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of organic compounds in air. It is extremely important in detoxification since hydroxylation converts lipophilic compounds into water-soluble (hydrophilic) products that are more readily removed by the kidneys or liver and excreted.

b.Pterin is a heterocyclic compound composed of a pteridine ring system, with a "keto group" (a lactam) and an amino group on positions 4 and 2 respectively. It is structurally related to the parent bicyclic heterocycle called pteridine. Pterins, as a group, are compounds related to pterin with additional substituents. Pterin itself is of no biological significance. 8






1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817353/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278043/
3. https://royalsocietypublishing.org/doi/10.1098/rstb.2012.0258
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311541/
5. https://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S0010854508000167
6. https://academic.oup.com/femsre/article/40/1/1/2467802
7. https://hal.archives-ouvertes.fr/hal-01413222/document
8. https://en.wikipedia.org/wiki/Pterin
9. https://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S0010854511000671
10. https://link.springer.com/chapter/10.1007/1-4020-2179-8_10
11. https://www.sciencedirect.com/science/article/pii/S0167488906001017
12. https://academic.oup.com/jxb/article/58/9/2289/542465
13. https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1007518#pgen.1007518.ref051
14. https://www.nature.com/articles/srep00263
15. https://blogs.scientificamerican.com/thoughtomics/a-spoonful-of-molybdenum-some-ulysses-and-the-origin-of-life/
16. https://www.imoa.info/essentiality/molybdenum_trace_element.php
17. https://boa.unimib.it/retrieve/handle/10281/53873/82319/phd_unimib_057957.pdf

A Spoonful of Molybdenum, some Ulysses and the Origin of Life

Methanogens. These bacteria and archaea make a living by stripping electrons from hydrogen (H2) and attaching them onto carbon dioxide (CO2) in several steps, generating methane (CH4) and water (H2O) in the process. That might sound easy enough, but carbon dioxide is not a thankful molecule: it's stable as it is, and very reluctant to accept additional electrons.

This is where molybdenum comes in. Or rather could come in, for while molybdenum plays a role in the conversion of carbon dioxide to methane, the mechanism that Russell proposes has not yet been proven for this particular reaction. Russell's argument boils down to a single point: molybdenum can ease difficult electron transfers because it usually has not one, but two electrons to give away. There's nothing stopping molybdenum from donating these electrons to two different molecules. In this way, the molybdenum ion could compensate for the effort of imposing one electron onto a stubborn naysayer (such as carbon dioxide), by donating the other one to a more willing recipient.

The forking of electrons works because the electron that rolls 'downhill' releases energy that is channelled into pushing the other electron 'up the slope'. Some molybdenum enzymes are known to perform this trick, but no one really knows how widespread such crossed electron transfers really are in biochemistry. In an article published last year, Russell and Wolfgang Nitschke write that electron bifurcation 'is an old, but almost forgotten friend of research'.

How old? In Russell's most recent paper (the one with 'ineluctable' in the title), he and his team suggest as old as life itself. Previous investigations into the age of the molybdenum family were based on genetic sequences alone, and pointed towards a more recent origin. But comparisons between bare genes can paint a misleading picture. Genetic sequences are to proteins what recipes are to cooking: shallow descriptions that lack the finer subtleties of texture and form. This is why Russell and his colleagues compared the three dimensional structure of different molybdenum proteins instead. Structure is more conserved than sequence, which is perfect for exploring ancient relationships.

They found that the roots of molybdenum enzymes run deep. According to structure, most molybdenum proteins can be sorted into two piles: those of archaea and bacteria. The oldest divide in life. Archaea are microorganisms, just like bacteria, but their biochemistry differs like day and night. According to Russell, the presence of molybdenum proteins in both archaea and bacteria means they were also present in the last common ancestor of all life on earth. His conclusion: to master the molecules of metabolism, including both electron lovers and haters, life needed molybdenum (and tungsten, which is chemically similar).

Does the ancient origin of molybdenum enzymes really prove that electron bifurcation by molybdenum was 'ineluctable' for the origin of life? No. When it comes to life's earliest beginnings, some degree of speculation is inevitable. "Some of the time, you're just going to be wrong", says Russell. "We try to approach the truth, but we have to face up with the fact that we cannot be right all the time." While molybdenum might not be the key to our origins, it still holds a clue to the larger riddle, a tiny puzzle all in itself.

Russell has one more argument to persuade me that molybdenum really does hold the answer to life, the universe and everything. Near the end of our conversation, he asks whether I have read the Hitchhiker's Guide to the Galaxy. "The atomic number of Molybdenum is 42." I can almost hear the grin on the other end of the line.

https://blogs.scientificamerican.com/thoughtomics/a-spoonful-of-molybdenum-some-ulysses-and-the-origin-of-life/

The structure of the molybdenum cofactor

The pterin structure of Moco is unique in nature and is required in order to control and maintain the special redox properties of Mo.

All Mo-dependent enzymes (molybdoenzymes) use this metal in the form of the Mo cofactor (Moco), which consists of Mo coordinated to an organic tricyclic pyranopterin moiety, referred to as molybdopterin 1
The cofactor appears in four basic configurations. The cofactor typically has a tricyclic pyranopterin structure, with pyrimidine, pyrazine, and pyran rings, respectively labelled a, b, and c
The tricyclic pterin structure may have evolved in order to position the catalytic metal correctly within the active center of a given Mo-enzyme.


The alternative form of the pterin is the bicyclic molybdopterin form, which contains the pyrimidine and pyrazine rings but contains an open pyran ring (see Fig. 1D, distal pterin). Mo-PPT is the form of the cofactor found in eukaryotes and in essentially all sulfite oxidase enzymes (SUOX) and some xanthine dehydrogenase enzymes (XDH). PPT can be modified by the addition of a nucleotide (typically cytosine or guanine) at the phosphomethyl position to generate a dinucleotide form (Fig. 1C and D). Intriguingly, two PPTs can coordinate a single W atom via a pair of dithiolenes (Fig. 1B). This form of the cofactor is the bis-mononucleotide form. The vast majority of bacterial
molybdoenzymes contain the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) (Fig. 1D) cofactor or its tungsto derivative (W-bisPGD). A minority of the Mo-bisPGD cofactors contain both a tricyclic pyranopterin (‘Proximal pterin’ in Fig. 1D) and a bicyclic molybdopterin (‘Distal pterin’ in Fig. 1D)

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3311541/

Cellular uptake of molybdenum and tungsten in prokaryotes

It requires specific uptake systems to scavenge molybdate in the presence of competing anions.

ABC transporters are arguably the most important molecular pumps that transport small solute molecules into and out of prokaryotic and eukaryotic cells.  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. The genes encoding these three proteins of the molybdate transporter are usually found in the same operon, in the order modA, modB, modC.

The molybdate-binding proteins from A. vinelandii and E. coli contain 232 and 233 residues, respectively. Molybdate and tungstate both bind in a completely dehydrated site at the interface between the two domains. Side-chain hydroxyls, a side-chain amide, and backbone amides donate hydrogen bonds to the anions. A predominance of backbone amide hydrogen bonds provides a rigid binding site. In addition to the binding protein, ModA, the molybdate transporter consists of an integral membrane protein or permease, and its associated ATP-binding protein. ModB, is a 24 kDa protein with five potential transmembrane helices. 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

In most organisms, ABC transporters constitute one of the largest families of membrane proteins. 4 
Moco biosynthesis and its insertion into molybdoenzymes depend on transport of molybdate anion, activation of molybdate and incorporation of Mo into molybdopterin (MPT). 5
In bacteria, molybdate is mainly taken up through the high-affinity ModABC system, which is the first identified Mo transporter in this group of organisms. Commonly, the ABC transporter consists of a periplasmic molybdate-binding protein ModA, two integral membrane proteins of the ModB family and two hydrophilic peripheral membrane proteins of the ModC family that are ATPases. ModA may bind both molybdate and tungstate in the periplasm via several conserved amino acid residues. 

Two additional ABC transporters, WtpABC (Mo/W) and TupABC (W-specific), have been reported in different organisms. 

Bacteria have a high-affinity molybdenum uptake system belonging to the ABCsuperfamily of transporters.Components of this system include a periplasmic binding protein (ModA), an integralmembrane protein (ModB), and an energizer protein (ModC). 6

The ABCtransporter binds the oxoanion with high specificity, then uses an ATP hydrolysis driven conformational change tomoveit through the membrane, and finally releases it on the other site. 1

Amajor point emphasized in this review is the existence of three different prokaryotic transporter systems whose oxoanion binding proteins ModA, WtpA, and TupA do not only differ in their binding affinity for molybdate versus tungstate, but are also essentially unrelated at the primary sequence level, and furthermore appear to have very different oxoanion coordination chemistry. It is therefore imperative to reverse common practice of mixing up the labels Mod, Wtp, Tup.

Cells acquire molybdenum and tungsten as their highly soluble oxoanions, MoVIO42− orWVIO42−, which they internalize by means of an active (i.e. energy requiring) transmembrane importer, for subsequent conversion into the metalloenzyme cofactors Moco or Wco (and FeMoco in nitrogen fixers). The complex exhibits interesting variants, known as the microbial Mod, Tup, and Wtp system, and the – less well defined – eukaryotic MOT1 system, which mutually differ in oxoanion coordination chemistry and in the control of intracellular Mo/W levels.

For the synthesis of molybdoenzymes, bacteria need to transport molybdate, activate it to an appropriate form, and incorporate it into the organic part of the molybdenum cofactor. In nature, the predominant form of Mo is molybdate oxyanion, which is transported by an ABC-type transport system into the Cell.

The  cellular transport system for oxoanions like tungstate, molybdate, sulphate and phosphate are all systems are members of the adenosine triphosphate (ATP) binding cassette (ABC) transporter family. The majority of these oxoanion transporters consist of three proteins; the ‘A’ protein is responsible for the recognition and binding of the substrate. This protein is located in the periplasm, which is the space between the cytoplasmic membrane and: 

(i) the cell wall in Gram-positive bacteria, 
(ii) the outer membrane in Gram-negative bacteria, or 
(iii) the S-layer in archaea. For some ABC transporters the first component is linked to the outer surface of the cellular membrane with a so-called ‘lipotail’, which is a lipid-modified cysteine residue. 

The B component forms the transmembrane pore through which the substrate is transported into the cell, and this transport is facilitated by the ATP hydrolyzing activity of component C on the inner surface of the membrane.

For some ABC transporters the first component is linked to the outer surface of the cellular membrane with a so-called ‘lipotail’, which is a lipid-modified cysteine residue. The B component forms the transmembrane pore through which the substrate is transported into the cell, and this transport is facilitated by the ATP hydrolyzing activity of component C on the inner surface of the membrane. The periplasmic molybdate-binding protein in E. coli, referred to as ModA, specifically binds molybdate. ModA is also able to bind tungstate with a similar affinity. After binding to the periplasmic component the molybdate or tungstate is actively transported against a concentration gradient into the cell through the transmembrane unit ModB energized by the ATP hydrolyzing activity of ModC.

There is a tungsten-specific transporter: Tungsten uptake protein ABC (TupABC),

The molybdate system Mod
The modABC genes encode the proteins ModA, ModB, and ModC, which together assemble into the ModABC transporter.


Schematic drawing of a molybdate ABC transporter.
The soluble, periplasmic A-protein scavenges the oxoanion and then binds to the membrane complex which consists of a pair of translocating, transmembrane B-subunits bound to a cytoplasmic pair of C-subunits with ATPase activity.


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. 2


The structure of the A. fulgidus molybdate transporter. 
Left, the overall structure of the ModAB2C2 heteropentamer, with ModA in blue, ModB in gray, and ModC in red; the two subunits of ModB and ModC are indicated in dark and light shading. Right, the molybdate binding site of ModA, as seen from the face that interacts with the ModB2 dimer. The anion binding site consists of Asp 153 and Glu 218, both bound in a bidentate fashion, plus Tyr 236 and Ser 42 that hydrogen bond to the bound anion (in the case of the crystal structure tungstate, rather than molybdate, from the crystallization mother liquor).

ABC transporters consist of two transmembrane domains that provide a translocation pathway, and two cytoplasmic nucleotide- binding domains  that hydrolyse ATP and drive the transport reaction. ABC importers contain between 10 and 20 transmembrane helices. 

Several charged amino acid residues on the ModA surface contribute to the interface. Mutating such residues abolishes transport. 
Bacterial periplasmic transport systems consist of multiple components, the interaction between which is necessary to accomplish transport. In the absence of the periplasmic binding protein, the membrane complex is incapable of supporting transport. Because of this requirement for binding protein, it is clear that an interaction between the binding protein and the membrane complex must take place to yield transport. Genetically isolated transport mutants  have been shown to be defective in binding to the membrane complex  
https://sci hub.tw/https://www.jbc.org/content/266/15/9673

Without having a functional Molybdenum transport system, which depends on the correctness of even a tiny fraction of amino acid residues which contribute to the functionality of the ABC transporter, there would be no import, its malfunction would abolish transport, and molybdenum or tungsten enzymes could not be imported and assembled and used in the Cell, and remarkably, that would have catastrophic consequences, and life as a whole would not be possible to emerge. That is one tiny example, there could be cited thousands, where a small sub-unit of a given protein which helps to make a life essential protein must be just right and have the correct amino-acids sequences from the beginning, or nothing goes. It is comparable to a huge factory, where a tiny malfunction of a  sub-part of a robot in a production line might stop the production process of the huge part of the factory, or its entirety. That's an all or nothing business. This raises also the question in the first place, how such ABC transporters emerged on a pre-biotic earth in the first place. There was no evolution at this stage, so they had to self-assemble in a random unguided fashion. That's a hard sell. 

ATP hydrolysis in the ModC subunits drives a conformational change in the membrane-spanning ModB2 dimer that alternately opens it to the periplasm and cytoplasm, thus effecting ion transport. Once in the cell, molybdate is stored bound to small (∼7 kDa) binding proteins. 


The NBDs (ModC subunits) contain the highly conserved P-loops and LSGGQ motifs involved in ATP binding and hydrolysis.

ATP is the energy currency employed to make the ABC Transporters perform their opening and closing conformation , and transport the goods.

Precise positioning of the P-loops and of the LSGGQ motifs is required in the ATP-bound state of ABC transporters. The ModC–ModB interface transmits critical conformational changes, thus coupling ATP binding and hydrolysis to transport. 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.


NBD conformations and interfaces. 
a, Schematic summarizing the conformational changes. 
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 cytoplasmic nucleotide-binding domains (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://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S0010854511000671
2. https://sci-hub.tw/https://www.nature.com/articles/nature05626?draft=marketing
3. https://sci-hub.tw/https://www.jbc.org/content/266/15/9673
4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652935/
5. Molybdenum and Tungsten Enzymes Biochemistry page 97
6. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/11080635

Biosynthesis of the Molybdenum cofactor
The organic cofactor that accompanies molybdenum is variously called molybdopterin or pyranopterin. 


Active site structures for the three families of mononuclear molybdenum enzymes. The structures shown are, from left to right, for xanthine oxidase, sulfite oxidase, and DMSO reductase. The structure of the pyranopterin cofactor common to all of these enzymes (as well as the tungsten-containing enzymes) is given at the bottom of the illustration above.

Mo is complexed by a pterin compound thus forming the biologically active molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes.


A general scheme of Mo uptake and Moco biosynthesis pathway in bacteria and eukaryotes is shown in Figure above. 

Biosynthesis of the most basic form of the molybdenum center involves three steps: 

1. cyclization of GTP to form a cyclopyranoperin monophosphate (cPMP) intermediate; 
2. sulfuration of cPMP to give the now mature pyranopterin (often abbreviated MPT for historical reasons); and 
3. coordination of molybdate to the enedithiolate thus generated.

In all organisms studied so far, Moco is synthesized by a conserved biosynthetic pathway that can be divided into four steps, according to the biosynthetic intermediates

1. precursor Z,
2. MPT,
3. adenlyated MPT,
4. and Moco

In eukaryotes always six gene products catalyzing Moco biosynthesis have been identified

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.

For instance, in E. coli, nine proteins with known function are directly involved in Moco biosynthesis 

MoaA 
MoaC
MobA
MocA
MoaD
MoaE
MoeA
MoeB
MogA

and are encoded by genes organized in distinct gene loci.  In all prokaryotes, Moco is synthesized by a conserved pathway which can be divided into four general steps.

1. the synthesis of cyclic pyranopterin monophosphate (cPMP) from 5′GTP  
2. insertion of two sulfur atoms into cPMP and formation of MPT
3. formation of Moco by insertion of molybdate to the sulfur atoms of MPT
4. further modification of Moco by the attachment of GMP or CMP to the phosphate group of MPT, forming the MGD cofactor  or MCD cofactor

The final step in the biosynthesis of the basic form of the molybdenum cofactor is the insertion of the metal itself. By contrast to the biology of most first-row transition metals, which are typically present in the cell as cations, that of
molybdenum involves the anionic (and highly water-soluble) molybdate ion. Molybdate enters the cell via a specific transporter system, in the case of E. coli ModABC, a member of the ATP-binding cassette (ABC) superfamily of membrane transporters. 

Mutations in the genes for Moco biosynthesis result in the pleiotropic a loss of all molybdenum-dependent cellular processes. 2

Step one:  conversion of GTP into precursor Z
The first step in the process, conversion of GTP to cPMP (referred to in the earlier literature as “Precursor Z”), is catalyzed by two enzymes ( In E.Coli, MoaA and MoaC); During the first stage, a guanosine derivative (probably GTP) is transformed into a sulfur-free pterin compound, the precursor Z, possessing already the Moco-typical four carbon side chain. The detailed mechanism of this reaction step remains unclear, yet hypothetical multistep-reactions have been suggested

The MoaA enzyme
MoaA is a member of the large radical- SAM family of enzymes. The MoaA and MoaC proteins catalyze the first step during molybdenum cofactor biosynthesis, the conversion of a guanosine derivative to precursor Z. 1
MoaA belongs to the S-adenosylmethionine (SAM)-dependent radical enzyme superfamily, members of which catalyze the formation of protein and
or substrate radicals by reductive cleavage of SAM by a [4Fe–4S] cluster.


Overall structure of MoaA. 
(A) Ribbon diagram of the monomer. The N-terminal TIM barrel structure is shown in maroon, and the C-terminal part is shown in gold. 
(B) Ribbon diagram of the dimer. The two monomers are colored in blue and orange. 
(C) Molecular surface of the dimer colored as in B viewed along the hydrophilic channel in one of the monomers. FeS clusters are shown in CPK (Fe, green; S, yellow), and SAM is shown in stick representation


The active site of MoaA. 
(A) Stereoview of the FeS clusters and SAM. SAM is shown in a FoFc omit map contoured at 3 . Hydrogen bonds are indicated as dashed lines. 
(B) Closer view of the active-site pocket created by predominantly positively charged amino acids. Conserved amino acids are labeled in red. Secondary structural elements and cofactors are rendered transparent. 
(C) Surface representation viewed from both sides into the active site channel. Conserved residues are mapped onto the molecular surface. 
(D) A weighted 2FoFc map of the C-terminal FeS cluster with a DTT molecule as the fourth ligand contoured at 1 . Surrounding amino acids are shown as a C trace colored in orange. Amino acids, FeS clusters, and SAM are shown in ball and stick.

MoaA Staphylococcus aureus is typical, being a 2 × 41 kDa homodimer with each subunit possessing a canonical [4Fe-4S] cluster in its amino-terminal domain, and a second in its C-terminal domain


The structure of MoaA from S. aureus. 
Top, the structure of the dimeric enzyme, with the N- and C-terminal domains of the subunit on the left shaded in blue and green, respectively. Bottom left, a close-up of the active site with S-adenosylmethionine bound to the N-terminal [4Fe-4S] cluster and dithiothreitol bound to the C-terminal [4Fe-4S]. Bottom right, a close-up of the active site of GTP bound to the C-terminal cluster, and methionine bound at the N-terminal cluster; 5′deoxyadenosine is also present in the active site

The MoaC enzyme 
MoaC  has also been crystallographically characterized


The structure of MoaC in complex with GTP and citrate. Left, overall structure of the dimer, with the two subunits in blue and gray. The GTP/citrate binding sites are at the top and bottom. Right, a close-up of one binding site, showing the bound GTP and citrate in teal.


Illustration of the reaction catalyzed. 



The reactions catalyzed by MoaA and MoaC. 
It is possible that the MoaA reaction proceeds without opening of the ribose ring.

Step two: synthesis of molybdopterin
In the second stage, sulphur is transferred to precursor Z in order to generate MPT. This reaction is catalysed by the enzyme MPT synthase. After MPT synthase has transferred the two sulphurs to precursor Z, it has to be resulphurated in order to reactivate the enzyme for the next reaction cycle of precursor Z conversion. This resulphuration is catalysed by Cnx5 involving an adenylation of MPT synthase followed by sulphur transfer. Cnx5 is a two-domain protein consisting of an N-terminal domain responsible for adenylating MPT synthase and a C-terminal rhodanese-like domain where the sulphur is bound to a conserved cysteine in the form of persulphide. The identity of the donor for the reactive mobile sulphur is as yet unknown.

Step 3: adenlyation of molybdopterin
in the biosynthetic pathway involves sulfuration of cPMP to form the mature pyranopterin (molybdopterin) cofactor. The MPT synthase catalyzing the reaction has been examined crystallographically.
The bacterial enzymes are (αβ)2 heterotetramers consisting of two equivalents each of the MoaD and MoaE gene products.


The MPT synthase

The structure of S. aureus MoaDE in complex with cPMP .
Left, the overall structure of the (αβ)2 heterotetramer, with the two MoaD subunits in gray, and the two MoaE subunits, with cPMP bound in each active site, in blue and green. The C-terminal tails of the MoaD subunits, ending in a highly conserved GG, are seen extending into the MoaD active sites. Right, a close-up of one active site showing the several amino acid residues interacting with the bound cPMP, and the proximity of the MoaD C-terminus to the positions to become sulfurated (indicated by asterisks).



The  reaction mechanism of MPT synthase. 
In the second sulfuration step (bottom), the stereochemistry of the pyran ring is inferred from the spatial disposition of cPMP relative to the C-terminus of MoaD

It is evident from the crystal structure that the sulfuration process must proceed sequentially, with two successive equivalents of sulfur-charged MoaD binding to each active site of the (MoaE)2 core in the course of turnover.

Several gene loci are involved in Moco biosynthesis. They point to an evolutionarily conserved biosynthetic pathway that can be divided into three steps, according to the stable biosynthetic intermediates. The synthesis of cyclic pyranopterin monophosphate (cPMP), conversion of cPMP into MPT by introduction of two sulfur atoms and insertion of molybdate to form Moco. In bacteria, a fourth step involves the further modification of Moco by the addition of nucleotide monophosphates to the phosphate group of MPT.

After MPT synthase has transferred the two sulphurs to precursor Z, it has to be resulphurated in order to reactivate the enzyme for the next reaction cycle of precursor Z conversion. This resulphuration is catalysed by Cnx5 involving an adenylation of MPT synthase followed by sulphur transfer. Cnx5 is a two-domain protein consisting of an N-terminal domain responsible for adenylating MPT synthase and a C-terminal rhodanese-like domain where the sulphur is bound to a conserved cysteine in the form of persulphide.


Step 4: molybdenum insertion
The crystal structure of the Cnx1-G revealed another unexpected finding, namely a copper bound to the MPT dithiolate sulphurs. Up to now, the function of this novel MPT ligand is unknown but copper might play a role in sulphur transfer to precursor Z, in protecting the MPT dithiolate from oxidation, and/or presenting a suitable leaving group for Mo insertion. The origin of this copper is still unclear, but it is reasonable to assume that it binds to the dithiolate group just after the latter has been formed, i.e. at the end of step 2 of Moco biosynthesis. In the final step of Moco biosynthesis, MPT-AMP is transferred to the N-terminal domain of Cnx1 (=Cnx1-E) that cleaves the adenylate in a molybdate-dependent way, releases copper, and inserts Mo, thus yielding active Moco.












Biosynthesis of eukaryotic molybdenum cofactor.
The pathway of Moco synthesis can be divided into four steps, each being characterized by its main features as given in italics on the right side. For MPT and MPT-AMP, the ligands of the dithiolate sulfurs are indicated by an “R” as it is currently unknown at which state copper is bound to the dithiolate. Upon Mo insertion, it is also not clear how many oxo ligands are bound to the metal. Therefore, two Mo-oxo ligands are depicted and a third line indicates an additional ligand. The proteins from plants and humans catalyzing the respective steps are depicted and their names are given in green (plants) and red (humans). Functional properties like [Fe–S] clusters in Cnx2 and Mocs1A, the use of S-adenosyl methionine (SAM), adenylation and sulfuration of the small subunit of MPT synthase (Cnx7 and Mocs2B, respectively) are indicated. All substrates/co-substrates are indicated in blue. The in vivo source of sulfur (X–S) for Cnx5 and Mocs3 is not known yet. Steps three and four in plants and humans are catalyzed by the individual domains of Cnx1 (G and E) or Gephyrin (G and E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

a Pleiotropic: Producing or having multiple effects from a single gene. For example, the Marfan gene is pleiotropic, potentially causing such diverse effects as long fingers and toes (arachnodactyly), dislocation of the lens of the eye, and dissecting aneurysm of the aorta.

1. https://sci-hub.tw/https://www.pnas.org/content/101/35/12870
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650355/

Intracellular Trafficking of the Molybdenum Cofactor

The cofactor is extremely sensitive to airoxidation, therefore in eukaryotes, the newly synthesized cofactor does not exist in free solution in the cell but is maintained bound to a storage protein to protect it from oxidation.

Cofactor Insertion into Apoenzyme

Cofactor insertion in the case of members of the sulfite oxidase family appears to be straightforward, as the cofactor is not particularly deeply buried in the holoenzyme. As illustrated by the structure of the A. thaliana sulfite oxidase, it is evident from an examination of the overall protein fold (common to all family members) that the two helices indicated in yellow and blue, possibly along with the N-terminal subdomain in green, could easily move apart transiently in a largely folded apoprotein to accommodate the cofactor;

thermal energy alone might be sufficient to induce the necessary conformational changes. Indeed, the cofactor has been shown to bind spontaneously to apo sulfite oxidase in vitro, although the (most likely passive) involvement of carrier proteins may occur in the case of apo nitrate reductase. Very recently, it has been demonstrated that in vitro cofactor insertion into apo nitrate reductase from Neurospora crassa also occurs spontaneously.98 Under physiological conditions, however, it cannot be excluded that the Cnx1 (and its orthologues in other organisms) that catalyze the final molybdenum insertion step into the cofactor may be involved in a specific protein−protein interaction with apoenzymes in the course of cofactor insertion.

The insertion process is necessarily more complex for members of the DMSO reductase family, as the bis pyranopterin form of the cofactor is not simply more deeply buried but also significantly more complicated (particularly when present as the dinucleotide). Cofactor insertion involves MobA and usually (but not always) a chaperone; again MobA acts on the bispyranopterin version of the cofactor. The available data suggest that the chaperone stabilizes a distinct apoprotein configuration that enables cofactor insertion.

Insertion of the molybdenum center into enzymes of the xanthine oxidase family constitutes the greatest challenge. The molybdenum center is not simply deeply buried, but the two protein domains that line the narrow, 15 Å-long solvent access channel to the active site are laced together by multiple passes of the polypeptide chain, and there is no readily identifiable domain motion that might provide better access to the active site of apoenzyme. In both prokaryotic and eukaryotic systems, however, it has become increasingly clear that the enzymes responsible for sulfuration of the cofactor destined for members of the xanthine oxidase family of enzymes participate directly in
its insertion once the sulfur has been incorporated.

The Xanthine Oxidase Family

Xanthine dehydrogenase/oxidase(XDH/XO) is a molybdenum-containing enzyme which is involved with hydroxylation a a number of sp2-hybridized centers including purines, heterocycles, and aldehydes.  Its main role in the cell is to convert hypoxanthine to xanthine and xanthine to uric acid. 1



a Hydroxylation is a chemical process that introduces a hydroxyl group (-OH) into an organic compound. In biochemistry, hydroxylation reactions are often facilitated by enzymes called hydroxylases. Hydroxylation is the first step in the oxidative degradation of organic compounds in air. It is extremely important in detoxification since hydroxylation converts lipophilic compounds into water-soluble (hydrophilic) products that are more readily removed by the kidneys or liver and excreted.

1. https://escholarship.org/uc/item/8bh4v2zd#article_main

Storage and Transfer of the Molybdenum Cofactor
Cellular storage systems maintain intracellular levels of essential elements such as transition metals. Proteins are often used in metal storage since they can bind and release metals under specific conditions via protein-protein
interactions or cellular changes. In addition, proteins provide a suitable environment to control metal solubility and reactivity.

For most cofactors, it is not known how, after completion of biosynthesis, these groups are directed to their various cellular destinations and how they ultimately find the way into their correct cognate proteins, or whether they are stored after synthesis. Intricate mechanisms can be assumed to control distribution, trafficking, and insertion into proteins, as most of these prosthetic groups are extremely “fragile” and oxygen-sensitive. For Moco in higher organisms, some pieces of such a sorting machinery became known in recent years. Moco is extremely sensitive to oxidation and therefore is assumed to occur permanently protein bound in the cell.
Moreover, the fast flow of Moco to its target enzymes is an essential prerequisite to reduce the threat of Moco degradation. Both preconditions may be met by Moco-binding proteins (MoBP) ensuring Moco binding as well as its directed transfer to cognate target enzymes. Thus, a pool of insertion-competent Moco may be stored and provided on demand. The availability of sufficient amounts of Moco is essential for the cell to meet its changing demand for Moco arising from newly synthesized Mo-enzymes. Among eukaryotes, a first MoBP named Moco carrier protein (MCP) was identified in the green algae C. reinhardtii. The protein is able to bind and protect Moco against oxidation and the atomic structure showed that it forms a homotetramer capable of holding four molecules of Moco. Without any denaturing procedure, subsequent transfer of Moco from the carrier protein to apo-NR from the Moco-free Neurospora crassa mutant nit-1 was possible, indicating that carrier-bound Moco was readily transferable. These properties of the Chlamydomonas carrier protein make it a promising candidate for being part of a cellular Moco delivery system. It is however unknown whether MCP is also able to donate Moco to Mo-enzymes other than NR. Preliminary data suggest that Mo is bound in a tri-oxo coordinated form in MCP. However, a complex structure of MCP with Moco is still missing.

In bacteria, two families of molybdate storage proteins have been reported.
The first is the molbindin family, present in bacteria and archaea. Members have one or two molybdate-binding (Mop) domains. Both molybdate and tungstate bind to molbindins via multiple hydrogen bonds derived from protein main-chain and side-chain atoms. 
The second group, denoted Mo/WSto, binds both anions via polyoxometalate clusters.97 Crystallization of Mo/Wsto to form Azotobacter vinelandii revealed a hexameric structure formed by a trimer of hetero-dimers. Such a hexamer appears to be able to store up to 100 Mo or W atoms.

The Moco carrier protein (MCP) from C. reinhardtii (16 kDa) forms a tetramer that binds up to four Moco molecules. MCP discriminates between Moco and the Mo-free molybdopterin (MPT), suggesting specific binding and recognition by the protein of the pterin-bound molybdenum centre.

Dimethyl sulfoxide (DMSO) reductase
DMSO reductase is a molybdenum-containing enzyme that catalyzes reduction of dimethyl sulfoxide (DMSO) to dimethyl sulfide (DMS). This enzyme serves as the terminal reductase under anaerobic conditions in some bacteria, with DMSO being the terminal electron acceptor. During the course of the reaction, the oxygen atom in DMSO is transferred to molybdenum, and then reduced to water. 5

Dimethyl sulfoxide reductases (DMSO) are molybdoenzymes widespread in all domains of life. They catalyse not only redox reactions, but also hydroxylation/hydration a and oxygen transfer processes. 

DMSO reductase is a CISM enzyme comprised of three modular subunits, DmsABC.
 
DmsA, the RR-leader containing subunit of the enzyme, functions as the catalytic subunit that coordinates the MobisPGD catalytic cofactor in addition to one [4Fe-4S] 
DmsB serves as the electron conduit subunit coordinating four [4Fe-4S] clusters through conserved Cys residues, and is essential for anchoring to the cytoplasmic membrane via the integral membrane protein DmsC.
Importantly, insertion of DmsC into the cytoplasmic membrane in the absence of DmsAB is lethal or suppresses growth. DmsC is the subunit responsible for subunit anchoring to the membrane and is involved with binding and oxidation 4



Maturation pathway model for DMSO reductase. The cartooned pathway shows stages (10–15). 
1 and 2. The nascent chain exiting the ribosome and the RR-leader interacts with DnaK and trigger factor (TF) 
2. The REMP chaperone DmsD
3. In addition to DnaJ and GrpE, 
4. join the interactome. DmsD interaction with GroEL 
5. allows insertion of the molybdopterin cofactor through contact with MoeB. 
6. After DmsA is fully folded,
7 and 8. the 4[4Fe-4S] center containing DmsB must fold and interact with DmsA. 
9,10,11. With DmsAB assembled, it is targeted to the Tat translocon 
12,13,14. and translocated. 
15. Finally, DmsAB docks to the membrane anchor subunit DmsC to complete the accomplishment of securing a fully functional respiratory enzyme.

4. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/28688222
5. https://en.wikipedia.org/wiki/DMSO_reductase

(A) 3D structure-based NJ-phylogenetic tree  of the CISM-enzymes 

nitrate reductase (Nar), 
DMSO/TMAO reductase (Dms/Dor/Tor), 
arsenate reductase (Arr), 
formate dehydrogenase (Fdh), 
arsenite oxidase (Aro) 
polysulfide reductase (Psr).

Violet and orange denote eury- and crenarchaeal branches, dark green, cyan and light green stand for Proteobacteria, Firmicutes and other Bacteria, respectively. Open and closed dots indicate bootstrap values for the deep branchings exceeding 70 and 90%, respectively. In 3D structures, the Mo-subunit is in violet. (B) Redox potential range of relevant substrates and corresponding enzymes.

The clades corresponding to

formate dehydrogenase (Fdh),
polysulfide reductase (Psr),
arsenite oxidase (Aro)
nitrate reductase (Nar)

by and large resemble species trees, feature a prominent Archaea/Bacteria cleavage and their roots fall in between the archaeal and bacterial subtrees. The combined occurrence of these features strongly suggests the enzymes making up these clades to have been present in LUCA.

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.

This redox versatility in part certainly arises from the fact that molybdenum and tungsten are 2-electron redox compounds, that is, they can shuttle between the +4/+5 and the +5/+6 redox couples. However, it is precisely the property of Mo and W to feature 2-electron transitions which allows these two elements to perform energetically challenging redox conversions.

Several 2-electron compounds under certain circumstances feature so-called crossed-over individual redox transitions which allows them to redox bifurcate electrons with one of the two reducing equivalents going seemingly uphill towards very low potential electron acceptors

It is tempting to hypothesize that the possibility to redox bifurcate electrons may play a crucial role in energetically challenging redox reactions such as the reduction of CO2 to formate in aceto- and methanogens. Both these reactions indeed rely on members of the CISM superfamily.

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3278043/

Metalloproteins/metalloenzymes for the synthesis of acetyl-CoA in the Wood-Ljungdahl pathway

Most models of the primitive atmosphere around the time life originated suggest that the atmosphere was dominated by carbon dioxide, largely based on the notion that the atmosphere was derived via volcanic outgassing, and that those gases were similar to those found in modern volcanic effluent. 4

The atmosphere in the early days of our planet was highly reducing and did not contain oxygen but gases such as molecular hydrogen and carbon dioxide. How life evolved under these conditions is a matter of debate but some researchers favour a pathway that combines two essential features: carbon fixation into organic molecules and, at the same time, generation of ATP [1]. The Wood-Ljungdahl pathway of carbon dioxide fixation is such a pathway and, therefore, could be considered to have evolved on earth very early 6

Conceptually, the simplest way to synthesize an organic molecule is to construct it one carbon at a time. The Wood-Ljungdahl pathway of CO2 fixation involves this type of stepwise process. The biochemical events that underlie the condensation of two one-carbon units to form the two-carbon compound, acetate, have intrigued chemists, biochemists, and microbiologists for many decades. Acetogens are obligately anaerobic bacteria that use the reductive acetyl-CoA or Wood-Ljungdahl pathway as their main mechanism for energy conservation and for synthesis of acetyl-CoA and cell carbon from CO2

When methanogens grow on hydrogen gas H2 + carbon dioxide CO2, they use the Wood-Ljungdahl pathway in the reductive direction (like acetogens) for carbon dioxide CO2 fixation.
Elucidation of the biochemical steps in the Wood-Ljungdahl pathway took many years and required and the use of many biochemical and biophysical methods.

Anaerobic organisms using the Wood-Ljungdahl pathway may have been the first autotrophs, using inorganic compounds like CO and H2 as an energy source and CO2 as an electron acceptor approximately 1 billion years before O2 appeared. Acquisition of the core Wood-Ljungdahl genes would allow a wide variety of organisms, including archaea and a broad range of bacterial phyla to fix CO and CO2 by the reductive acetyl-CoA pathway. The simple strategy of the Wood-Ljungdahl pathway of successively joining two one-carbon compounds to make a two-carbon compound has been envisioned to be the earliest form of metabolism. 5

The only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfill the thermodynamic requirements for life to come into being. Wood–Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism.

The reduction of CO2 with H2 to formate is not an insurmountable task in thermodynamic terms, but the route taken to get there is important, owing to the kinetic stability of CO2. 3

This is about the group of metalloproteins/metalloenzymes in the acetyl−coenzyme A synthesis pathway of anaerobic microbes called Wood-Ljungdahl pathway, including formate dehydrogenase (FDH), corrinoid iron-sulfur protein (CoFeSP), acetyl-CoA synthase (ACS) and CO dehydrogenase (CODH). FDH, a key metalloenzyme involved in the conversion of carbon dioxide to methyltetrahydrofolate, catalyzes the reversible oxidation of formate to carbon dioxide. CoFeSP, as a methyl group transformer, accepts the methyl group from CH3−H4 folate and then transfers it to ACS. CODH reversibly catalyzes the reduction of CO2 to CO and ACS functions for acetyl−coenzyme A synthesis through condensation of the methyl group, CO and coenzyme A, to finish the whole pathway. 1



In this pathway, CO2 is first reduced to formate by the catalysis of formate dehydrogenase (FDH).
Formate is a kind of metabolite existing widely in almost all life forms, and its production or consumption involves the enzyme FDH in most cases. FDH has been found in strict anaerobic microbes such as methanogenic bacteria in archaea and several clostridia. When growing under anaerobic conditions, it can also be produced by multiple facultative enterobacteria including Escherichia coli, Salmonella typhimurium, Proteus strains and Serratia. FDH catalyzes the reversible two-electron oxidation of formate to CO. Sequence analysis and other researches show that FDH is an extremely conserved enzyme. 60 aa residues are absolutely conserved among enzymes from all sources, and the homology scores are even higher than 75% within some individual groups.



Schematic of the main reaction steps and enzymes involved in acetogenic (a) and methanogenic (b) WL-type pathways.
Steps restricted to Bacteria are marked in bright blue colour, whereas those only found in Archaea are shown in violet. Dark blue stands for reactions and enzymes observed in both prokaryotic domains. Proton motif force (pmf)-generating steps are boxed in red in both reactions.

Formate dehydrogenase (FDH)
Formate is an important energy substrate for sulfate-reducing bacteria in natural environments, and both molybdenum- and tungsten-containing formate dehydrogenases have been reported in these organisms. 9 Formate is a key metabolite in anaerobic habitats, arising as a metabolic product of bacterial fermentations and functioning as a growth substrate for many microorganisms (for example, methanogens and sulfate-reducing bacteria [SRB]). Formate is also an intermediate in the energy metabolism of several prokaryotes and a crucial compound in many syntrophic ( cross-feeding ) associations. Formate plays an even more important role in anaerobic microbial metabolism than previously considered.

The key enzyme in formate metabolism is formate dehydrogenase (FDH), a member of the dimethyl sulfoxide (DMSO) reductase family. It catalyzes the reversible two-electron oxidation of formate or reduction of CO2 and plays a role in energy metabolism and carbon fixation. In anaerobic microorganisms, FDH includes a molybdenum or tungsten bis-(pyranopterin guanidine dinucleotide) cofactor and iron-sulfur clusters and shows great variability in quaternary structure, physiological redox partner, and cellular location.

Many prokaryotes are flexible organisms that can thrive under hostile conditions, such as anoxic environments. Under these conditions, formate, produced from pyruvate during anaerobic respiration, serves as a major electron donor to a variety of inducible respiratory pathways that use terminal acceptors other than molecular oxygen. In this case, FDHs are NAD+-independent enzymes containing a complex inventory of redox centres with active sites sensitive to oxygen. These include transition metals, such as molybdenum (Mo), tungsten and non-haem iron, and molybdopterin guanine dinucleotide (MGD) cofactors. FDHs also contain an intrinsic selenocysteine (SeCys) residue. Escherichia coli contains two structurally related, but differentially expressed, molybdopterin dependent respiratory FDHs: formate dehydrogenase-O and formate dehydrogenase-N (Fdh-N). Each is a membrane-bound heterotrimer consisting of two cofactor binding peripheral membrane subunits that associate with a third, integral membrane subunit.

Composition and overall quaternary structure of formate dehydrogenase-N
Fdh-N consists of three subunits: the catalytic a subunit coordinates a bis-MGD cofactor and a [4Fe–4S] cluster, as well as harboring the intrinsic SeCys residue; the b subunit contains one transmembrane helix (TMH) and
coordinates four [4Fe–4S] clusters, which mediate electron transfer between the a and g subunits; and the integral membrane g subunit, which has four TMHs coordinating two haem b groups. The crystal structure of Fdh-N revealed a trimer (a3b3g3) with an overall ‘mushroom’ shape 8


The trimer of Fdh-N viewed parallel to the membrane. 
The catalytic a subunit is in dark blue, the b subunit is shown in red and the integral membrane g subunit is coloured light blue. The catalytic subunit is situated in the periplasmic space (P-side), from where electrons are transferred through the Mo atom (green) and five [4Fe–4S] clusters (grey/yellow) before they cross the membrane through the two haem b groups (black) and then on to the quinone (purple) reduction site on the cytoplasmic side of the membrane (N-side). Cardiolipin molecules (yellow), derived from the native E. coli membrane, can be seen situated in the trimer interface.


Structure and homology of the b subunit. 
(a) Subunit and redox centres coloured as in Figure 1. The two subdomains of the subunit can be discerned, with two [4Fe–4S] clusters each. 
(b) The subunit is highly homologous to other ferredoxin-like proteins, such as the corresponding subunit from hydrogenase (HYBA_ECOLI) and DMSO reductase (DMSB_ECOLI). Cysteines coordinating the [4Fe–4S] clusters are indicated (FeS-1, blue circle; FeS-2, red circle; FeS-3, green circle; FeS-4, black circle).

Molybdenum or tungsten-dependent formate dehydrogenases (FDH) can reduce carbon dioxide (CO2) to formate under ordinary conditions. 7

Molybdenum is a widespread metal in biological systems and it is mainly contained in two classes of enzymes: the nitrogen- fixating nitrogenases, which have an iron-molybdenum cofactor, and the mononuclear molybdenum enzymes (molybdoenzymes). The latter catalyze oxygen-atom transfer reactions to and from biological substrates in the nitrogen, sulfur and carbon cycles. FDH contains a selenocysteine residue bound to the Mo centre which is crucial for its catalytic activity. The structure is made up by four a/b domains, and among them domain I coordinates a Fe4S4 cluster, which is involved in the electron flow. Domains II and III bind the two MGD cofactors. The molybdenum, which is coordinated to two cofactors, the Fe4S4 cluster and the selenocysteine residue, are central to the catalytic activity. In the reduced MoIV form, the molybdenum in the active site is coordinated by four sulfur atoms, which come from the cofactors, and by the selenium atom of the SeCys140 residue. Other residues, His141 and Arg333, are well conserved in all Mo-containing formate dehydrogenases.

FDH belongs to the dymethil sulfoxide reductase family. Unlike other members of the DMSO reductase family, the FDH catalytic reaction involves direct proton transfer, which is the rate-determining step of the whole catalytic process. Bacterial formate dehydrogenases are part of intermembrane complexes, which feature several cubane clusters that carry electrons to the active site of another enzyme, where reduction of a substrate takes place. Several variants of these complexes exist, binding different FDHs which carry out the reduction of different species and are expressed according to different cellular conditions. For instance, three different isoforms a , FDH-H, FDH-N, FDH-O are coupled with, respectively, hydrogen evolution, nitrate reduction and ubiquinone reduction.

In anaerobic organisms, FDH is an NAD+-independent enzyme containing a complex list of metal centers sensitive to oxygen, including tungsten (W), molybdenum (Mo), non-haem iron and molybdopterin guanine dinucleotide (MGD) cofactors





Overall structure of Fdh-N. 
The figures are based on the native Fdh-N structure except for the position of HQNO, which is determined in the Fdh-N and HQNO complex structure. The a, b, and g subunits are shown in brown, blue, and magenta, respectively. Heme groups and MGD cofactors are shown in green, and Mo, cardiolipin (CL), and HQNO are colored in magenta, yellow, and navy, respectively. Five [4Fe-4S] clusters are painted as red (Fe atoms) and yellow (S atoms). (A) Fdh-N trimer viewed parallel to the membrane. (B) Fdh-N trimer viewed from the periplasm along the membrane normal. (C) View of the Fdh-N monomer parallel to the membrane. Center-to-center and edgeto- edge (in parentheses)  distances in angstroms between each of the redox centers are also shown. The edge-to-edge distance for HQNO to heme bC is 0 because HQNO is directly binding to the histidine ligand of heme bC. 2





a  isoform: any of two or more functionally similar proteins that have a similar but not identical amino acid sequence and are either encoded by different genes or by RNA transcripts from the same gene which have had different exons removed.


1. https://sci-hub.tw/https://link.springer.com/article/10.1007/s11426-009-0082-3
2. https://sci-hub.tw/https://www.ncbi.nlm.nih.gov/pubmed/11884747
3. https://sci-hub.tw/https://royalsocietypublishing.org/doi/full/10.1098/rstb.2006.1881
4. https://www.pnas.org/content/99/23/14628
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2646786/
6. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033439
7. https://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S1010603015002403
8. https://sci-hub.tw/https://www.sciencedirect.com/science/article/abs/pii/S0959440X03000988?via%3Dihub
9. https://jb.asm.org/content/193/12/2909