Nitrogenase

The Nitrogenase enzyme,  the molecular sledgehammer  

https://reasonandscience.catsboard.com/t1585-nitrogenase#2406

Currently, there are four genetically distinct nitrogenases, three of which are closely related. The best-studied of the three related enzymes is the conventional molybdenum-based nitrogenase (Mo-nitrogenase). The other two in this group are the alternative nitrogenases, the vanadium-based enzyme (V-nitrogenase), and an enzyme system based on iron alone (Fe-nitrogenase). Although each enzyme has a different heterometal (Mo, V, or Fe), they are otherwise so similar that they must have arisen from a common ancestor. In contrast, the fourth and so far unique nitrogenase, which was isolated from the thermophilic organism, Streptomyces thermoautotrophicus, is so different that it may well have been an evolutionary “independent invention” 8

The amazing story of how scientists struggled for years to duplicate an important bit of chemistry. 1 Great human inventions are usually recognized, with due fame and honour given to those whose work they are. The awarding of the Nobel Prizes is a yearly reminder to us that great achievements are worthy of recognition and reward.

The light-harnessing ability of the chlorophylls (the chemicals that utilize the sun's energy in green plants) might also find a place of honour. Another tiny but marvellous bit of biochemistry which could be nominated to such a position is a mechanism which might be termed ‘the molecular sledgehammer’.



Schematic presentation showing the interaction between the two component proteins of Mo-nitrogenase during catalysis.

To appreciate the work done by this ‘sledgehammer’, it is important to understand the role of the element nitrogen in the living world. The two main constituents of our atmosphere, oxygen (21%) and nitrogen (78%), both play important roles in the makeup of living things. Both are integral parts of the amino acids which join together in long chains to make all proteins, and of the nucleotides which do the same thing to form DNA and RNA. Getting elemental oxygen (O2) to split apart into atoms and take part in the reactions and structures of life is not hard; in fact, oxygen is so reactive that keeping it from getting into where it's not wanted becomes the more challenging job. However, elemental nitrogen poses the opposite problem. Like oxygen, it is diatomic (each molecule contains two N atoms) in its pure form (N2); but, unlike oxygen, each of its atoms is triple-bonded to the other. This is one of the hardest chemical bonds of all to break. So, how can nitrogen be brought out of its tremendous reserves in the atmosphere and into a state where it can be used by living things?

Perhaps this problem can be better appreciated by putting it into terms of human engineering. We need nitrogen for our bodies, to form amino acids and nucleic acids. We must get this nitrogen from our food, whether plant or animal. The animals we eat must rely on plant sources, and the plants must get it from the soil. Nitrogen forms the basis for most fertilizers used in agriculture, both natural and artificial. Natural animal wastes are rich in nitrogen, and it is largely this property that makes them enrich the soil for plant growth. In the late 1800s, a growing population created a great need for nitrogen compounds that could be used in agriculture. At the time, the search for more usable nitrogen was considered a race to stave off Malthusian1 predictions of mass starvation as population outgrew food supply. So chemists wrestled for years with the problem of how to convert the plentiful nitrogen in the air into a form suitable for use in agriculture.

Since naturally occurring, mineable deposits of nitrates were rare, and involved transportation over large distances, an industrial process was greatly needed. Finally, around 1910, a German, Fritz Haber, discovered a workable large-scale process whereby atmospheric nitrogen could be converted to ammonia (NH3). His process required drastic conditions, using an iron-based catalyst with around 1000oF (540oC) heat and about 300 atmospheres of pressure. Haber was given the 1918 Nobel Prize for chemistry because of the great usefulness of his nitrogen-splitting process to humanity.

One might ask, if elemental gaseous nitrogen is such a tough nut to crack, how do atoms of nitrogen ever get into the soil naturally? Some nitrogen is split and added to the soil by lightning strikes. Again, it is a reminder of the force necessary to split the N=N bond that the intense heat and electricity of lightning are needed to do it. Still, only a relatively minor amount of nitrogen is added to the Earth’s topsoil yearly by thunderstorms. How is the remainder produced?

The searching chemists of a century ago did not realize that an ingenious method for cracking nitrogen molecules was already in operation. This process did not require high temperatures or pressures, and was already working efficiently and quietly to supply the Earth's topsoil with an estimated 100 million tons of nitrogen every year. This process’ inventor was not awarded a Nobel Prize, nor was it acclaimed with much fanfare as the work of genius that it is. This process is humbly carried on by a few species of the ‘lowest’ forms of life on Earth—bacteria and blue-green algae (Cyanobacteria).

Some of these tiny yet amazingly sophisticated organisms live in symbiosis (mutually beneficial ‘living together’) with certain ‘higher’ plants, known as legumes. The leguminous plants include peas, soybeans and alfalfa, long valued as crops because of their unique ability to enrich the soil. The microbes invade their roots, forming visible nodules in which the process of nitrogen cracking is carried on.

Modern biochemistry has given us a glimpse of the enzyme system used in this process. The chief enzyme is nitrogenase, which, like hemoglobin, is a large metalloprotein complex.2 Like Fritz Haber’s process, and like catalytic converters in cars today, it uses the principle of metal catalysis. However, like all biological enzymatic processes, it works in a more exact and efficient way than the clumsy chemical processes of human invention. Several atoms of iron and molybdenum are held in an organic lattice to form the active chemical site. With assistance from an energy source (ATP) and a powerful and specific complementary reducing agent (ferredoxin), nitrogen molecules are bound and cleaved with surgical precision. In this way, a ‘molecular sledgehammer’ is applied to the NN bond, and a single nitrogen molecule yields two molecules of ammonia. The ammonia then ascends the ‘food chain’, and is used as amino groups in protein synthesis for plants and animals. This is a very tiny mechanism, but multiplied on a large scale it is of critical importance in allowing plant growth and food production on our planet to continue.

One author summed up the situation well by remarking, ‘Nature is really good at it (nitrogen-splitting), so good in fact that we've had difficulty in copying chemically the essence of what bacteria do so well.’4 If one merely substitutes the name of God for the word 'nature', the real picture emerges.

One thing is certain—that matter obeying existing laws of chemistry could not have created, on its own, such a masterpiece of chemical engineering. To believe that it was worked out by a wise and caring Creator, who provides all necessary things for the life of His creatures, is far more reasonable than the mystical evolutionary alternative. One grows tired of hearing the same monotonous mantra that ‘we know evolution did it, we just don’t know how.’

The system is so complex that elaborate controls are required to regulate when and how rapidly each reaction occurs. The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, or the citric acid cycle, all needing complex enzyme catalytic pathways. A living cell, even the most primitive ones, contain thousands of these enzymes, many of which operate at the same time and in the same small volume of the cytosol( the liquid inside the cell ) .  Not only do you need a encoder to produce the coded information to make the enzymes, but you need the machinery all in place right since the beginning : how could otherwise the machinery be built in a step up fashion, one enzyme after the other, if the end product is only made with all the machines in place and working in a ensemble, and the end product are actually the building blocks of these machines, that make amino acids and ATP ? that is a interdependent system. If one enzyme is not in place, the whole machinery will not work. No amino acids, no ATP ( the fuel in the cell ), no life. Without cyanobacteria - no fixed nitrogen is available. Without fixed nitrogen, no DNA, no amino-acids, no protein can be synthesised. Without DNA, no amino-acids,protein, or cyanobacteria are possible. 


Nitrogen fixing bacteria possess a nitrogenase enzyme complex that catalyses the reduction of molecular nitrogen to ammonia. The nitrogenase enzyme complex consists of two components:

  Component I is nitrogenase MoFe protein or dinitrogenase, which contains 2 molecules each of 2 non-identical subunits.
  Component II is nitrogenase Fe protein or dinitrogenase reductase, which is a homodimer. The monomer is encoded by the nifH gene [PMID: 6327620].

the subunits are unique , and cannot be used in other proteins :

Since the Nitrongenase enzyme is composed of two subunits, set of well-matched, mutually interacting, nonarbitrarily individuated parts such that each part in the set is indispensable to maintaining the system's basic  it can be considered  irreducible complex :


Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase
The iron-molybdenum cofactor (FeMo-co), located at the active site of the molybdenum nitrogenase, is one of the most complex metal cofactors known to date. 2 During the past several years, an intensive effort has been made to purify the proteins involved in FeMo-co synthesis and incorporation into nitrogenase. This effort is starting to provide insights into the structures of the FeMo-co biosynthetic intermediates and into the biochemical details of FeMo-co synthesis. Most biological nitrogen fixation is carried out by the activity of the molybdenum nitrogenase, which is found in all diazotrophs.  The molybdenum nitrogenase enzyme complex has two component proteins encoded by the nifDK and the nifH genes

Nitrogen Fixation: The Mechanism of the Mo-Dependent Nitrogenase
This review focuses on recent developments elucidating the mechanism of the Mo-dependent nitrogenase. This enzyme, responsible for the majority of biological nitrogen fixation, is composed of two component proteins called the MoFe protein and the Fe protein. 3 Recent progress in understanding the mechanism of this enzyme has focused on elucidating the structures of the active site metal clusters and of the proteins, understanding substrate interactions with the active site, defining the flow of electron transfer between the metal clusters, and defining the various roles of MgATP hydrolysis.

Our investigation provides ample support to the fact that NifH protein and BchL share robust structural similarities and have probably deviated from a common ancestor followed by divergence in functional properties possibly due to gene duplication 4

There are at least three different types of nitrogenase known including both Vanadium (V) nitrogenase and iron (Fe) nitrogenase2. These forms of nitrogenase are often found in bacteria.3 The commonly studied and used form of the metalloenzyme is the molybdenum (Mo) nitrogenase.2 It involves a Fe protein and MoFe protein.6 The Fe protein is composed of a [4Fe-4S] cluster and MgATP proteins that help send electrons to the MoFe protein.Meanwhile, the MoFe protein consists of a FeMo active site and P-cluster [8Fe-7S] metals that serve as an intermediate for transferring electrons.7 Equation (1) illustrates the complete reaction of the reduction of N2  5 

Fifteen nitrogen fixation or nitrogen fixation-related genes, including the structural genes for nitrogenase,nifHDK, are clustered together as follows:nifB-fdxN-nifS-nifU-nifH-nifD- nifK-nifE-nifN-nifX-orf2-nifW-hesA-hesB-fdxH. These genes are organized in at least six transcriptional units:nifB-fdxN-nifS-nifU, nifHDK, nifEN,nifX-orf2, nifW-hesA-hesB, and fdxH  6

Nitrogenases are enzymes used by some organisms to fix atmospheric nitrogen gas (N2). There is only one known family of enzymes that accomplishes this process. Dinitrogen is quite inert because of the strength of its N≡N triple bond. 7

In addition to reducing agents, such as dithionite in vitro, or ferredoxin or flavodoxin in vivo, the enzymatic reduction of dinitrogen to ammonia therefore also requires an input of chemical energy, released from the hydrolysis of ATP, to overcome the activation energy barrier. The enzyme is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein. Electrons for the reduction of nitrogen are supplied to nitrogenase when it associates with the reduced, nucleotide-bound homodimeric Fe protein. The heterocomplex undergoes cycles of association and disassociation to transfer one electron, which is the rate-limiting step in nitrogen reduction[citation needed]. ATP supplies the energy to drive the transfer of electrons from the Fe protein to the MoFe protein. The reduction potential of each electron transferred to the MoFe protein is sufficient to break one of dinitrogen's chemical bonds, though it has not yet been shown that exactly three cycles are sufficient to convert one molecule of N2 to ammonia. Nitrogenase ultimately bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH3), which is in turn bonded to glutamate to form glutamine. The nitrogenase reaction additionally produces molecular hydrogen as a side product.

The exact mechanism of catalysis is unknown due to the difficulty in obtaining crystals of nitrogen bound to nitrogenase. This is because the resting state of the MoFe protein does not bind nitrogen and also requires at least three electron transfers to perform catalysis. Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which binds to the enzyme and thereby prevents binding of dinitrogen. Dinitrogen will prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and requires only one electron for reduction to ethylene. All nitrogenases have an iron- and sulfur-containing cofactor that includes a heterometal complex in the active site (e.g., FeMoCo). In most, this heterometal complex has a central molybdenum atom, though in some species it is replaced by a vanadium or iron atom.

Due to the oxidative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to protect nitrogenase from oxygen in vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen. Although the ability of some nitrogen fixers such as Azotobacteraceae to employ an oxygen-labile nitrogenase under aerobic conditions has been attributed to a high metabolic rate, allowing oxygen reduction at the cell membrane, the effectiveness of such a mechanism has been questioned at oxygen concentrations above 70 µM (ambient concentration is 230 µM O2), as well as during additional nutrient limitations.


Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes 
Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, (  Diazotrophs are bacteria and archaea that fix atmospheric nitrogen gas into a more usable form such as ammonia )  including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. 7

Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.

All known diazotrophs contain at least one of the three closely related sub-types of nitrogenase: Nif, Vnf, and Anf. Despite differences in their metal content, these nitrogenase sub-types are structurally, mechanistically, and phylogenetically related. Their catalytic components include two distinct proteins: dinitrogenase (comprising the D and K component proteins) and dinitrogenase reductase (the H protein)

The best studied sub-type is the molybdenum-dependent (Mo-dependent) nitrogenase, the structural components of which are encoded by nifH, nifD, and nifK

The high level of complexity of nitrogenase metalloclusters results in a laborious pathway for the assembly and insertion of the active site metal-cofactor, FeMoco, into dinitrogenase. Apart from the catalytic components, additional gene products are required to produce a fully functional enzyme Although the number of proteins involved in the activation of nitrogenase seems to be species-specific and varies according to the physiology of the organism and environmental niche , so far over a dozen genes have been identified as being involved in this process. Despite variations in the precise inventory of proteins required for nitrogen fixation, it is well acknowledged that the separate expression of the catalytic components is not enough to sustain nitrogen fixation, thus indicating that the FeMoco biosynthetic enzymes play a crucial role in dinitrogenase activation
The current biosynthetic scheme involves a consortium of proteins that assembles the individual components, iron and sulfur, into Fe-S cluster modules for subsequent transformation into precursors of higher nuclearity, and addition of the heteroatom (Mo) and organic component (homocitrat

A method to fix nitrogen was absolutely critical for the early species to fluorish on the early earth; otherwise life at best could only falter along using the scarce fixed nitrogen found naturally. A major task of this early life was to spread fixed nitrogen as food worldwide so that it could be used by more advanced life, and so it had to have an abundant supply. There appears to be only one way to fix nitrogen naturally, and that is with the use of the complex nitrogenase molecule. The nitrogenase molecule is so complex that to date (2010) the procedure that it uses is not fully understood. In any case the process is very slow (taking 1.3 seconds to fix a single nitrogen molecule), and requires not only a very complex molecular process, but it also requires a specialized cell in which oxygen is excluded.

How is such a molecule to be developed by purely natural, undirected processes? As with photosynthesis, the molecule is so complex and unique that it is inconceivable that the molecule could have arisen naturally more than one time in the history of life -- and I would argue that it stretches credulity to think that it could have arisen even one time without a creator's hand.

Nitrogenase is also very scarce. All the world's supply of nitrogenase could be carried in a single bucket. It's not surprising that nitrogen-fixing bacteria had to work for billions of years to make enough nitrogen available for higher plants and animals to thrive. It was a vital task for the early cyanobacteria, along with building the earth's supply of atmospheric oxygen.

There is an irony here: It was vital that cyanobacteria produce oxygen, but oxygen is lethal to the nitrogen-fixing process.  The solution is that the cyanobacteria had to conduct nitrogen-fixing in a specialized cell, called a heterocyst, that was isolated from the photosynthetic activity. The heterocyst has a thick wall to isolate its contents, and it is dependent on other cells for food and energy, which it needs in abundance. In a typical nitrogen-starved medium, about one in 15 cells in a (modern) cyanobacteria chain is a heterocyst .



https://www.youtube.com/watch?v=o5mGO7njcM0


Before we get into the bulk of amino acid biosynthesis there are some things that we really have to understand that don't occur in humans but they are absolutely necessary for human function, what we're going to be talking about is them is the nitrogen cycle okay so nitrogen we always have this idea that  you know wwe eat a steak and we get amino acids well  the amino acids are alread made but somebody had to put the nitrogen in a form where amino acids could be made from that it turned out that nitrogen ultimately comes from the atmosphere. dinitrogen that's just diatomic nitrogen n2 it's two nitrogen atoms joined by a triple bond  that's this n douching right in the middle of the nitrogen cycle it turns out that nitrogen from the atmosphere n2 can undergo what is called nitrogen fixation this arrow right here is going to be very very important for our function because the way that we are ultimately or and I'm going to say indirectly also going to have to use the nitrogen is not in the form of dinitrogen as an in N2 to its in ammonia

The ammonia can then be made into amino acids n2 doesn't do any good for us we need the nitrogen in the form of ammonia  indirectly  the process of going from dinitrogen to ammonia is called nitrogen fixation and in general the organisms the list of them that do this process is very limited the main ones that we're usually concerned with are Klebsiella asoto bacterin rhizobian and those are a lot of bacteria in the soil and these bacteria are known to be obligate anaerobes they don't like oxygen they need to be in an anaerobic environment  they're in the soil and so it's going to become very important to protect the soil because we want to protect these organisms because they give us the nitrogen that we need so we need to kind of save them however ammonia does not just stay like that it's either taken up by certain other organisms and convert it to amino acids or it's put right back into nitrogen and that's done through a series of steps it turns out there's a process called nitrification that can convert ammonia to nitrite and then more nitrification can convert the nitrite to nitrate and the nitrate can undergo a process called denitrification where it goes back into the atmosphere as end to the latter processes.

Nitrification and denitrification don't do any good for us we really need to be thankful for these nitrogen-fixing bacteria because they give us the ammonia and the amino acids indirectly. the nitrogen cycle is very important because we have a balance between taking nitrogen from the atmosphere and putting it into a usable form for us but then there's other organisms that take that usable form for us and convert it back to atmospheric dinitrogen so what we really want to focus is that conversion of nitrogen die nitrogen that is to ammonia.

The conversion is catalyzed by an enzyme called nitrogenase I have here an exercise in activation energy this is an unusual reaction.  Notice one molecule of nitrogen die nitrogen will be converted to ammonia so ever it requires 16 ATP's to do this so for the organisms that do this as in asoto bacter Klebsiella etc.  this is an extremely energetically costly reaction but they do it  so they have to burn 16 ATP to do this process once and also they have to get a total of 8 electrons from already activated molecules, so it turns out this occurs in several stages number one:

we're going to have either a ferrodoxin  or a flavodoxin these are electron carrying proteins and they're going to pick up electrons from usually four Coenzyme a's and for pyruvates now from the purpose of the bacteria it's not as much of a waste for the 16 ATP because they get out four acetyl co a's but the electrons are going to go into either ferredoxin or flavodoxin then those electrons are transferred to an enzyme that is part of the whole enzyme complex so it turns out nitrogenase has two parts of it it has a dinitrogenase and a dinitrogenase reductase the dinitrogenase on the bottom is the part that actually catalyzes the conversion of nitrogen to ammonia the dinitrogenase reductase gives the electrons to dinitrogens, the electrons come to the reductase part through the ferredoxin or flavodoxin so in other words dinitrogenase reductase is going to pick up electrons from ferredoxin or flavodoxin and it's going to be reduced then because it got an electron it's then going to bind 16 ATP now a lot of the ATP's function here is binding energy because it turns out this reaction is very unfavorable. 

Notice it has an activation energy over 940 kilojoules per mole that's enormous for an activation energy  so the ATP binding the reason you have to have so much of it is you're really trying to put some binding energy in there to lower the activation energy ok so that ATP binding is actually very important once the ATP is bound the dinitrogenase reductase can transfer electrons one at a time to dinitrogen. Once that ATP is bound , dinitrogenase reductase can transfer electrons one at a time to dinitrogenase. in which case dinitrogenous reductase is going to be oxidized back to its oxidized form and then that ATP is going to be hydrolyzed to ADP and phosphate and you're back to no ATP bound oxidize dinitrogenase reductase which can pick up more electrons from the ferredoxin or flavodoxin and but back down here to the bottom remember we had with ATP bound die nitrogenous reductase transferred electrons to dinitrogenase so now die nitrosegenous is in the reduced state and dinitrogenase is directly the species that transfers the electrons to nitrogen okay so it's going to take a total of eight cycles like this eight electrons - totally reduce nitrogen to the two ammonia's so to completely get rid of an atmosphere of dinitrogen it takes eight electrons and 16 ATP and that's partly because the activation energy is so massive. 

So just to do a quick recap the dinitrogen gets two electrons from dinitrogenase that gets its electrons from dinitrogenase reductase that gets its electrons from either ferredoxin or flavodoxin which gets its electrons from pyruvate and coenzyme a. So this is another example of what we call an electron transport chain but this particular one we don't do this is not in US this is only a nitrogen-fixing bacteria ie Klebsiella  Rhizobium.

The triple NN bond is strong (942 kJ/mol) and its cleavage requires a six-electron reduction. 2

1. http://creation.com/the-molecular-sledgehammer
2. http://www.annualreviews.org/doi/full/10.1146/annurev.micro.62.081307.162737
3. http://informahealthcare.com/doi/pdf/10.1080/10409230391036766
4. http://www.ias.ac.in/jbiosci/nov2013/733.pdf
5. http://chemwiki.ucdavis.edu/Wikitexts/UC_Davis/UCD_Chem_124A%3A_Berben/Nitrogenase/Nitrogenase_2
6. http://jb.asm.org/content/183/2/411.full#xref-ref-110-1
7. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207485/
8. Hermann Bothe: The book Biology of the Nitrogen Cycle 2007 https://www.sciencedirect.com/book/9780444528575/biology-of-the-nitrogen-cycle

More info:
https://rationalwiki.org/wiki/Nitrogen_fixation

The Natural History of Nitrogen Fixation.

http://www.ncbi.nlm.nih.gov/pubmed/14694078/

Abstract

In recent years, our understanding of biological nitrogen fixation has been bolstered by a diverse array of scientific techniques. Still, the origin and extant distribution of nitrogen fixation has been perplexing from a phylogenetic perspective, largely because of factors that confound molecular phylogeny such as sequence divergence, paralogy, and horizontal gene transfer. Here, we make use of 110 publicly available complete genome sequences to understand how the core components of nitrogenase, including NifH, NifD, NifK, NifE, and NifN proteins, have evolved.These genes are universal in nitrogen fixing organisms-typically found within highly conserved operons-and, overall, have remarkably congruent phylogenetic histories. Additional clues to the early origins of this system are available from two distinct clades of nitrogenase paralogs: a group composed of genes essential to photosynthetic pigment biosynthesis and a group of uncharacterized genes present in methanogens and in some photosynthetic bacteria. We explore the complex genetic history of the nitrogenase family, which is replete with gene duplication, recruitment, fusion, and horizontal gene transfer and discuss these events in light of the hypothesized presence of nitrogenase in the last common ancestor of modern organisms, as well as the additional possibility that nitrogen fixation might have evolved later, perhaps in methanogenic archaea, and was subsequently transferred into the bacterial domain.

So, basically, no answer of how these genes could have been result of evolution.... Just guesswork, as always...

Biologically available nitrogen, also called fixed nitrogen, is essential for life. All known nitrogen-fixing organisms (diazatrophs) are prokaryotes, and the ability to fix nitrogen is widely, though paraphyletically, distributed across both the bacterial and archaeal domains . The capacity for nitrogen fixation in these organisms relies solely upon the nitrogenase enzyme system, which, at 16 ATPs hydrolyzed per N2 fixed, carries out one of the most metabolically expensive processes in biology

Maintaining the ability to fix nitrogen in the presence of exogenous or endogenous sources of O2 has necessitated innovative biochemical and physiological mechanisms for segregation.  Certain cyanobacteria contribute substantial amounts of fixed nitrogen in marine environments and do so because of exquisite controls on temporal and spatial separation of the two processes.


Based on phylogenetic reconstruction as well the presence of nitrogenase in diverse archaea as well as bacteria, it has been inferred that the nitrogenase family had already evolved in the last common ancestor (LCA) of the three domains of life

Wow... thats telling.....

http://www.asu.edu/news/research/bacterial_genomes_040704.htm


Of all of evolution's great biochemical developments, the ability of life to break up and "fix" atmospheric nitrogen  was one of the most important accomplishments, and perhaps one of the most challenging.

"At some point though, things reached a food crisis - you either find some way to get the atmosphere's molecular nitrogen into the cycle or you die. A minimum input of nitrogen can't sustain a big biosphere," he noted.

Thats funny. So evolution had the goal to develop and sustain a big biosphere ??!!

"But it is hard to do. Nitrogen fixation is one of the most interesting biological processes because it's so difficult to do chemically. Nitrogenase is a very complex enzyme system that actually breaks molecular nitrogen's triple bond -- one of the strongest bonds in nature," he said.

The nitrogenase system is so sophisticated and complex that it is difficult to reconstruct its evolutionary development.


How about just admit its impossible ??!!

In some of its most sophisticated forms, such as versions incorporating the rare metal molybdenum, the system uses a network of complex enzymes to control and regulate the process and make it energy efficient.

Such a system could have evolved gradually through a series of small changes, but the analysis suggests instead that it might have developed through duplication of the gene for a more primitive enzyme that has just now been discovered.

Why would it do that ? The selection pressure to produce a big biosphere just won't cut it. And how about the intermediate steps which would have no function ?

However, even the simplest enzyme capable of breaking nitrogen's triple bond requires great structural complexity that could not have evolved without earlier stages.

What early stages ??

"Breaking molecular nitrogen required a lot of energy and was an evolutionarily complex transition," Blankenship notes. "Even the most basic nitrogenase complex that we have today is amazingly sophisticated and energetically a very expensive system. It's not something that would have just popped up out of nowhere."

Its that sophisticated, that even today, scientists with all their knowledge and intelligence are unable to understand, even less to copy the mechanism...... Id did not pop up out of nowhere, neither could it have arised by a step up evolutionary mechanism. Design is the only reasonable explanation.

What selection pressure at all would have existed, to produce such a extremely complex enzyme, and the  P-cluster and FeMo-co which are among the most complex metalloclusters known ? What selective advantage would have a unfinished metallocluster after all ? It is only fully operational when fully assembled, and put in the right place .

"Evolution is a great recycler, a junkman who takes some piece that was invented for some other purpose and reuses it in a new way," Blankenship said. "Once you do that, you can end up with some amazing things, like the nitrogenase complex - a stunningly complicated molecular machine that does extremely difficult chemistry."

Thats the funny part of the paper. Some sort of scientific joke..... A proposal like Millers of the Flagellum :


co-opting parts from other biological systems. That copying, modifying, and combining together preexisting parts , already operating in other systems, would do the job. But, is it really ? Could it be, that super evolutionary mechanisms would act that way, borrowing parts from other biological systems and assemble them to a nitrogenase enzyme with a new function , perfectly ordered, with perfect fits, and new functions,with the help of saint time , that would do that miracle ? Even thinking, that time in this case would rather be detrimental, than help ? Would it really be, that such a extremely complex and  and energetically a very expensive system could arise by copy/pasta , by a supernatural pick and add , a molecular quilt and patchwork mechanism? and you believe in Santa Claus, as well ? Thats not only insane, but completely impossible.


On the whole, these ideas suggest that the extraneous (e.g., regulatory and assembly) components of the nitrogenase system will provide an important and informative test bed for understanding the evolutionary history of nitrogenase.

http://www.annualreviews.org/doi/full/10.1146/annurev.micro.62.081307.162737

The P-cluster and FeMo-co are among the most complex metalloclusters known

The structural genes for the MoFe protein, nifD and nifK, are not required for the biosynthesis of FeMo-co

It is accepted that FeMo-co is assembled separately in the cells and is finally incorporated into a FeMo-co-deficient apo-MoFe protein.

How could this process be result of evolutionary mechanisms ? The nitrogenase enzyme functions only, if fully assembled .

A number of nitrogen fixation (nif) genes are required for the biosynthesis of FeMo-co and maturation of the nitrogenase component proteins from folded polypeptides to their metallocluster-containing catalytically active forms.




When synthesized, the nitrogenase components are not immediately competent for nitrogen fixation. Rather, they become mature by the actions of several nif and non-nif gene products to achieve catalytic competency.

How did evolutionary mechanisms forsee the necessity of maturing  and evolve  several gene products for doing so  ?  

Due to its enormous complexity, the study of the maturation of the apo-MoFe protein is especially challenging to biochemists. Many pieces of this puzzle have been already placed. We know that the Fe protein is required for the synthesis of functional P-clusters of the apo-MoFe protein and that the reaction directed by the Fe protein promotes a conformational change within the apo-MoFe protein that leaves the FeMo-co insertion site accessible

Overview of the Mo-dependent Nitrogenase Mechanism
To understand the nitrogenase mechanism, we must understand the workings of each of the two component proteins, both alone and when associated with each other. The Mo-dependent nitrogenase is composed of two separable component proteins. The Fe-protein component binds two equivalents of ATP (usually with Mg2+ as the counter ion). The Fe protein, with bound MgATP, associates with one ab-dimer half of the MoFe protein, at an ab interface. Each half of the MoFe protein acts as a catalytic unit, with two Fe proteins binding to each MoFe protein. Following docking of an Fe protein to an ab-dimer half of the MoFe protein, the MgATP molecules bound to the Fe protein are hydrolyzed to MgADP plus Pi, and a single electron is transferred from the Fe protein [4Fe-4S]1+ cluster to the MoFe protein. The current model is that the electron is first accepted in the MoFe protein by the P-cluster and then is ultimately transferred to the FeMo-cofactor (called FeMoco), where substrates are bound. Following electron transfer and MgATP hydrolysis, the Fe protein probably dissociates from the MoFe protein . 

The MgADP molecules bound to the dissociated Fe protein are exchanged with MgATP and the oxidized [4Fe-4S]2+ cluster is reduced again to the 1+ oxidation state. The Fe protein is then poised for another round of binding to the MoFe protein, electron transfer, MgATP hydrolysis, and component-protein dissociation. Given the need for six electrons for the reduction of N2, plus the need for two additional electrons for the obligate reduction of two protons to give H2, this cycle of Fe-protein binding and dissociating must occur a minimum of eight times for each equivalent of N2 reduced. There are two intersecting catalytic cycles, with one cycle for the Fe protein and one for the MoFe protein.

The steps of each cycle represent discrete chemical events (e.g., electron transfer, MgATP hydrolysis, component protein association, etc.) and the intersection of the two protein cycles occurs when the two proteins are associated. The overall goal of the field is to define all of the steps of these cycles at a molecular level, thus yielding a molecular mechanism for nitrogenase. As we shall see, some of the steps are well understood, whereas for others, much remains to be elucidated. We start with the Fe-protein cycle and then proceed onto the MoFe-protein cycle. Finally, we finish with a summary of some of the open issues remaining in understanding this enzyme.

What sets nitrogenase apart from essentially all other enzymatically catalyzed redox processes is the number of electrons (eight) that must ultimately be delivered to the substrates each turnover cycle, with the consequent demand for precise timing of the underlying electron transfer events. 3

The first part of this mechanism consists of a cycle involving the ATP-dependent electron transfer between the two protein components of nitrogenase, named Fe protein and MoFe protein.
The second part of the process  involves substrate reduction on the MoFe protein when sufficient cycles of intermolecular electron transfer have occurred.

All nitrogen-fixing prokaryotic organisms utilize relatively large amounts of ATP and an enzyme known as nitrogenase to fix nitrogen (Figure below). This process occurs in three steps. In the first step, a molecule of nitrogen gas (N2) binds to nitrogenase. In the second step, the bound nitrogen is reduced by the addition of two hydrogen atoms (2 H), a reaction powered by the breakdown of ATP. Such a reduction occurs three times, with the addition of a total of three hydrogen atoms to each nitrogen atom. In a third and final step, two molecules of ammonia (NH3) are released and dissolve in cell water to form NH4+. The nitrogenase enzyme is then free to bind more N2. Because the O2 molecule resembles N2, oxygen can bind to the active site of nitrogenase. Oxygen binding disables nitrogenase, thereby stopping nitrogen fixation.. 

In the nitrogenase reaction , electrons from reduced ferredoxin pass to nitrogenase reductase, which serves as electron donor to nitrogenase, the enzyme that actually catalyzes N2 fixation. Electron transfer from nitrogenase reductase to nitrogenase takes place through docking of nitrogenase reductase with an ab-subunit pair of nitrogenase. Nitrogenase reductase transfers e+ to nitrogenase one electron at a time. N=N (or the substrate analog CqO) binding to FeMoCo displaces one of the nine S atoms in FeMoCo;  Fe7MoS8, with the C atom of CO occupying the site where the ninth S atom would be in the substrate-free complex. N2 is bound within the FeMo-cofactor metal cluster until all electrons and protons are added.

During substrate turnover, the Fe protein forms a transient complex with the MoFe protein and transfers electrons from its [Fe4S4] cluster to the P-cluster of MoFe protein in an ATP-dependent manner.  The electrons are subsequently transferred to FeMoco, where substrates are eventually reduced after the accumulation of certain amounts of electrons.


Nitrogen only binds to the cofactor when it has undergone three electron transfer reactions (i.e. three single electron reductions). One mol of ammonia is released when the cofactor has undergone five electron transfer reactions, and the second is released after seven electron transfer reactions.

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. 4 A multiplicity of the genes are involved, the oxygen sensitivity of nitrogenase, plus a high demand for energy and reducing power. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power.

The overall reaction mechanism of biological nitrogen fixation  may be divided into two parts:

(i) the control or regulation of electron transfer to the substrate reduction site and
(ii) the substrate reduction process itself.

Electron Transport in Nitrogenase: 
The association of nucleotide-bound Fe-protein subunits to the MoFe protein subunit leads to the release of an electron from ATP towards the first Fe4S4 cluster, which shuttles the electron towards the larger P-cluster. The P-cluster gains further electrons when another Fe-protein binds, eventually passing them on towards the FeMo-co reactive center, which, by the way, has a central carbon atom with 6 bonds to it. Note that this entire process goes on simultaneously on the other half of the protein, making nitrogenase that much more efficient. 15

Prevention of Oxidation:
For a successful dinitrogen reduction to occur, it is imperative that any, and all oxidizing agents are kept well clear of the catalytic site. Rather conveniently, the binding of Fe-proteins subunits to the Fe-Mo protein, using a 'β-grasp' structure, seals off the Fe-Mo protein from oxidation. Even if oxidation were to occur, it would be at the interface of the smaller FeS cluster, thereby protecting the Fe-Mo-co cluster and facilitating further enzymatic reactions to occur.  

Nitrogenase Pathway
The FeMo protein binds substrate and reduces H+ and N2 to H2 and ammonia, while the Fe protein receives electrons from ferredoxin, hydrolyzes ATP, and reduces the FeMo protein.  12
The Lowe and Thorneley (LT) model has been proposed as a mechanism for dinitrogen reduction.  In this model an electron and proton are added to the oxidized form of the enzyme (Eo) to produce E1.  This is repeated 3 more times to form sequentially, E2, E3 and E4.   Only then does N2 bind and the reduction of N2 occur.  Two of the added electrons are accepted by H+ ions which form H2, which is liberated on N2 binding. 18



Exploring new frontiers of nitrogenase structure and mechanism 16
28th February 2006
The mechanism of the complex enzyme nitrogenase has long been one of the most challenging problems in bioinorganic chemistry. The complexity of the metal centers of nitrogenase has stretched the boundaries of biochemical, physical and computational tools for providing insights into its structure and chemical function. Nitrogen fixation has always been considered of fundamental importance, not only for its significance in global nutrition, but also because of the relevance of nitrogenase as a model system for examining processes such as multiple electron oxidation-reduction reactions, complex biological metal assembly, and even nucleotide-dependent signal transduction. Nitrogenase occurs in molybdenum, vanadium and iron forms, with unique metal centers located at the sites of nitrogen binding and reduction. Molybdenum nitrogenase is by far the most extensively characterized. The Fe protein is a
60 kD homodimer that contains a single [4Fe–4S] cubane and functions in MgATP hydrolysis and electron transfer to the substrate reduction component, the
230 kD MoFe protein heterotetramer. 

An essential step in the nitrogenase mechanism occurs when the Fe protein, with two bound MgATP molecules, associates with the MoFe protein. This associated complex is fleeting, existing for about 1 s during normal substrate reduction. Several events occur while the two proteins are associated, including the hydrolysis of the two MgATP molecules to two MgADP and two Pi molecules and the transfer of one electron from the Fe protein to the MoFe protein. The order of these two events has not been definitively established and is the subject of current studies.

Docking occurs so that the two-fold symmetry axis surrounding the Fe protein’s [4Fe-4S] cluster becomes paired with the surface of the MoFe protein’s pseudosymmetrical ab-interface. This places the [4Fe-4S] cluster close to the P-cluster of the MoFe protein and places the P-cluster between the Fe protein [4Fe-4S] cluster and the FeMo cofactor. This arrangement indicates that the P-clusters role is to broker electron transfer between the Fe protein and the substrate reduction site provided by the FeMo cofactor.




Evidence for functionally relevant encounter complexes in nitrogenase catalysis 14
September 11, 2015
Here, we provide evidence for the first time that encounter complexes between FeP and MoFeP play a functional role in nitrogenase turnover. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the β-subunit of MoFeP. There is mounting evidence that electron transfer (ET) reactions between protein partners are complex, multi-step processes that proceed through the initial formation of a dynamic ensemble of encounter complexes. These complexes then lead to a specific, functionally active docking geometry. ATP binding and hydrolysis not only provide a timing mechanism for the successive ETinteractions between FeP and MoFeP, but likely also coordinate the downstream, multi-electron catalytic reactions.



We examined the functional significance of the positively charged β399-401 patch on the MoFeP surface. In summary, our results indicate the following:
1) FeP interaction with the β399-401 patch of MoFeP is largely electrostatic in nature, as evidenced by the NaCl inhibition experiments and the structure of the DG1 complex itself.
2) Interactions involving the MoFeP β399-401 patch and FeP are functionally important for nitrogenase catalysis and are populated along a productive reaction pathway. They serve to increase the association rate constant between FeP and MoFeP.
3) Interactions between the MoFeP β399-401 patch and FeP are not operative during ATP/ET coupling processes that take place within the activated complex.

All of these features are characteristic of dynamic encounter complexes, which have been implicated in many protein-protein interactions of electron transfer ET partners, as well as those that are regulated by nucleotide binding and hydrolysis.Our data, coupled with the available structural information, suggest a new picture regarding the initial steps of nitrogenase turnover (Figure above). docking geometries DG1 conformation is likely a multistep process. We postulate that FeP and MoFeP first form an ensemble of loosely bound, electrostatically guided complexes centered at the β399-401 patch, with the corresponding association and dissociation rates. The DG1 complex then transitions to the activated DG2 conformation. 

During catalysis, the Fe protein delivers electrons, one at a time, to the MoFe protein in a process that couples MgATP binding and hydrolysis to the association and dissociation of the two-component proteins and concomitant electron transfer. Both component proteins are required for MgATP hydrolysis, and neither component protein reduces any substrate in the absence of its catalytic partner.  So they are interdependent. The process by which electrons are sequentially delivered to the MoFe protein and subsequently to substrate has been described by a kinetic model that involves two interlocking cycles called the Fe protein cycle and the MoFe protein cycle.



 The Fe protein cycle involves oxidizing and reducing the Fe protein’s [4Fe-4S] cluster between the 1+ and 2+ redox states as it sequentially delivers electrons to the MoFe protein and is re-reduced by other electron transfer proteins (usually a ferredoxin or flavodoxin ). The MoFe protein cycle involves the progressive reduction of the MoFe protein, which ultimately leads to N2 binding and reduction. Because eight electrons and eight protons are required for N2 reduction and H2 evolution, each MoFe protein cycle requires eight Fe protein cycles and storage of the electrons. Kinetic studies have also shown that N2 does not become bound to the active site until at least two, and probably more, electrons have been accumulated within the MoFe protein. It is not yet known where and how the electrons delivered to the MoFe protein are stored before to the binding and reduction of the substrate. 13

The P-cluster is located at the ab interface of the MoFe protein subunits and, in its fully reduced form, is constructed from two [4Fe-4S] subclusters that share a central sulfide. Upon oxidation of the P-cluster, a structural rearrangement occurs involving movement of two Fe atoms and a change in the ligand arrangement around the cluster. Such redox-dependent structural changes within the P-cluster might be mechanistically related to its role in accepting electrons from the Fe protein and delivering them to the FeMo cofactor.

Nitrogenase demonstrates the amazing catalytic ability of iron-sulfur clusters in biological systems. Nitrogen fixation is carried out by a highly reduced form of dinitrogenase and requires eight electrons: six for the reduction of N2 and two to produce one molecule of hydrogen H2. Production of H2 is an obligate part of the reaction mechanism, but its biological role in the process is not understood. Dinitrogenase is reduced by the transfer of electrons from dinitrogenase reductase (Fig. below). The dinitrogenase tetramer has two binding sites for the reductase. The required eight electrons are transferred from reductase to dinitrogenase one at a time: a reduced reductase molecule binds to the dinitrogenase and transfers a single electron, then the oxidized reductase dissociates from dinitrogenase, in a repeating cycle. Each turn of the cycle requires the hydrolysis of two ATP molecules by the dimeric reductase. The immediate source of electrons to reduce dinitrogenase reductase varies, with reduced ferredoxin, reduced flavodoxin, and perhaps other sources playing a role. In at least one species, the ultimate source of electrons to reduce ferredoxin is pyruvate.The role of ATP in this process is somewhat unusual.  

In the reaction carried out by dinitrogenase reductase, both ATP binding and ATP hydrolysis bring about protein conformational changes that help overcome the high activation energy of nitrogen fixation. The binding of two ATP molecules to the reductase shifts the reduction potential (E′°) of this protein from −300 to −420 mV, an enhancement of its reducing power that is required to transfer electrons through dinitrogenase to N2; the standard reduction potential for the half-reaction N2 + 6H+ + 6e− → 2NH3 is −0.34 V. ATP binding produces a conformational change that brings the 4Fe-4S center of the reductase closer to the P cluster of dinitrogenase (from 18 Å to 14 Å away), which facilitates electron transfer between the reductase and dinitrogenase.




Electron path in nitrogen fixation by the nitrogenase complex.
Electrons are transferred from pyruvate to dinitrogenase via ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase reductase reduces dinitrogenase one electron at a time, with at least six electrons required to fix one molecule of N2. Two additional electrons are used to reduce 2H+ to H2 in a process that obligatorily accompanies nitrogen fixation in anaerobes, making a total of eight electrons required per N2 molecule.


The details of electron transfer from the P cluster to the FeMo cofactor, and the means by which eight electrons are accumulated by nitrogenase, are not known. Nor are the intermediates in the reaction known with certainty;  two reasonable hypotheses are being tested, both involving the Mo atom as a central player. 




Two reasonable hypotheses for the intermediates involved in N2 reduction. 
In both scenarios, the FeMo cofactor (abbreviated as M here) plays a central role, binding directly to one of the nitrogen atoms of N2 and remaining bound throughout the sequence of reduction steps.

The nitrogenase complex is remarkably unstable in the presence of oxygen. The reductase is inactivated in air, with a half-life of 30 seconds; dinitrogenase has a half-life of only 10 minutes in air. Free-living bacteria that fix nitrogen cope with this problem in a variety of ways. Some live only anaerobically or repress nitrogenase synthesis when oxygen is present. Some aerobic species, such as A. vinelandii, partially uncouple electron transfer from ATP synthesis so that oxygen is burned off as rapidly as it enters the cell. 












A general catalytic mechanism scheme for nitrogenase.
A) Components I and II are dissociated; II is ready for reduction. 
B) ATP binds to component II, which receives electrons from an electron donor (ferredoxin or flavodoxin); binding of ATP induces an allosteric conformational change which allows association of the two proteins. Electrons flow from the [4Fe-4S] cluster on II to the P cluster on I. 
C) Electrons are further shuttled to the iron-molybdenum cofactor (FeMoco), and ATP is hydrolised to ADP. This step is repeated several times before a molecule of N2 can bind to FeMoco. 
D) The protein complex dissociates, and nitrogenase reduces dinitrogen to ammonia and dihydrogen. I: component I (dinitrogenase; MoFe protein); II: component II (dinitrogen reductase; Fe protein); ATP: adenosine triphosphate; ADP: adenosine diphosphate; Fdx: ferredoxin; Fld: flavodoxin.

The precise order of some of these steps is not totally understood, and therefore this scheme should not be seen as an established mechanism. 1

[4Fe-4S] cluster was traced from PDB file 2NIP; P cluster and FeMoco were traced from PDB file 1M1N.


Fe and MoFe protein catalytic cycles. 
Shown is a three state cycle for the Fe protein (top) and an eight state cycle for the MoFe protein (bottom). For the Fe protein (abbreviated FeP), the [4Fe-4S] cluster can exist in the +1 reduced state (Red) or the 2+ oxidized state (Ox). The Fe protein either has two MgATP molecules bound (ATP) or two MgADP with two Pi (ADP+Pi). The exchange of an electron occurs upon association of the Fe protein with the MoFe protein at the bottom of the cycle. In the MoFe protein cycle, the MoFe protein is successively reduced by one electron, with reduced states represented by En, where n is the total number of electrons donated by the Fe protein. Acetylene (C2H2) is shown binding to E2, while N2 is shown binding to E3 and E4. N2 binding is accompanied by the displacement of H2. The two ammonia molecules are shown being liberated from later E states.

In this cycle, the reduced Fe protein, with its [4Fe-4S] cluster in the 1+ oxidation state, has two bound MgATP molecules. It is this state that transiently associates with the MoFe protein. During this association, the two MgATP molecules are hydrolyzed to two MgADP molecules, and a single electron is transferred from the Fe protein [4Fe-4S] cluster into the MoFe protein. The oxidized Fe protein ([4Fe-4S]2+), now with two bound MgADP molecules, then dissociates from the MoFe protein in what is the overall rate-limiting step for nitrogenase catalysis. The released Fe protein is then regenerated in two steps. The MgADP molecules are replaced by MgATP, and the [4Fe-4S]2+ cluster is reduced to the 1+ oxidation state, with the order of these two events being unclear. The physiological reductant for the Fe protein depends on the organism, with reduced ferredoxin or flavodoxin being the most common immediate electron donor. Many questions remain about mechanistic details of the Fe protein cycle. For example, the specific nature of the communication between the nucleotide binding sites within the Fe protein and MoFe protein interface remain obscure. Prior to association with the MoFe protein, the Fe protein shows undetectable rates of MgATP hydrolysis. Once bound to the MoFe protein, the hydrolysis of MgATP is activated. Given that the MgATP binding sites within the Fe protein are located some distance away (> 10 Å) from the MoFe protein docking interface, this situation demands protein conformational changes induced within the Fe protein that activate MgATP hydrolysis. Two specific “switch” regions (stretches of amino acids) within the Fe protein have been proposed to function to communicate between the docking interface and the MgATP binding site, resulting in movement of key catalytic residues that result in activation of nucleotide hydrolysis. Although broad strokes of this process have been defined, details of the mechanism remain unresolved. Likewise, little is known about how MgATP binding and hydrolysis within the Fe protein is communicated into the MoFe protein. That such a communication must occur is suggested by the observation that the Fe protein is the only known reductant for the MoFe protein that will support substrate reduction. An ability of small-molecule electron donors to reduce the MoFe protein without an attendant ability to support substrate reduction indicates that changes within the MoFe protein are induced by the Fe protein and must be coupled to MgATP hydrolysis. Other reviews provide a detailed historical perspective on the role of the Fe protein in nitrogenase catalysis.

HOW ARE SUBSTRATES REDUCED?
Using the reductant sodium dithionite in vitro, the Fe protein is alternately oxidized and re-reduced as it delivers single electrons to the MoFe protein in a process that couples MgATP binding and hydrolysis to Fe protein–MoFe protein
electron transfer and the association and dissociation of the two component proteins (see Fig. below). 


A modified Lowe–Thorneley scheme for Mo-nitrogenase catalysis. 
(a) The Fe-protein cycle describes the one-electron redox reactions of the Fe protein’s [4Fe-4S] cluster, nucleotide exchange of the spent MgADP and phosphate (Pi) for MgATP, and complex formation with and electron transfer to the MoFe protein, when dithionite is used as reductant. Each turn of this cycle adds one electron to the MoFe protein. Fe represents the Fe protein in its oxidized (ox) or one-electron reduced (red) state; MoFe represents the MoFe protein with the number of electrons accepted shown as (n) or (n+1); the dashed arrow indicates that the MoFe(n+1) protein can reenter this cycle and accept additional electrons and protons. Source: Modified from Lowe and Thorneley, 1984. 
(b) The MoFe-protein cycle for N2 reduction. The dithionite-reduced (resting state) of the MoFe protein is designated as E0 and, with each turn of the Fe-protein cycle, the MoFe protein goes through a succession of increasingly reduced states (E1, E2, …, E7) as electrons and protons are accepted until sufficient and are accumulated for substrate reduction. An important concept of this scheme is that different substrates bind reversibly to different MoFe-protein redox states, for example, N2 binds (as shown) at either E3 or E4 most likely by displacing H2, whereas C2H2 binds at either E1 or E2. H2 may be evolved from several redox states (as shown). The reduced-nitrogen intermediates shown are postulated and not proved. Each solid arrow (−→) represents one turn of the Fe-protein cycle, i.e., one hydrogenation (H+∕e−) event with concomitant MgATP hydrolysis and each dashed arrow represents a substrate-binding or product-release event. Source: Modified from Lowe and Thorneley, 1984.

The Fe protein alone is capable of binding MgATP, but both component proteins are required for MgATP hydrolysis. Neither component protein alone, with or without MgATP and/or reductant, will reduce substrate under the usual assay conditions. Use of a different reductant, for example, either Ti(III) or the in vivo reductant, flavodoxin hydroquinone, may impact one or more of the steps in this process. The overall reduction of N2 to yield two molecules of NH3 is thermodynamically favorable. So, why is MgATP required? If it is not a thermodynamic requirement, it must be for kinetic reasons. Most likely, MgATP binding helps drive electron transfer toward substrate reduction by increasing the difference in redox potential of the electron donor and acceptor and its subsequent hydrolysis, and complex dissociation ensures the irreversibility of the reaction. This so-called gating mechanism allows no backflow of electrons to the Fe protein and prevents energy-wasting futile cycling of electrons. In this way, multiple electrons are accumulated within the MoFe protein and its bound substrate. This view of the catalytic process actually describes only one part of the process, the so-called Fe-protein cycle. A more detailed description has been developed that involves two interconnecting processes: the Fe-protein cycle (Fig. a above) and theMoFe-protein cycle (Fig.b above). Here, a mechanistic simplification, which treats each αβ-subunit pair (with its encapsulated prosthetic groups) of the MoFe protein as an independently operating catalytic entity, is employed, even though long-range interactions between the two αβ-subunit pairs have been detected.

 The MoFe-protein cycle involves the progressive reduction of the MoFe protein (plus bound substrate) by up to eight electrons for N2 binding and reduction, which therefore requires eight turns of the Fe-protein cycle. Partially reduced-nitrogen intermediates must remain on the enzyme until the reduction cycle is finished because NH3 is the only product of catalyzed N2 reduction by Mo-nitrogenase. Major concerns are how and where the eight electrons necessary for the reduction of each N2 (accompanied by one H2) are accommodated within the MoFe protein and how the required protons are delivered. This situation has been simplified by the assertion that the metal core of the FeMo-cofactor has only two accessible redox states, the EPR-active resting state (called MN) and the one-electron-reduced EPR-sil14ent state (calledMR). All other electrons delivered from the Fe protein must, therefore, reside on either partially reduced substrate or inhibitor. For N2 to be bound to the active site, either three or four electrons must have been accumulated within the MoFe protein. If only one electron can be accommodated by the FeMo-cofactor, which would then become EPR-silent, where do the other two electrons reside before N2 binds? Most likely, they reside as either a hydride (H−) or hydrogen atom (H•) that subsequently becomes either H2 or used to partially reduce N2. Similarly, the four-electron-reduced MoFe protein (labeled as E4H4 in Fig.above) would equate to an oxidized EPR-active FeMo-cofactor with two bound hydrides. In fact, this latter conclusion has been experimentally demonstrated with both hydrides proposed as μ2-bridges between Fe atoms (as Fe−H−Fe) on the FeMo-cofactor rather than as either Mo−H−Fe or on μ2-/μ3-sulfur atoms (as>S−H)

THE MECHANISM OF Mo-DEPENDENT NITROGENASE: THERMODYNAMICS AND KINETICS
The biological fixation of N2 occurs in select prokaryotes, with representatives in both the bacteria and the archaea. In all cases, the initial reduction of N2 to ammonia is catalyzed by the enzyme nitrogenase. There are two distinct classes of nitrogenase currently known. One class is represented by the recently discovered superoxide-utilizing nitrogenase from Streptomyces thermoautotrophicus. The other class of nitrogenase is composed of two component proteins, with one component being a homodimer with a single [4Fe-4S] cluster bridging the subunits and containing a nucleotide (e.g., ATP) binding site on each subunit. This protein has been called the Fe protein, component II, dinitrogenase reductase, or azoferredoxin. An alternative nomenclature for this component employs a two-letter abbreviation of the source organism followed by 2 (e.g., Cp2 for the Fe protein from Clostridium pasteurianum). The Fe protein serves as a one-electron reductant of the other nitrogenase component protein in a reaction that is coupled to the hydrolysis of at least two molecules of MgATP. The other nitrogenase component protein has a core tetrameric subunit arrangement and contains two copies of two unique Fe-S clusters. An additional subunit  is associated with some nitrogenases.  One Fe-S cluster, called the P-cluster, is a [8Fe-7S] cluster. The P-cluster probably functions as the immediate electron acceptor from the Fe-protein component, delivering electrons to the active-site metal cluster. An active-site cluster is located in each subunit and is a M/Fe/S/homocitrate-containing metal cluster (where M = Mo, V, or Fe). Nitrogenases, where M is either V or Fe, are often referred to as “alternative” nitrogenases. The present review focuses on the Modependent nitrogenase system, where M is Mo, and the active-site metal cluster has the composition [Mo-7Fe-9S-homocitrate-X] and is called the FeMo-cofactor. The presence of an unknown atom X (suggested to be N, C, or O) at the center of the FeMo-cofactor has recently been discovered in a high resolution X-ray crystal structure (Einsle et al., 2002). The nitrogenase component protein that binds the FeMo-cofactor is called the MoFe protein, component I, dinitrogenase, or molybdoferredoxin. The alternative nomenclature uses a two-letter abbreviation for the source organism followed by 1 (e.g., Cp1 for the MoFe protein from C. pasteurianum). Although many features of the Mo-dependent nitrogenase mechanism will be transferable to our understanding of the alternative nitrogenases, it is clear that these latter enzymes have many unique properties as well. 

At a minimum, 16 equivalents of MgATP are hydrolyzed for each equivalent of N2 fixed. Using concentrations of nucleotides typically found in bacteria, the free energy change for the hydrolysis of MgATP to MgADP plus Pi is calculated to be ca. -45 kJ/mol. Thus, the hydrolysis of 16 equivalent of MgATP per N2 fixed amounts to a tremendous energy expenditure for the cell. How is this energy “utilized” in the nitrogenase reaction? The reduction of N2 as catalyzed by nitrogenase is likely to proceed through the two- and fourelectron/ proton-reduced intermediates, diazene and hydrazine.  As can be seen from the enthalpies of formation for both of these compounds, these steps are substantially uphill from N2. A first analysis might suggest that the energy liberated from MgATP hydrolysis is simply “coupled” to the unfavorable steps in the reduction of N2, making the overall reaction thermodynamically favorable. Our current understanding of the nitrogenase mechanism suggests a different view, one involving a much more complex usage of energy.


The Role of MgATP in Nitrogenase Catalysis
The reduction of N2 to yield 2 NH3 is thermodynamically favorable. Thus, the need for MgATP binding and hydrolysis during nitrogenase catalysis is kinetic so that electron transfer toward substrate reduction is favored and the flow of electrons back toward the Fe protein is prevented. One way of envisioning this process is to consider that the energy released through MgATP binding and hydrolysis could be used to open and close electron gates to ensure that multiple electrons are accumulated within the MoFe protein before they are donated to substrates. In support of this model, primary sequence and structural comparisons have revealed that the Fe protein is a member of a large class of signal transduction proteins that undergo conformational changes upon MgATP binding and hydrolysis. A consensus view of the events that occur during a single turn of the Fe protein cycle and which accounts for the role of MgATP in nitrogenase catalysis is as follows. 

Intercomponent interaction is initiated when the reduced Fe protein binds two MgATP molecules. This elicits a conformational change in the Fe protein that makes it competent to interact with the MoFe protein. Upon complex formation, changes occur in the midpoint potentials of the respective clusters such that electron flow toward the FeMo cofactor is energetically favorable. For example, in the complexed form, the Fe protein’s [4Fe-4S] cluster has a redox potential of about -620mV, the P-cluster a potential of about -390mV, and the FeMo cofactor a potential of about -40mV. In addition to eliciting redox changes that lead to electron transfer, the component docking event also triggers MgATP hydrolysis at about the same time as, or shortly after, electron transfer. Conversion of the Fe protein from the MgATP-bound state to the MgADP-bound state subsequently causes complex dissociation, which is believed to be the rate-limiting step in nitrogenase catalysis. Thus, the accumulation of electrons within the MoFe protein is a dynamic process that involves transmitting signals back and forth between the Fe protein and MoFe protein. Then the role of MgATP is to synchronize these events through sequential conformational changes induced by MgATP binding, component protein interaction, and nucleotide hydrolysis.

Breaking the N2 triple bond: insights into the nitrogenase mechanism 5
2006 May 21
Understanding how the nitrogenase active site metal cofactor (FeMo-cofactor) catalyzes the cleavage of the N2 triple bond has been the focus of intense study for more than 50 years. Goals have included the determination of where and how substrates interact with the FeMo-cofactor, and the nature of reaction intermediates along the reduction pathway. Progress has included the trapping of intermediates formed during turnover of non-physiological substrates (e.g., alkynes, CS2) providing insights into how these molecules interact with the nitrogenase FeMo-cofactor active site. More recently, substrate-derived species have been trapped at high concentrations during the reduction of N2, a diazene, and hydrazine, providing the first insights into binding modes and possible mechanisms for N2 reduction. A comparison of the current state of knowledge of the trapped species arising from non-physiological substrates and nitrogenous substrates is beginning to reveal some of the intricacies of how nitrogenase breaks the N2 triple bond. Reduction of the N2 triple bond to form two ammonia molecules (Chart below) is a chemically demanding reaction1 requiring six electrons and six protons. This reduction reaction constitutes the key step in the global biogeochemical nitrogen cycle, and is thus essential to all life



A major contributor to the global reduction of N2 is biological nitrogen fixation, which occurs exclusively in a select group of microbes. These microbes contain a complex metalloenzyme called nitrogenase that catalyzes the optimal reaction. At least four different types of nitrogenase have been characterized, with the most significant difference between the systems being the metal constitution of the active site metallocofactor where N2 binds and is reduced. The most widely distributed and studied type of nitrogenase is composed of two component proteins, one called the Fe protein (also called component II or dinitrogenase reductase) and the other called the MoFe protein (also called component I or dinitrogenase), which work together as a molecular machine to catalyze the reduction of N2 at ambient temperature and pressure. The Fe protein functions as a reductant of the MoFe protein, transferring one electron at a time from its [4Fe–4S] cluster to the MoFe protein in a reaction linked to the hydrolysis of MgATP. As part of the dynamics of the process, the Fe protein dissociates from its partner MoFe protein following each electron transfer event, allowing the Fe protein to be recharged by reduction and replacement of the spent nucleotides with MgATP. A minimum of eight such association/ dissociation events is required for each N2 reduced. The MoFe protein contains the site of N2 binding and reduction,  a complex metal cluster called FeMo-cofactor [7Fe–9S–Mo–Xhomocitrate].

None of the eight intermediate stages meets the goal to split nitrogen. Let me outline this. The author of above paper mentions a " optimal reaction", which is achieved by two components working together as a molecular machine !! That raises the question : Why would natural selection or any evolutionary mechanism evolve such intermediate stages, if there is no survival advantage to be gained by it ? This is an all or nothing business. Either the whole process goes through, eight times, or nitrogen is not split. 

TheMoFe protein contains an additional metal cluster called the P-cluster [8Fe–7S] that mediates electron transfer from the Fe protein to FeMo-cofactor. Many questions remain to be answered about how nitrogenase catalyzes the difficult N2 reduction reaction, including how N2 binds to FeMo - cofactor and the nature of the intermediates formed along the reduction pathway to products. Progress over the last few years has provided insights into the binding and mechanism of reduction for  non-physiological substrates (e.g., alkynes),15 but it has only been over the last year that substrate-derived species have been trapped on FeMo-cofactor during the reduction of N2 and other nitrogenous substrates. These newly trapped species are providing insights into the mechanism of this complex reaction, and are guiding future research directions. This review focuses on the current state of knowledge of states trapped during N2 reduction and what these bound species are telling us about how nitrogenase breaks the N2 triple bond.

Nitrogenase active-site FeMo-cofactor
It has been known for many years that N2 binds and is reduced at the complex metallocluster called FeMo-cofactor. A significant step forward in understanding substrate reduction by nitrogenase came from solution of X-ray crystal structures of the nitrogenase component proteins, first individually, and more recently as the complex. From these structures, a molecular model of FeMo cofactor has evolved. This cofactor is composed of an [4Fe–3S] subcluster bridged by three l2-sulfides to a [Mo–3Fe–3S] subcluster. The organic acid (R)-homocitrate provides two O-ligands to the Mo and the cofactor is covalently attached to the protein through coordination of the terminal Fe by the side chain of a-275Cys and coordination of the Mo by the side-chain of a-442His (the numbering of amino acids is indexed to the proteins from Azotobacter vinelandii). A central atom of unknown identity (X) was more recently identified as a constituent of FeMo-cofactor based on electron density observed in a higher resolution X-ray structure of the MoFe protein than previously available. The electron density of X is compatible with it being N, C, or O and a number of calculations on fragments of FeMo-cofactor have favored X = N. Contrary to these results has been recent experimental evidence indicating that X is unlikely to be N. In these latter studies, bacteria were grown on 14N- or 15N-urea as the exclusive nitrogen source and the nitrogenase proteins and FeMo-cofactors were isolated from these bacteria. ENDOR analysis of these protein and FeMo-cofactor samples revealed no 15N-that stayed associated with FeMo-cofactor, indicating that X was not N. The model of the FeMo-cofactor provided by the X-ray structures of the MoFe protein suggests a number of possible binding sites for substrates, including one or more of the Fe atoms arranged in three symmetrically related [4Fe–4S] faces at the central waist region, the Mo, or some combination of sites. Unfortunately, the structure does not differentiate between the many possible binding modes and provides no information about possible intermediates in the reaction pathway. This has prompted a number of theoretical studies of varying degrees of sophistication to predict possible substrate binding sites and mechanisms of reduction. These calculations have yielded a wide range of possibilities, and no consensus has yet emerged. Recent experimental work with nitrogenase indicates that N2, hydrazine, and alkynes interact with a common 4Fe–4S face in the waist region of FeMo-cofactor composed of Fe atoms. The strategy and experimental results that have led to this conclusion have been reviewed recently. While these studies suggest a general location for substrate binding, a remaining challenge is to further refine the specific site of interaction of substrates and to define the reduction intermediates along the reaction pathway.







New insights into the evolutionary history of biological nitrogen fixation  1

Nitrogenase, which catalyzes the ATP-dependent reduction of dinitrogen (N2) to ammonia (NH3), accounts for roughly half of the bioavailable nitrogen supporting extant life. The fundamental requirement for fixed forms of nitrogen for life on Earth, both at present and in the past, has led to broad and significant interest in the origin and evolution of biological N2 fixation. One key question is whether the limited availability of fixed nitrogen was a factor in life's origin or whether there were ample sources of fixed nitrogen produced by abiotic processes or delivered through the weathering of bolide impact materials to support this early life. If the latter, the key questions become what were the characteristics of the environment that precipitated the evolution of this oxygen sensitive process, when did this occur, and how was its subsequent evolutionary history impacted by the advent of oxygenic photosynthesis and the rise of oxygen in the Earth's biosphere. Since the availability of fixed sources of nitrogen capable of supporting early life is difficult to glean from the geologic record, there are limited means to get direct insights into these questions. Indirect insights, however, can be gained through phylogenetic studies of nitrogenase structural gene products and additional gene products involved in the biosynthesis of the complex metal-containing prosthetic groups associated with this enzyme complex. Insights gained from such studies, as reviewed herein, challenge traditional models for the evolution of biological nitrogen fixation and provide the basis for the development of new conceptual models that explain the stepwise evolution of this highly complex life sustaining process.

All life requires fixed sources of nitrogen (N) and its availability is what often limits productivity in natural systems . Most N on Earth is in the form of dinitrogen (N2), which is not bio-available. On early Earth, fixed sources of N may have been supplied by abiotic processes such as electrical (i.e., lightning) based oxidation of N2 to nitric oxide (NO) or mineral (e.g., ferrous sulfide) based reduction of N2 , nitrous oxide , or nitrite (NO−2)/nitrate (NO−3)  to NH3. Abiotic sources of fixed N (e.g., NO, NO−2, NO−3, NH3) are thought to have become limiting to an expanding global biome , which may have precipitated the innovation of biological mechanisms to reduce N2.

The primary enzyme that catalyzes the reduction of N2 to bio-available NH3 today is the molybdenum (Mo)-dependent nitrogenase (Nif) although other phylogenetically-related forms of nitrogenase that differ in their active site metal composition (termed alternative nitrogenase, or Vnf & Anf) may also contribute NH3 in environments that are limiting in Mo . Nitrogenase catalyzes the production of half, if not more, of all of the fixed nitrogen on Earth today.. As such, this process functions to relieve fixed N limitation in natural ecosystems  and is likely to have a disproportionate effect on the functioning of an ecosystem, relative to inputs from other populations. Thus, organisms which fix nitrogen in natural communities have been described as keystone species .

The taxonomic distribution of nitrogenase is curiously restricted to bacteria and archaea, with no known examples of the genes encoding for this process occurring within the eukarya . Within the archaea, nitrogenase has a narrow distribution and is restricted to methanogens (Euryarcheota) within the orders Methanococcales, Methanobacteriales, Methanosarcinales and has yet to be identified among members of the Crenarchaeota, Thaumarchaeota, or Nanoarchaeota. Likewise, nif exhibits a limited distribution among bacteria. For example, nif has been identified in a number of aerobic soil bacteria and has been identified in the genomes of 21 of the 44 sequenced cyanobacterial genomes, including those that inhabit terrestrial (e.g., Cyanothece and Synechococcus strains) and marine (Crocosphaera watsonii) environments. In addition, nif gene clusters are commonly detected in the genomes of Firmicutes, Chloroflexi, Chlorobi, and Bacteroidetes and in several lineages of Actinobacteria and Proteobacteria.

non-filamentous cyanobacteria tend to operate on a diurnal cycle where N2 fixation is up-regulated at night when oxygen tensions have dropped due to concomitant decreases in the production of photosynthetic O2 and increased O2 consumption by co-inhabiting heterotrophic populations. Alternatively, the co-occurrence of N2 fixation and O2 production in filamentous cyanobacteria is made possible by spatial segregation of nitrogenase in anaerobic heterocyst structures where increased protection of the nitrogenase complex is achieved through the photoreduction of O2 to H2O in photosystem I

Question: had this segregation not have to be present in the bacteria right from the start, otherwise one process would poison the other ?

In contrast, in obligate aerobes the nitrogen fixation apparatus is protected by what has been described as a cytochrome-dependent respiratory protection mechanism whereby high rates of respiration ensure the consumption of oxygen at the cell membrane thereby maintaining low intracellular oxygen tensions

It is likely that these mechanisms emerged later in the evolutionary history of biological nitrogen fixation due to the increased complexity of nif gene clusters associated with microorganisms adapted to fixing nitrogen in an oxygenated atmosphere

The simplest assemblages of specific genes associated with nitrogen fixation occur in strict anaerobes. Nevertheless, tracing the evolutionary trajectory of this process and identifying the most ancient nitrogen fixers present in extant biology has been a challenge.

New insights into the evolutionary history of biological nitrogen fixation
Biological nitrogen fixation has been suggested to be an ancient and perhaps even primordial process. This prevailing view is based on simulations of Archaean atmospheric chemistry that contend that decreasing CO2 concentrations and concomitant decreases in abiotic N2 oxidation to NO led to a nitrogen crises at ~3.5 Ga. However, using the same logic, Navarro-González argue that the nitrogen crisis could have ensued much later, even as late as 2.2 Ga. Abiotic sources of nitrogen produced through mechanisms such as lightning discharge or mineral based catalysis are thought to have become limiting to an expanding global biome. Since extant nitrogenase functions to relieve N limitation in ecosystems, the imbalance in the supply and demand for fixed N is thought to have represented a strong selective pressure that may have precipitated the emergence of nitrogen fixation. Little direct evidence exists, however, with respect to the availability of ammonia or other reduced forms of nitrogen over the course of geological time, although several recent isotopic analyses of shale kerogens have suggested ample enough supply of ammonia to support nitrifying populations in the late archean, >2.5 Ga.

While the geologic record cannot yet definitively reconcile when fixed sources of nitrogen became limiting, one can ask the general question of whether the overall distribution and phylogenetic history of nitrogenase and its associated functionalities in extant biology are consistent with a primordial process or a property of the Last Universal Common Ancestor (LUCA). Although widely distributed among bacteria, the distribution of the process is far from universal among archaea, and as previously mentioned has never been identified among members of the eukarya. Moreover, unlike processes and functionalities that we ascribe to properties of LUCA, nitrogenase is not generally (note caveat below) associated with deeply rooted lineages identified by 16S ribosomal RNA evolutionary trajectories.

Our recent screening of two representative Aquificales genomes [i.e., Thermocrinis albus and Hydrogenobacter thermophilus] reveal the presence of nitrogenase gene clusters. The identification of nif gene clusters in the genomes of thermophilic members of the Aquificales, regarded by many as the most deeply rooted bacterial lineage, prompted a re-analysis of the distribution of nif on a depiction of the taxonomic tree of life. Although this analysis suggests that deeply rooted bacteria encode for nif (e.g., Aquificales) the limited distribution of nif among deeply branching archaea (e.g., Thaumarchaeota, Nanoarchaeota lineages) and deeply branching bacteria (Thermus/Deinococcus) suggests that nif may have been subject to extensive gene loss/lateral gene transfer or was not a property of the Last Universal Common Ancestor (LUCA). If Nif was a property of LUCA, then phylogenetic analyses of nif gene or protein sequences, would be expected to reveal reciprocally monophyletic bacterial and archaeal lineages (e.g., subtrees containing just archaeal homologs and bacterial homologs joined at LUCA). However, our previous maximum likelihood and Bayesian phylogenetic analyses of a concatenation of the structure proteins required for nitrogen fixation (homologs of H, D, and K,) indicate that archaea are paraphyletic with respect to bacteria, suggesting that Nif emerged after the divergence of archaea and bacteria. Additionally, our current maximum likelihood analysis of a concatenated HDK protein alignment block (Figure (Figure2)2) indicates that Nif proteins from deeply rooted thermophilic members of the Aquificales were acquired recently through a lateral gene transfer with a more recently evolved and thermophilic member of the bacterial phylum Deferribacteres (e.g., ancestor of Calditerrivibrio nitroreducens or Denitrovibrio acetiphilus) (Figure (Figure2).2). This suggests that Aquificales acquired nif in the recent evolutionary past from an exchange with a bacterial partner in a thermal environment. In further support of this hypothesis, numerous Aquificales genera (e.g., Hydrogenobaculum) do not encode nif (Romano et al., 2013), despite branching more basal than Thermocrinis and Hydrogenobacter in 16S rRNA gene phylogenetic reconstructions (Eder and Huber, 2002). Hydrogenobaculum spp. tend to populate acidic geothermal environments where NH+4 produced from magmatic degassing is in much higher supply (Holloway et al., 2011) whereas Thermocrinis and Hydrogenobacter tend to populate circumneutral to alkaline environments that are N limited (Reysenbach et al., 2005). Thus, the recent diversification of Aquificales into N limited environments may have been facilitated by acquisition of nif. Together, these findings add to a growing body of evidence suggesting that lateral gene transfer has played a significant role in expanding the taxonomic and ecological distribution of N2 fixation (Raymond et al., 2004; Kechris et al., 2006; Bolhuis et al., 2009).



Although this analysis suggests that deeply rooted bacteria encode for nif (e.g., Aquificales) the limited distribution of nif among deeply branching archaea (e.g., Thaumarchaeota, Nanoarchaeota lineages) and deeply branching bacteria (Thermus/Deinococcus) suggests that nif may have been subject to extensive gene loss/lateral gene transfer or was not a property of the Last Universal Common Ancestor (LUCA).

Thats interesting. Can't trace phylogeny back to a common ancestor, invent a different mechanism ( lateral gene transfer ) , that justifies the evolutionary framework.

WHEN DID BIOLOGICAL NITROGEN FIXATION APPEAR?
The ability of organisms to fix N2 and so grow on N2 as the sole nitrogen source is called diazotrophy. Most likely, diazotrophy did not evolve until the geochemical fixed-nitrogen reserves of the biosphere were depleted. Do we have any idea of when this was in geological time? Unfortunately, we do not. The major problem here is the considerable uncertainty about the composition of the pre-biological paleo-atmosphere, which makes it very difficult to estimate how long any fixed nitrogen source would persist after becoming accessible to the biosphere. One view is that the atmosphere may have been strongly reducing, containing ammonia, methane, carbon monoxide and hydrogen sulfide ; another view is that it may have been more mildly reducing, containing mostly N2, carbon dioxide and water vapor. Whatever the situation with respect to the atmosphere, the appearance of free O2 on the planet would have had a major impact. Its presence would result in any geochemical ammonia being converted into N2 and nitrogen oxides. Eventually, these nitrogen oxides would become limiting for growth and the resulting selective pressure would produce diazotrophy. In such a scenario, assimilatory nitrate/nitrite reduction would pre-date diazotrophy. However, if so, the bigger question is where would the free O2 have come from? If it had a biological origin, such as O2-producing photosynthesis, this source would help date the rise of diazotrophy. However, very low levels of free O2 might have been produced non-biologically by photolysis of water at a much earlier geological time than the appearance of O2-producing photosynthesis. 2

One final scenario takes account of the facts that diazotrophy is limited to the most primitive organisms on Earth and that the three classical nitrogenases are so very similar wherever they are found. This approach suggests that classical diazotrophy is an ancient process that arose just once in evolutionary time. If so, the known haphazard distribution of diazotrophy among prokaryotes could be explained as a common ancestral property that is being lost randomly during divergent evolution. This suggestion is supported by some of the earliest phylogenic studies based on both 16S rRNA and nifH/D sequences. Those results suggest that the nitrogen-fixation genes are established components of their genomes and that they have been present there for as long as those backgrounds have existed.

But would diazotrophy be needed in these ancient times when the atmosphere may have contained sources of fixed nitrogen? And, if they are so old, why do nitrogenases show relatively little divergence in their composition and structure? And why then have plants (or other higher life forms) not acquired the ability to fix N2? The reason has to involve more than the presence of O2 because cyanobacteria, which also perform plant-type O2-generating photosynthesis, and other aerobic organisms have developed strategies that allow them to fix N2. An alternative explanation is that nitrogen fixation is of more recent origin and its observed distribution could be due to lateral gene transfer among diverse prokaryotic genera, much like the spread of antibiotic resistance. This argument is consistent with the structural similarity of nitrogenases, even across the Eubacteria and Archaea kingdoms, the location of nitrogen-fixation genes on plasmids in many species, and the ease with which nitrogen-fixation genes are transferred among bacterial species in the laboratory. It is likely that both arguments have merit; they are certainly not mutually exclusive.

The evolutionary roots of nitrogen fixation, inferred from molecular phylogenetic analyses of nifH genes, suggest that the genes encoding for nitrogenases are ancient and underwent horizontal gene transfer, gene duplication, recruitment and fusion 3  Indeed, phylogenic analyses do not rule out the possibility that nitrogenase was present in the last universal common ancestor (LUCA) and, therefore predates the divergence of the archaea and bacteria.

Nitrogen is an essential nutrient for all organisms that must have been available since the origin of life. Abiotic processes including hydrothermal reduction, photochemical reactions, or lightning discharge could have converted atmospheric N2 into assimilable NH4 1, HCN, or NOx species, collectively termed fixed nitrogen. But these sources may have been small on the early Earth, severely limiting the size of the primordial biosphere. The evolution of the nitrogen-fixing enzyme nitrogenase, which reduces atmospheric N2 to organic NH4 1, thus represented a major breakthrough in the radiation of life, but its timing is uncertain5,6. Here we present nitrogen isotope ratios with a mean of 0.0 6 1.2% from marine and fluvial sedimentary rocks of prehnite–pumpellyite to greenschist metamorphic grade between 3.2 and 2.75 billion years ago. These data cannot readily be explained by abiotic processes and therefore suggest biological nitrogen fixation, most probably using molybdenum-based nitrogenase as opposed to other variants that impart significant negative fractionations. Our data place a minimum age constraint of 3.2 billion years on the origin of biological nitrogen fixation and suggest that molybdenum was bioavailable in the mid-Archaean ocean long before the Great Oxidation Event. 4

We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. 5 While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O2 in metabolism, the increase is not correlated with any of the known O2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles.

The genes encoding the two nitrogenase subunits are highly conserved but are widely dispersed across many phyla of bacteria and archaea, which suggests that nitrogen fixation evolved once and subsequently spread by vertical inheritance and by horizontal gene transfer.  Is that a suggestion based on evidence, or just guesswork ?

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733012/
2. Nitrogen Fixation: Origins, Applications, and Research Progress VOLUME 1, page 3
3. http://ocean.mit.edu/~mick/Papers/BermanFrank-etal-NitrogenBook-2008.pdf
4. http://www.nature.com.https.sci-hub.hk/articles/nature14180
5. http://jb.asm.org/content/197/9/1690.full
6. http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.465.3125&rep=rep1&type=pdf

NITROGEN FIXATION

NITROGEN FIXATION: A CASCADE OF GENE AND OPERON DUPLICATION

Nitrogen fixation is widespread in bacteria and archaea.  The enzyme responsible for nitrogen fixation is called nitrogenase. All known Mo-nitrogenase consist of two components: the dinitrogenase, a 22 tetramer encoded by nifD and nifK genes, and the dinitrogenase reductase, a homodimer coded for by the nifH gene. Nitrogenase contains two metal clusters, one of which is the iron-molybdenum cofactor (FeMo-co), the site of dinitrogen reduction and whose synthesis requires the activity of an another tetrameric enzymatic complex (Nif N2E2) whose subunits are encoded by nifE and nifN. The detailed analysis of the nifDK and nifEN pairs of genes showed that their products share common features (Fani et al., 2000): they code for tetrameric complexes, and the products of nifE and nifN are structurally related to the nifD and nifK products, respectively. Finally, those diazotrophs in which nifDK and/or nifEN have been characterized share the same gene organisation. The four genes are clustered in operons where the two genes of each pair are contiguous and arranged in the same order (nifDK and nifEN). Moreover, the four genes shared a high degree of sequence similarity suggesting that they belong to a paralogous gene family. Fani et al (2000) proposed a two-steps evolutionary model leading to these genes. The model proposes the existence of a single ancestral gene that underwent an in-tandem gene duplication event, which gave rise to a bicistronic operon. Then, this ancestral operon duplicated leading to the ancestors of the present-day nifDK and nifEN operons.

If the ability to fix nitrogen was a primordial property, then the duplication events leading to the two operons predated the appearance of the last common ancestor of Archaea and Bacteria. Thus the function(s) performed by the primordial enzyme would have evolved because of the composition of the atmosphere. Theories vary from strongly reducing to neutral; but it is generally accepted that O2 was absent, an essential prerequisite for the evolution of an ancestral nitrogenase, as free oxygen inactivates it. The first living organisms were probably heterotrophic anaerobes ( heterotrophic anaerobes" means they were creatures which ate some naturally occurring food and did not breathe oxygen )  and dependent on abiotically produced organic matter for their metabolism. Depending on the composition of the early atmosphere (neutral or reducing), the ancestor gene coded for an enzyme with a nitrogenase or a detoxyase activity, respectively. The first duplication event, leading to the ancestral bicistronic operon, followed by divergence, refined the specificity of the primitive nitrogenase/detoxyase, which might have also been involved in the biosynthesis of a Fe-Mo cofactor. Successively the ancestral operon duplicated and the following divergence lead to the appearance of the ancestors of the present day nifDK and nifEN operons which encoded different proteins involved in reducing substrates and biosynthesize FeMo cofactor, respectively. Thus, the ability to fix nitrogen appears to be an ancient property and might have arisen during an early period of cellular evolution. The corresponding genetic information could have been lost in many strains, possibly in the course of adaptation to changing environmental conditions and the associated selective pressures upon microorganisms. Nevertheless, the high degree of conservation of nitrogenase genes within archaeal and bacterial diazotrophs suggests that lateral transfer of nif genes might have occurred frequently.

Two pathways of electron transport to nitrogenase in Rhodospirillum rubrum: the major pathway is dependent on the fix gene products 1
19 May 2006
We have shown that reductant for nitrogenase in R. rubrum can be generated through two different pathways, one directly dependent on PMF and the other not utilizing PMF. In the former the Fix proteins catalyze the transfer of reductant generated in the carbon metabolism to Ferredoxin N that we have shown is the primary electron donor to dinitrogenase reductase. In our model for the electron transfer to nitrogenase shown in Fig. 3, we suggest that reductant in this pathway is supplied by NAD(P)H or possibly by direct coupling between FixAB and dehydrogenases as has been suggested in A. caulinodans. We have previously shown that FdI can substitute for FdN but we have not identified under what physiological conditions this may occur. The NifJ-dependent transfer of reductant to nitrogenase we suggest plays a more central role under dark anaerobic conditions, although, nitrogenase activity is less than 15% of the activity in the light in wild-type R. rubrum.



1. http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00297.x/full

Demolishing the Myth of Intelligent Design

First, why was the nitrogen-fixing propensity conferred only upon primitive microbes and not on vascular plants, which are desperate for a supply of nitrate? Some vascular plants have the ability to fix atmospheric nitrogen because of bacteria resident in their root nodules. Yet the only plants that possess root nodules are the Leguminosae. If nitrogen fixation has been intelligently designed, then it should be present in all forms of plant that require it. Even if the propensity has to be restricted to bacteria, and nothing larger, then why have these organisms formed an association with a single family of vascular plants? Surely, the Rosaceae and Graminae should also have root nodules. If cereal plants could fix nitrogen naturally, as do the legumes with their resident bacterial partners, then grain crops would always have been more abundant and their productivity increased. Whoever decided to exclude the ability from
every other family was the antithesis of an intelligent designer.

https://www.academia.edu/3633314/Critical_Focus_13_Demolishing_the_Myth_of_Intelligent_Design

The nitrogenase enzyme

http://reasonandscience.heavenforum.org/t2590-molecular-biochemistry-biology-the-origin-of-life-and-biodiversity-sistematically-analyzed-from-a-universal-perspective#5867

If the foundation of a house is not solid, the whole house is subject and exposed to crumble. In the quest of origins, before someone debates, if Darwinian evolution is credible, it must be elucidated, if it makes sense to believe, that the organisms, responsible to synthesize the basic building blocks of life could have emerged through evolution.  As is comes out, remarkably, the most complex biosynthesis pathway, photosynthesis,  and the most complex proteins and metal-clusters and co-factors, namely Nitrogenases, required to make ammonia, used to make amino acids, are the most ancient proteins, going back 2,5 - 3 billion years by evolutionary timescale, or even back to the Last Universal Common Ancestor ( Luca ), and did not change afterwards.  How does that fit to an evolutionary scenario, where everything supposedly got more complex gradually? Nitrogenase is rarely debated, no wonder: to understand how the mechanism works, it takes weeks, if not month of intense study. And then to understand, how it is synthesized and assembled, even more. Science is working over half a century to understand the details of its structure, functions, and its mechanism, and many questions are still open. But one thing is already clear: Evolution hardly explains its origins. Nitrogenase requires eight rounds to catalyze the reaction, and split dinitrogen.  

Because 78% of the Earth’s atmosphere consists of nitrogen gas (N2), it may seem that nitrogen should not be in short supply for organisms. However, nitrogen is often a limiting factor in ecosystems because N2 molecules must be broken apart before the individual nitrogen atoms are available to combine with other elements. Because of its triple bond, N2 is very stable, and only certain bacteria can break it apart and incorporate its atoms into usable forms such as ammonia (NH3). This process, called nitrogen fixation, is a critical component of the five-part nitrogen cycle

The conversion of dinitrogen gas (N2) to ammonia (NH3) is called nitrogen fixation. Because ammonia is necessary for the formation of biologically essential, nitrogen-containing compounds, such as amino acids and nucleic acids, a fixed nitrogen source is necessary to sustain life on earth.

Some have called Nitrogenase a monstrously complicated  enzyme;
" Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase make it the ‘Everest of enzymes’. " ( Hoffman, 2009)

Nitrogenase performs the most biologically difficult reactions there is: a reduction of dinitrogen. It so happens, that this reaction is absolutely vital for every living organism. In order to perform this reaction, nitrogenase employs a number of novel structural features, such as two FeS clusters, a molybdenum metal-center, and a carbide, with 6 bonds to carbon. In unison, these unusual features enable nitrogenase to perform some simply phenomenal chemistry.Should you choose not to vote for it, consider the fate of every protein, DNA & RNA molecule in your body without nitrogen. None of it would polymerize and any bonds that formed would be totally insufficient to support life... all you would be is a pile of oxidized carbon. 16 ATPs are consumed per N2 reduced.  Substantial energy input is needed to overcome this large activation energy and break the N=N triple bond.

Some see the possibility that nitrogenase was present in the last universal common ancestor (LUCA) and, therefore predates the divergence of the archaea and bacteria. Others put it back between 3.2 and 2.75 billion years ago.

Several interesting aspects of nitrogenase structure and mechanism  include the biosynthesis of the nitrogenase and insertion of the FeMo-co, the specific role of the nitrogenase P cluster, the role of MgATP in nitrogenase catalysis, the mode and manner of substrate binding and the nitrogenase mechanism. 15 To meet the kinetic challenge, the biological process of nitrogen fixation requires a complex enzyme with multiple redox centers. The nitrogenase complex, which carries out this fundamental transformation, consists of two proteins: a reductase (also called the iron protein or Fe protein), which provides electrons with high reducing power, and nitrogenase (also called the molybdenum-iron protein or MoFe protein), which uses these electrons to reduce N 2 to NH 3 . The transfer of electrons from the reductase to the nitrogenase component is coupled to the hydrolysis of ATP by the reductase.

In most nitrogen-fixing microorganisms, the eight high-potential electrons come from reduced ferredoxin, generated by oxidative processes. Two molecules of ATP are hydrolyzed for each electron transferred. Thus, at least 16 molecules of ATP are hydrolyzed for each molecule of N2 reduced. O2 is required for oxidative phosphorylation to generate the ATP necessary for nitrogen fixation. However, the nitrogenase complex is exquisitely sensitive to inactivation by O2 . To allow ATP synthesis and nitrogenase to function simultaneously, leguminous plants maintain a very low concentration of free O2 in their root nodules, the location of the nitrogenase. This is accomplished by binding O2 to leghemoglobin, a homolog of hemoglobin.

The three FeS clusters of nitrogenase are essential for catalysis. Neither component protein reduces any substrate in the absence of its catalytic partner.  So they are interdependent.   The iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation.

The mechanism of the complex enzyme nitrogenase has long been one of the most challenging problems in bioinorganic chemistry. Nitrogen fixation has always been considered of fundamental importance, not only for its significance in global nutrition, but also because of the relevance of nitrogenase as a model system for examining processes such as multiple electron oxidation-reduction reactions, complex biological metal assembly, and even nucleotide-dependent signal transduction.

The MoFe protein cycle involves the progressive reduction of the MoFe protein, which ultimately leads to N2 binding and reduction. Because eight electrons and eight protons are required for N2 reduction and H2 evolution, each MoFe protein cycle requires eight Fe protein cycles and storage of the electrons. Kinetic studies have also shown that N2 does not become bound to the active site until at least two, and probably more, electrons have been accumulated within the MoFe protein.

What sets nitrogenase apart from essentially all other enzymatically catalyzed redox processes is the number of electrons (eight) that must ultimately be delivered to the substrates each turnover cycle, with the consequent demand for precise timing of the underlying electron transfer events.

Question: How could Nitrogenase have evolved, if an intermediate stage would not have broken the triple bond? None of the eight intermediate stages meets the goal to split nitrogen. Let me outline this. The author of above paper mentions an " optimal reaction", which is achieved by two components working together as a molecular machine !! That raises the question: Why would natural selection or any evolutionary mechanism evolve such intermediate stages if there is no survival advantage to be gained by it? This is an all or nothing business. Either the whole process goes through eight times, or nitrogen is not split. 

The make of Nitrogenase enzymes, essential for life on earth, is a monstrously complex process

https://reasonandscience.catsboard.com/t1585-nitrogenase#5922

Following is the resume of several month of detailled inquiry and investigation.

Here, I will outline the complex process to make Nitrogenase, a monstrously complicated enzyme - and essential for life on earth. 

Overview of the Nitrogenase enzyme complex
https://reasonandscience.catsboard.com/t2590-origins-what-cause-explains-best-our-existence-and-why#5867



Nitrogenase transforms Dinitrogen into ammonia, used in all life forms in their molecular machinery, to make amino acids, RNA, DNA etc.  Secular science has tried to avoid to explain how this complex machinery emerged on early earth, by claiming, that HCN or ammonia was initially readily available, and then only later by becoming scarce, would have triggered the evolutionary requirement to make the machinery, outlined below, but these suppositions do not withstand closer scrutiny. I have examined the evidence, and as it seems, Nitrogen was only available in gas form, even on early earth:

Availability of ammonia in a prebiotic earth
https://reasonandscience.catsboard.com/t2689-availability-of-ammonia-in-a-prebiotic-earth

In reducing atmospheres, such compounds are readily formed by electrical discharges but geochemical evidence suggests that the early Earth had a non-reducing atmosphere in which discharges would have instead produced NO ( Nitrogen oxide may refer to a binary compound of oxygen and nitrogen or a mixture of such compounds ).

HCN is a form of reduced nitrogen that can enter directly into prebiotic chemistry, but recent results make such a composition unlikely. The possibility that reactions of hydrogen cyanide (HCN) might form the basis for a complex cyclic organization has been proposed, but there is as yet no experimental evidence to support this proposal.

Abelson (1966) first suggested that the lifetime of ammonia NH3 would be short because of its photochemical dissociation. We find that if the initial NH3 mixing ratio was 10-4 then in only 40 years the NH3 would have been destroyed through photolysis. If the mixing ratio were 10-5, then the lifetime was less than 10 years.

There is every reason to suspect that a biological capacity for nitrogen fixation would have been a valuable and needed commodity even on the relatively oxygenic case 2 earth.

So, there are good reasons to doubt that nitrogen in biologically usable form was available before life beban on earth to form the first molecular compounds, essential for life, like amino acids, RNA, DNA etc. So, the only possible pathway was by the emergence of nitrogen fixation through nitrogenase enzymes. The biosynthesis of nitrogenase is extremely complex, even today, after over 50 years of intensive research not fully understood, and depending on a large number of enzymes, many using and requiring Iron-Sulfur clusters as reaction centers for electron transport, energy generation, in order for  the enzymatic biochemical reactions to become possible - enzymes which by themselves depend on the availability of ammonia to make their own basic building blocks  and DNA which encodes the instructions to make the right polypeptides,  sequences- which origin is precisely what we try to explain, and so, creating a catch22 situation - either everything was there right from the beginning, or - no deal.

Nitrogenase is composed of two main subunits, called Component I, and Component II ( component I is the subunit where the main nitrogen splitting and fixing reaction occurs, so the logic of events goes from Component II to Component I ) :

Component II contains 1 [4Fe–4S] cluster ( redox centers ), while Component I contains one  P cluster (Fe-S center) , and one  FeMo-cofactor [7Fe–9S–Mo–Xhomocitrate], which is  one of the most complex metalloclusters known in biology:

Overview of the Nitrogenase enzyme complex
https://reasonandscience.catsboard.com/t2590-origins-what-cause-explains-best-our-existence-and-why#5867

The biosynthesis of FeMo-cofactors is indeed a daunting task, requiring the genetic information to make the two subunits, the metal clusters, homocitrate, and all the biosynthesis machinery, including import of iron, molybdate, and sulfur proteins, the substrates, and the machinery to make it available for import into the cell.

Biosynthesis of the Cofactors of Nitrogenase  
https://reasonandscience.catsboard.com/t2429-biosynthesis-of-the-cofactors-of-nitrogenase

The biosynthesis of the MoFe protein is extremely complex. At least six gene products are involved in the biosynthesis of FeMoco: the products of nifQ, nifB, nifV, nifN, nifE, and nifH.   FeS cluster assembly is a complex process involving the mobilization of Fe and S atoms from storage sources, their assembly into [Fe-S] form, their transport to specific cellular locations, and their transfer to recipient apoproteins. 30  Biological Fe-S cluster assembly is tightly regulated within cells. 29

Following is the biosynthetic process that leads to the formation of active MoFe protein.

Iron, Sulfur, and Molybdenum need to be imported into the internal cellular milieu:

Molybdenum:

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

Molybdate enters the cell through ABC Transporters and is processed by NifQ enzymes, or possibly just cystine, to form a putative Mo-S containing species.

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

Transport of molybdates into the cell
https://reasonandscience.catsboard.com/t2430-molybdenum-essential-for-life#5923

Sulfur:
Following are the enzymes required in the pathway:

Biosynthesis of the Cofactors of Nitrogenase  
https://reasonandscience.catsboard.com/t2429-biosynthesis-of-the-cofactors-of-nitrogenase

1. Sulfate permeases
2. ATP sulfurylase
3. APS kinase
4. PAPS reductase
5. Sulfite reductase
6. Cysteine Synthase Complex ( O-acetylserine (thiol)-lyase )

Iron:
Iron uptake in  bacteria involves four distinct steps:
(i) siderophore synthesis,
(ii) siderophore secretion into the extracellular space,
(iii) iron chelation by the siderophores, and
(iv) siderophore/ iron uptake via complexes in the outer membrane and the intermembrane space as well as in the plasma membrane, through:

Outer membrane transporters (TBDT)
ExbB/ExbD/TonB system
ATP-binding cassette (ABC) transporter
Periplasmic binding protein (PBP)

while

RND
P-type ATPase
CDF

are used and essential to expel overload of B12 and transition metals.

Biosynthesis of the Cofactors of Nitrogenase
https://reasonandscience.catsboard.com/t2429-biosynthesis-of-the-cofactors-of-nitrogenase#5924

To get Molybdenum Co-factors, which are the reactive centers in the nitrogenase enzyme, where the action occurs,  following is required:

1. A fine tuned strong atomic force
2. Molybden
3. Iron
4. Sulfur
5. The elements to make the cell membrane, that is to say: phospholipids, glycolipids, and sterols.
6. Molybdenum co-factors
6. FE/S clusters for maturation of Molybdenum cofactors (Moco)
7. All functions and biosynthesis pathways to make the cell membrane
8. sulfur import proteins
10. Nonribosomal peptide synthetases (NRPS) - siderophore assembly lines and all subunits to make them
11. Coenzyme A
12. siderophore/iron complex import proteins
13. Molybdenum ABC transporters for molybdate import  
14 Eighteen NIF genes to instruct the assembly of nitrogenase enzymes

Assembly of nitrogenase FeMo-co is a considerable chemical feat because of its complexity and intricacy. Elucidation of the biosynthesis of FeMo-co  is further complicated by the large ensemble of participating gene products.
the synthesis of FeMo-co requires :

nifS,
nifU,
nifB, 
nifE, 
nifN, 
nifV, 
nifH, 
nifD 
nifK
Cysteine desulfurase
NifB-co,
homocitrate,
Mo,
Mg-ATP,
DTH as reductant, and
the tetrameric protein NifEN, which acts as a molecular scaffold on which some of the FeMo-co assembly reactions take place
Iron (possibly from NifU) and sulfur (from NifS activity) are combined by NifB to form NifBco.
NifBco binds to NifN2E2 .
The next events are still obscure, but it is widely assumed that NifN2E2 acts as a scaffold for the combination of NifBco with the putative MoS species to form FeMoco.
In the final stage of activation, FeMoco is bound to the ‘‘apo- MoFe protein.’’ The ‘‘apo-MoFe proteins’’ must be bound to NifY or . NifY or dissociate after the activation of the MoFe protein by FeMoco. The role
of (NifY) may be to hold the ‘‘apo-MoFe protein’’ in an open conformation that will allow access of FeMoco to its binding site.


It takes all this machinery to make ammonia, but it takes ammonia to make the machinery that makes the Iron-sulfur clusters. This is another example that shows how everything had to begin fully setup right from the beginning, and so, evidence of intelligent design.

Eric S. Boyd: New insights into the evolutionary history of biological nitrogen fixation 2013 Aug 5 [url= https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733012/]1[/url]

The question of the ancestor of nitrogenase is intriguing and is framed by numerous paradigms that are not strongly supported by empirical observations gleaned from extant biology. It is likely that this anthropogenic-centered focus leads to the tendency to place the emergence of biological nitrogen fixation as a very early event and perhaps even a primordial process. However, even the simplest evolutionary observations such as the aforementioned limited association of biological nitrogen fixation in deeply rooted lineages are not consistent with this process being a property of LUCA. The history of nitrogen availability is not something that can, as of yet, be ascertained from the geologic record so the true time at which selective pressure was sufficient to affect the emergence of such a complicated biochemical process is unclear.

1. Eric S. Boyd: New insights into the evolutionary history of biological nitrogen fixation 2013 Aug 5 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733012/