Intelligent Design, the best explanation of Origins

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1 Nitrogenase on Sat Mar 08, 2014 2:01 pm


The Nitrogenase enzyme,  the molecular sledgehammer

The amazing story of how scientists struggled for years to duplicate an important bit of chemistry.

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’.

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 NN 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.

Creationist Christians are often accused of having the same easy answer for any question about specific origin of things in nature: the 'God of the gaps' did it. But this criticism can be easily turned around. What answers do evolutionists give to explain the origin of microscopic marvels like the molecular sledgehammer? They can't explain them scientifically, so they resort to a standard liturgy, worshipping the power of blind chance and natural selection.

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.’

How can natural processes and mechanisms  write a book ? compose a partiture ? write a morse code, or a computer code ? how can physics write a dna code to  make the machinery to produce the machines ( enzyme proteins made of amino acids ) , many of which operate at the same time and in the same small volume of the cytosol. By their catalytic action, these enzymes generate a complex web of metabolic pathways, each composed of chains of chemical reactions in which the product of one enzyme becomes the substrate of the next.

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. You can see the glycolytic pathway in the video :

Nitrogen fixing bacteria possess a nitrogenase enzyme complex that catalyses the reduction of molecular nitrogen to ammonia [PMID: 2672439, PMID: 6327620, ]. 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. 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.
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

There are at least three different types of nitrogenase known including both Vanadium (V) nitrogenase and iron (Fe) nitrogenase2, 5 . 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.7 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 .

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

Localization of a Catalytic Intermediate Bound to the FeMo-cofactor of Nitrogenase*

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.


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

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.[2]

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 [3] 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.[4] 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.[5

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.


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

Identification of a minimum gene set

The crucial involvement of the FeMoco biosynthesis enzymes prompted us to analyze the occurrence of nine additional nif genes in known diazotrophic species encoding NifK, NifE, NifN, NifB, VnfG, NifQ, NifV, NifS, and NifU. The involvement of eight of these proteins in FeMo-cofactor synthesis and nitrogenase maturation has been determined

Nevertheless, the identification of relevant hits (listed in the Additional file 2: Table S2) revealed that nearly all known diazotrophs contain a minimum of six conserved genes: nifH, nifD, nifK, nifE, nifN, and nifB

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. 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.

The active site of [NiFe]-hydrogenase has one carbon monoxide and two cyanide ligands coordinated to the iron atom, a feature that is to date unique in biology.

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[FOOTNOTE: David W. Wolfe, Tales from the Underground: A Natural History of Subterranean Life, Perseus, 2001, p. 78; Huxtable, Reflections: Fritz Haber (regarding the Haber process whish is the only inorganic way to fix nitrogen).]. 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 .

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2 The Natural History of Nitrogen Fixation. on Sat Mar 08, 2014 2:03 pm


The Natural History of Nitrogen Fixation.


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.....

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.

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

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Maturation of Nitrogenase: a Biochemical Puzzle

The nitrogenase enzyme catalyzes the reductive breakage of the very strong triple bond of N2 to generate NH3 in a process known as biological nitrogen fixation. Biological nitrogen fixation is an essential step in the nitrogen cycle in the biosphere, and it is a major contributor to the nitrogen available to agricultural crops. Nitrogenases are composed of two proteins that can be purified separately: dinitrogenase and dinitrogenase reductase. Dinitrogenase, also referred to as the MoFe protein or component I, is a 220- to 240-kDa tetramer of the nifD and nifK gene products that contains two pairs of two complex metalloclusters known as the P-cluster and the iron molybdenum cofactor (FeMo-co). Each αβ pair of subunits of NifD and NifK contains one P-cluster and one molecule of FeMo-co. FeMo-co is composed of homocitrate and a MoFe3-S3 cluster bridged to a Fe4-S3 cluster by three sulfur ligands. The Mo atom is coordinated to the C-2 carboxyl and hydroxyl groups of R-homocitrate. Recently, an electron-dense area has been observed within the core of Fe atoms of FeMo-co and has been proposed to be a low-atomic-weight species (O or N) (Fig. The P-cluster is a [8Fe-7S] cluster with a structure similar to that of FeMo-co, which consists of two [4Fe-3S] cubanes connected by a central S atom. The P-clusters are located at the αβ subunit interface and are coordinated by cysteinyl residues from both subunits. Dinitrogenase reductase, also referred to as the Fe protein or component II, is a 60-kDa dimer of the product of the nifH gene, which contains a single [4Fe-4S] cluster at the subunit interface and two Mg-ATP-binding sites, one at each subunit. Hereafter in this minireview, we will use the Fe protein and MoFe protein nomenclature when referring to the nitrogenase components. The Fe protein is the obligate electron donor to the MoFe protein; electrons are transferred from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and in turn to FeMo-co, the site for substrate reduction. In addition, the Fe protein functions in the biosynthesis of FeMo-co and in the maturation of apo-MoFe protein.

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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.

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.


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5 NITROGEN FIXATION on Fri Feb 10, 2017 5:08 pm




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.

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6 Re: Nitrogenase on Fri Feb 10, 2017 6:31 pm



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