Nitrogen fixation

[Principia]

In my googling around, I came across this little gem on the 'Net:
[...] the evidence to date indicates these gene products are system-dependent and specific. That is, there is no evidence that other cellular activities can substitute (as seen by the nif- phenotypes when any of these genes are lost) nor is there evidence (that I know of) that any of genes products are involved in processes other than biological nitrogen fixation. One can always imagine that these gene products are descendents of proteins that "did something else," but there just isn't any good evidence for this imaginary speculation.

[...]

Is there evidence of an evolutionary continuum with nitrogenase indicating sloppy simplicity?

No. You either have nitrogenase as we know it or have nothing. There are no simpler versions of nitrogenase and there is no reason to think it would function in a biologically significant manner in a much sloppier state.
Let's help put this one to rest, eh? ;)

[Principia]

Well, let me get it started. First, a brief overview provided here (http://www.spacedaily.com/news/life-03m.html): Two of the most important pieces of biochemical innovation that occurred in the early biosphere -- the development of photosynthesis (which made light energy available to life) and of nitrogen fixation (which made atmospheric nitrogen available to life) -- may be related to each other because some of their key enzymes appear to have evolved from a common ancestor that may be part of a third, significantly different, biochemical process.

[...]

Two new studies, to be presented at the February 2003 NASA Astrobiology Institute General Meeting by researchers at Arizona State University, provide evidence for the long-suspected relatedness of the two biochemical pathways, and find hints of other related pathways that may be key to understanding the evolutionary history of both.

A critical part of the emerging evolutionary picture seems to be "horizontal gene transfer" -- genetic change that occurs by the exchange of genetic material between bacteria. This process allows for sudden evolutionary leaps that are perhaps not possible through gradual genetic change and natural selection.

In a paper published in the November 22, 2002 issue of Science, Blankenship, ASU biochemist Jason Raymond and colleagues show through a comparative genomic analysis of five photosynthetic prokaryotic organisms that the genes that code for the intricate molecular complexes that perform photosynthesis seem to have originated through ancient genetic mixing that apparently combined a variety of independently evolved metabolic processes.

In one of the Astrobiology meeting papers, "Horizontal Gene Transfer in the Evolution of Nitrogen Fixation," Raymond, Blankenship and Rice University's Janet Siefert do an analysis of the genomes of a larger group of bacteria and archaea, comparing in particular similar genes that code for the protein nitrogenase, a critical enzyme in nitrogen fixation.

[...]

The researchers find that similar or "homologous" nitrogenase genes exist across a broad range of organisms, and appear to be related to other similar genes coding for proteins involved in photosynthesis, as well as to other genes in archaea and bacteria that do neither photosynthesis nor nitrogen fixation.

"We found a group of homologous genes that doesn't correspond to any genes that go with photosynthesis or any that we know in nitrogen fixation -- we found these in a wide range of organisms," said Raymond.

The analysis suggests that the related genes that code for neither nitrogenase nor enzymes in photosynthesis may be "relics," coding for metabolic pathways that are ancestral to both photosynthesis and nitrogen fixation.

Horizontal gene transfer appears to be responsible for the broad distribution of the original gene and for its subsequent divergence and specialization in the metabolic pathways of nitrogen fixation and photosynthesis.

In the second paper, "The Evolutionary Relationship between Nitrogen Fixation and Bacteriochlorophyll Synthesis," ASU's Christopher Staples, Blankenship, and Virginia Polytechnic Institute's Biswarup Mukhopadhyay examine the properties of enzymes created by these similar genes and finds that nitrogenase, the photosynthesis related enzymes, and other homologous enzymes all generally belong to a group of enzymes that break apart molecules and are known as reductase enzymes.

"We're purifying the proteins that the genes produce and will be looking at catalytic activity. We will test to see how activity differs and also to find what has been conserved and what has been changed in the active sites," said Staples. "Changes in the enzymes' active sites lead to differentiation in regard to what specific molecules they affect."

The less-specialized reductase enzymes appear to be ancestral to the others and were perhaps originally important in helping early prokaryotes neutralize toxic substances in their environment.

"There is a hypothesis that the ancient reductase, in the presence of a reducing atmosphere, may have been a hydrogen cyanide reductase," said Staples.

The team thinks that they have perhaps found a living model for this in Methanococcus jannaschii, a methane- producing archaea that performs neither nitrogen fixation nor photosynthesis but produces a reductase enzyme that the researchers suspect is used to break down hydrogen cyanide.

"We're testing to see if these organisms can grow in the presence of cyanide and if they can use cyanide as a nitrogen source," said Staples. "They don't appear to be able to use cyanide exclusively for nitrogen, but they can grow in concentrations of it that would be deadly to most organisms." [...]


I cannot find the papers, but I have the abstracts from NAI 2003 General Meeting (nai.arc.nasa.gov/institute/general_meeting_2003):
Abstract # 12890 - Horizontal Gene Transfer in the
Evolution of Nitrogen Fixation
Jason Raymond, Robert E. Blankenship, Janet L. Siefert

Whole genome sequences for nearly twenty nitrogen-fixing prokaryotes are now available, and we have analyzed each of these organisms for presence of one or more nif (nitrogenase-gene encoding) operons and conserved synteny. Additionally, we have used standard tools of sequence analysis to evaluate the evolutionary history of every characterized gene found in these nif operons and observe that the core genes segregate phylogenetically into the known metal-binding classes (iron-, vanadium-, or molybdenum-containing) of nitrogenase. We have also used molecular phylogenetic techniques to analyze a fourth group of yet-uncharacterized nitrogenase homologs, found in archaea and in a limited number of phototrophic bacteria, that appear to be ancestral to nitrogenase genes and to essential genetic components of chlorophyll and bacteriochlorophyll biosynthesis found in all known phototrophs. This fourth group of nitrogenase homologs may function as an electron sink or possibly as a non-specific detoxyase or reductase, and is likely a relic of some ancient metabolic trait. Here we trace the early origins of nitrogenase genes and the subsequent horizontal gene transfer events that have resulted in the current distribution of prokaryotic nitrogen fixation.
The Evolutionary Relationship Between Nitrogen Fixation and Bacteriochlorophyll Synthesis

Christopher Staples, Biswarup Mukhopadhyay, Robert E. Blankenship

Photosynthesis arguably developed relatively soon after the origin of life on Earth. Some reports of stromatolites suggest this development occurred as early as 3.5 Ga, just after the late-heavy meteoric bombardment period ended at 3.9 Ga. However, due to the complexity of photosynthesis, it is highly likely that the earliest life forms were not photosynthetic. It is thus proposed that photosynthesis developed as a favorable branch of a pre-existing, more fundamental pathway. From phylogenetic analyses and emerging biochemical data, it is possible that this pre-existing pathway was that currently utilized for nitrogen fixation (the Nif proteins). However, it is currently unclear if the Nif proteins were originally utilized in nitrogen fixation (in the case of a neutral atmosphere), or hydrocyanic acid detoxyase (in the case of a reducing atmosphere). If the atmosphere was indeed reducing, an enzyme like a hydrocyanic acid detoxyase might have independently been the precursor of both Nif and Bch proteins. In this study, work towards the study of the genetic, structural, functional, and mechanistic relationships between selected proteins in the anaerobic bacteriochlorophyll synthesis (bch) pathway and the nitrogen fixation (nif) pathway of Rhodobacter capsulatus are presented (BchNB [protochlorophyllide reductase, PR], BchYZ [chlorophyllide reductase, CR] with NifDK [dinitrogenase] and NifNE [FeMo-co synthesis protein]; BchL [PR reductase] and BchX [CR reductase] with NifH [dinitrogenase reductase]).

As shown in the presentation "Horizontal Gene Transfer in the Evolution of Nitrogen Fixation" by Jason Raymond, analysis of the whole genomes of several organisms for the presence of NifH or NifD/K homologs revealed the presence of several atypical sequences. A radial plot of the phylogenetic analyses shows that these sequences lie in between the Nif and Bch clades. One of the organisms containing these atypical sequences has not been shown to perform either photosynthesis or nitrogen fixation. This organism is Methanococcus jannaschii, a thermophilic methanogenic member of the euryarcheota. We are currently studying the functional relationships of the proteins encoded by these ìatypical Nif-likeî genes to their Nif and Bch homlogs. We are also looking into the physiological roles of these proteins in M. jannaschii. This includes testing under which conditions these proteins are expressed (e.g. growth in the presence of cyanide). It is possible that these ìatypical Nif-likeî sequences represent a relic precursor protein to both the Nif and Bch lines, or another branch off a more ancient, possibly extinct, precursor.

[Principia]

Now for some juicy tidbits:
Mol Biol Evol. 2003 Dec 23 [Epub ahead of print]

The Natural History of Nitrogen Fixation.

Raymond J, Siefert JL, Staples CR, Blankenship RE.

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 due to 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 Nif H, D, K, E, and N 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: one group comprised of genes essential to photosynthetic pigment biosynthesis, and an additional group of uncharacterized genes present in methanogens and 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.

The remaining group III sequences support an early paralogous origin for alternative nitrogenases. The divergence of Fe- and V-dependent nitrogenases clearly seems to have occurred subsequently, as separate monophyletic clades for these proteins are observed in NifD and NifK trees. One plausible scenario implied by this evidence is that an ancestral NifD homolog might have been less specific with respect to its metal cofactor. Such a nitrogenase would be responsive to environmental availability of vanadium, iron, and molybdenum that fluctuated with the changing redox state that characterized the Proterozoic Earth between one and two billion years ago (Normand and Bousquet 1989; Anbar and Knoll 2002). Empirical work supports this idea; under certain conditions FeMoco can be inserted into the Fe-dependent aponitrogenase (which normally requires FeFeco) and vice versa, resulting in a functional, albeit less efficient, enzyme hybrid (Gollan et al. 1993; Pau et al. 1993). Banded iron formations and paleosols indicate that the oceans of the early anoxic Earth were rich in soluble reduced iron whereas molybdenum, known to be much more soluble in its tetramolybdate (MnO4-2) form, was likely scarce (Anbar and Knoll 2002). Thus, under the presumably Mo-limiting conditions (or at least Fe-rich conditions) on the early Earth, a nitrogenase able to utilize alternative metals would have been advantageous. As the Precambrian Earth became progressively more oxic due to cyanobacterial photosynthesis, soluble iron became less available and soluble oxidized molybdenum (at least in oxic layers of the ocean) assumed its current role as the most abundant transition metal in the oceans (Anbar and Knoll 2002).

[...]

As a whole, groups IV and V include a diverse range of NifH and NifD homologs that are not known to be involved with fixing nitrogen. The pigment biosynthesis complexes protochlorophyllide reductase and chlorophyllide reductase, denoted herein as group V nitrogenase homologs, are not only homologous but are functionally analogous to nitrogenase, coupling ATP hydrolysis-driven electron transfer to substrate reduction (Burke, Hearst and Sidow 1993; Fujita and Bauer 2000). As with nitrogenase, electrons flow from a NifH-like ATPase (BchL and BchX) to a NifDK-like putative heterotetramer where the tetrapyrrole is bound (BchNB and BchYZ). These two enzymes catalyze independent reductions on opposing sides of a tetrapyrrole ring that are essential late steps in chlorophyll and bacteriochlorophyll biosynthesis. Based on sequence similarity, the BchLNB and BchXYZ complexes appear to have originated from a duplicated common ancestor that was less substrate-specific and able to catalyze both sequential ring reductions, albeit less efficiently (Fig. 2 shows the phylogenetic position of group V pigment biosynthesis genes). Interestingly, both complexes are found together only in anoxygenic photosynthetic bacteria; only one of the two complexes (denoted Bch or Chl LNB in anoxygenic or oxygenic phototrophs, respectively) is found in cyanobacteria, and consequently these organisms produce chlorophyll rather than more reduced bacteriochlorophyll (Xiong et al. 2000; Blankenship 2002). This fortuitous change, likely prompted by loss of the BchXYZ complex in the ancestor of modern cyanobacteria, resulted in a blue shift in primary pigment absorption wavelength, thereby incurring an increase in redox potential of the photosynthetic reaction center. This redox shift provided the necessary energy for the oxidation of water to O2 via oxygenic photosynthesis, common to cyanobacteria and photosynthetic eukaryotes (Blankenship and Hartman 1998). The subsequent oxidation of the Earth’s atmosphere had profound effects on Precambrian life and is widely held to have paved the way for the evolution of complex life (Blankenship and Hartman 1998; Des Marais 2000; Blankenship 2002). Detailed evolutionary trees for pigment biosynthesis genes have been constructed (Xiong et al. 2000) and will not be covered here.

Group IV consists of a subset of nitrogenase homologs (Nif-like proteins, herein designated NflH or NflD, depending on homology) that have yet to be characterized and, based on their breadth in this analysis, appear much more prevalent than previously known. Intriguingly, across the 101 genomes analyzed, these group IV homologs are found only in methanogens – not all of which are diazotrophs – and in some nitrogen-fixing bacteria, most of which are photosynthetic (the sole non-photosynthetic exception so far being D. hafniense, whose nitrogenase proteins are most similar to those from heliobacteria – the only known Gram + phototrophs). Conserved residues in alignments of NifH homologs (Fig. 6) from all five groups show that 4Fe-4S iron sulfur cluster-ligating cysteines and the P-loop/MgATP binding motif are invariant, suggesting that these proteins may function analogously to dinitrogenase reductase. Conversely, NifD homologs are highly diverged from both the nitrogenase subunits and the pigment biosynthesis genes. FeMoco-ligating residues (Fig. 7) are not conserved among group IV and V proteins, although several – but not all – conserved cysteines involved with P cluster coordination are found in NifD and NifK homologs. This suggests that a less complex FeS cluster, such as a 4Fe-4S, may be functioning in electron transfer in the Group IV and V proteins. In some bacterial genomes these genes are still found in putative operons, though the operon consists only of a NifH-homolog and one or two NifD-homologs. However, they are not found in operons among any methanogens, and taken with the unresolved paraphyly and low sequence identities that characterize these phylogenies, it is plausible that these Nif homologs have evolved to different functions in different domains. It is interesting to speculate that this group of proteins, apparently still utilized by a small subset of organisms, may represent the modern vestige of a primitive nitrogenase (a possible intermediary functional link to this enigmatic group of nitrogenase homologs may be provided by M. barkeri, in which one alternative nitrogenase operon has VnfH, D, and K proteins that cluster with other known vanadium-dependent nitrogenases, but whose VnfE and N proteins are only distantly similar to their group III counterparts and, phylogenetically, look like group IV proteins; see (Thiel 1996) for characterization and interesting discussion of what may be close homologs of these proteins in cyanobacterium Anabaena variabilis).

[Principia]

J Mol Evol. 2000 Jul;51(1):1-11

Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes.

Fani R, Gallo R, Lio P.

The pairs of nitrogen fixation genes nifDK and nifEN encode for the alpha and beta subunits of nitrogenase and for the two subunits of the NifNE protein complex, involved in the biosynthesis of the FeMo cofactor, respectively. Comparative analysis of the amino acid sequences of the four NifD, NifK, NifE, and NifN in several archaeal and bacterial diazotrophs showed extensive sequence similarity between them, suggesting that their encoding genes constitute a novel paralogous gene family. We propose a two-step model to reconstruct the possible evolutionary history of the four genes. Accordingly, an ancestor gene gave rise, by an in-tandem paralogous duplication event followed by divergence, to an ancestral bicistronic operon; the latter, in turn, underwent a paralogous operon duplication event followed by evolutionary divergence leading to the ancestors of the present-day nifDK and nifEN operons. Both these paralogous duplication events very likely predated the appearance of the last universal common ancestor. The possible role of the ancestral gene and operon in nitrogen fixation is also discussed.

The only data available concern a highly conserved modular domain of NifU (Hwang et al. 1996), a protein involved in the mobilization of iron and sulphur for nitrogenase specific iron–sulfur cluster formation (Zheng et al. 1998). In addition to this, duplicated copies of the same nif gene(s) were found within the same genome, as reported for the Rhodobacter capsulatus nifAB operon and nifU gene (Masepohl et al. 1988; Preker et al. 1992) and for Rhizobium phaseoli nitrogenase structural genes (Palacios et al. 1993 and references therein). They probably encode for proteins with related functions and can therefore substitute, albeit poorly, for their nif counterparts under certain conditions (Merrick 1992) and they might have originated by duplication of an ancestral gene.

The aim of this work was to try to shed some light on the origin and evolution of nif genes and on the mechanisms that could have played an important role in shaping the nitrogen fixation process. The comparative analysis of the structure and organization of nif genes in Archaea and Bacteria can suggest useful hints on these issues. The attention was focused on two operons, nifDK and nifEN, exhibiting some common features. First, both of them encode a tetrameric (a2b2 and N2E2) enzymatic complex. Second, nitrogenase contains two unusual rare metal clusters; one of them is the iron molybdenum cofactor (FeMo-co), which is considered to be the site of dinitrogen reduction (Kim and Rees 1992) and whose biosynthesis requires the products of nifNE and of some other nif genes (Ludden 1992; Dean et al. 1993). Roll et al. (1995) have proposed that NifNE might serve as a scaffold upon which FeMo-co is built and then inserted into component I. Moreover, it has also been reported that the products of Azotobacter vinelandii nifE and nifN genes are structurally homologous to the products of nifD and nifK (Dean and Brigle 1985; Brigle et al. 1987). Finally, in those diazotrophs where nifDK and/or nifEN have been identified, cloned and sequenced, they exhibit the same gene organization. In most cases they are clustered in operons where the two genes of each pair are contiguous and arranged in the same order (nifDK and nifEN) (Fig. 1). Moreover, in Anabaena variabilis, nifE and nifN are fused (Thiel et al. 1997). This conservation of operon organization is in agreement with the notion that, although the gene operon organization is not conserved in bacterial evolution and operon instability has been detected across a wide phylogenetic range (Itoh et al. 1999), the operon structure may be maintained for those genes encoding proteins which form a heteromeric component to give a functional enzymatic complex (Mushegian and Koonin 1996), such as nifDK and nifEN. In addition to this, the organization of nifDK and nifEN might also reflect a common evolutionary pathway of these genes. Therefore, to try to reconstruct the evolutionary history of nifDK and nifEN, a detailed analysis of their products was carried out.
ibid

Res Microbiol. 2003 Apr;154(3):157-64

Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria.

Berman-Frank I, Lundgren P, Falkowski P.

The biological reduction of N(2) is catalyzed by nitrogenase, which is irreversibly inhibited by molecular oxygen. Cyanobacteria are the only diazotrophs (nitrogen-fixing organisms) that produce oxygen as a by-product of the photosynthetic process, and which must negotiate the inevitable presence of molecular oxygen with an essentially anaerobic enzyme. In this review, we present an analysis of the geochemical conditions under which nitrogenase evolved and examine how the evolutionary history of the enzyme complex corresponds to the physiological, morphological, and developmental strategies for reducing damage by molecular oxygen. Our review highlights biogeochemical constraints on diazotrophic cyanobacteria in the contemporary world.

[Nic Tamzek]

This is a cool thread. I don't know anything about this particular topic, but I always amazed at what is available about systems that the IDists never think to mention.

[Dr.GH]

Good information. Thanks.

[theyeti]

Originally posted by Nic Tamzek
This is a cool thread. I don't know anything about this particular topic, but I always amazed at what is available about systems that the IDists never think to mention.

Even more than that, let's rewind the clock back before any of these articles were published. (In other words, not long ago.) The ID argument would be exactly the same, yet there wouldn't have been information available to counter it. How then would the IDists have known that the "mystery" they chose to hang their hat on would be solved? They would have no way of knowing, which goes to show that their methodology is incapable of ferreting out false positives. It's a good example of how utterly impotent the ID inference is.

theyeti

[Principia]

European Journal of Biochemistry
Volume 268 Issue 7 Page 1940 - April 2001
doi:10.1046/j.1432-1327.2001.02063.x

FeMo cofactor biosynthesis in a nifE mutant of Rhodobacter capsulatus

Stefan Siemann1,*, Klaus Schneider1, Kai Behrens2, Arndt Knöchel2, Werner Klipp3 and A. Müller1

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 µm). Moreover, in these strains a high proportion of ethane was produced from acetylene ( 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.

Various gene products, including the product of the nifE gene, have been implicated in the biosynthesis of the FeMoco in Mo-dependent nitrogenases [19]. Defects in nifE have been reported to cause the expression of a cofactorless, and therefore inactive, apo-dinitrogenase [24]. However, the results obtained here on a nifE mutant of the phototrophic non-sulfur purple bacterium R. capsulatus revealed that a defect in this gene renders the cells capable of expressing an active dinitrogenase that reduces acetylene as well as N2. Characterization of the MoFe protein isolated from the nifE mutant (specific activity, Fe/Mo content, demonstration of the typical S = 3/2 FeMoco EPR signal) confirmed the existence of a functionally intact, FeMoco-containing MoFe protein in this strain. The nifE mutant exhibited, however, three striking features that are in marked contrast with the results obtained from studies on Mo nitrogenases of wild-type strains of R. capsulatus and other organisms. (a) The rate of acetylene reduction observed with nifE mutant cells was only 5-7% (after growth with serine as nitrogen source) or maximally 15% (after growth with N2 as nitrogen source). This low activity, however, was not due to an altered less active cofactor, but correlated with the low amount of MoFe protein present in the cells. (b) Maximal expression of an active nitrogenase was dependent on relatively high concentrations of molybdate (20-50 µm) in the growth medium (Fig. 1). (c) A high proportion of ethane ( 10%) was produced from acetylene under in vivo conditions.

Several attempts to identify homologues to nifE (nifE II copies) that may theoretically be responsible for the formation of small amounts of catalytically active MoFe protein in the nifE mutant were not successful in R. capsulatus [55]. A. vinelandii contains two nifE-like genes (nifE and vnfE) coding for highly similar proteins (66% identity), which can (partially) substitute for each other [56]. However, unlike A. vinelandii, R. capsulatus does not harbour a vanadium nitrogenase [37,55]. Furthermore, on inspection of the (almost completed) genome sequence of this organism, no VnfE-like protein could be identified. An anfE gene, potentially involved in biosynthesis of the Fe-only nitrogenase cofactor, is obviously lacking as well [32]. However, it is noteworthy that there are three additional gene regions in R. capsulatus coding for proteins with about 20% sequence identity with NifE. Two of these regions are known to be involved in bacteriochlorophyll biosynthesis (bchXYZ, bchLNB), whereas the function of a third region (orf1294-1293-1292) remains unknown. However, in view of the low similarity between these proteins and NifE, it appears unlikely that the corresponding genes substitute for nifE.

It is interesting to note that residual nitrogenase activity has been reported previously for crude extracts of a nifE mutant strain from A. vinelandii [57] and also for whole cells and extracts of a nifE mutant from K. pneumoniae [24], but its origin has not been further investigated. It should be emphasized that K. pneumoniae contains neither a photosynthetic system (bacteriochlorophyll) nor an alternative Mo-independent nitrogenase.

Provided that a nifE homologue does not exist in R. capsulatus, the fundamental question arises as to why a strain of this organism with a mutation in nifE, a gene that encodes a protein previously hypothesized to play a crucial role in FeMoco biosynthesis, exhibits altered catalytic properties in vivo and why it displays nitrogenase activity at all. Undoubtedly, the nifE gene product of R. capsulatus is needed for maximal synthesis of cofactor and MoFe protein, but the occurrence of nitrogenase activity in a nifE mutant unambiguously proves that NifE is not required for these processes in principle.

[Principia]

Some oldies that may be relevant:
Plant Mol Biol. 1989 Nov;13(5):551-61

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha.

Fujita Y, Takahashi Y, Kohchi T, Ozeki H, Ohyama K, Matsubara H.

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572-574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31,000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31,945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67,000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.
J Bacteriol. 1993 Apr;175(8):2414-22

bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants.

Burke DH, Alberti M, Hearst JE.

We present the nucleotide and deduced amino acid sequences of four contiguous bacteriochlorophyll synthesis genes from Rhodobacter capsulatus. Three of these genes code for enzymes which catalyze reactions common to the chlorophyll synthesis pathway and therefore are likely to be found in plants and cyanobacteria as well. The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. The other three genes are required for portions of the pathway that are shared with chlorophyll synthesis, and they were expected to be common to both pathways. bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL), a reaction that has been observed in all major groups of photosynthetic organisms except angiosperms, where only the light-dependent reaction has been clearly established. The purple bacterial and plant enzymes show 35% identity between the amino acids coded by bchN and chlN (gidA) and 49% identity between the amino acids coded by bchL and chlL (frxC). Furthermore, bchB is 33% identical to ORF513 from the Marchantia polymorpha chloroplast. We present arguments in favor of the probable role of ORF513 (chlB) in protochlorophyllide reduction in the dark. The further similarities of all three subunits of protochlorophyllide reductase and the three subunits of chlorin reductase in bacteriochlorophyll synthesis suggest that the two reductase systems are derived from a common ancestor.

[lpetrich]

That's awfully interesting. It's nice to see all this effort put into unraveling metabolic pathways and trying to work out their origin.

It would also be interesting to see how nitrate and sulfate reductases compare to nitrogenase, as well as to final-step respiratory-chain enzymes (may be called oxygen reductases), and initial-step oxygenic-photosynthesis enzymes (may be called water oxidases). For instance, are those respiratory enzymes modified nitrate reductases?

It will be interesting to work out the metabolic capabilities of the Last Universal Common Ancestor. How much biosynthesis can reasonably be traced back to it? Was it capable of fixing carbon from CO2? If it could do complete biosynthesis and also fix carbon and nitrogen, then it would have been completely autotrophic.

I consider it unlikely that it was photosynthetic, since photosynthesis is somewhat restricted in distribution; it would therefore have been chemosynthetic -- making it dependent on some chemical disequilibrium. If the disequilibrium is from the Earth's interior being more reducing than the Earth's surface, then hot springs and hydrothermal vents are plausible habitats for this organism.

[Valentine Pontifex]

Originally posted by Nic Tamzek
This is a cool thread. I don't know anything about this particular topic, but I always amazed at what is available about systems that the IDists never think to mention.

Maybe Principia should consider turning this thread into an article that can be posted on Talk.Origins or TalkDesign?

[Principia]

Maybe Principia should consider turning this thread into an article that can be posted on Talk.Origins or TalkDesign?

VP, I think it'd be a great idea if anyone wrote this up -- after all, there's nothing here that is uniquely my work. Personally, I would wait to see if the hypothesized links with photosynthesis pan out. If they do, I think it could make for an interesting article to combine both N-fixation and photosynthesis systems. My guess is that there are many other II members (http://www.antievolution.org/cgi-bin/ikonboard/ikonboard.cgi?s=3ff214094173ffff;act=ST;f=9;t=5) besides myself who could contribute significantly to such a discussion paper.

[Mike H]

Nitrate reductases do contain molybdenum, as memory serves, not to mention a goodly number of Fe-S clusters. This cofactor complement is superficially similar to what you'd find in nitrogenase. An actual structural comparison is another thing, although that could be rather easily remedied by a visit to the PDB and some fiddling about with your preferred protein visualization software.

I do vaguely recall that the MoFe cluster in nitrogenase does appear rather similar to a [8Fe-8S] cluster, although with a Mo taking the place of one of the Fe atoms. The reason I mention this is that [3Fe-4S] clusters are found in nitrate reductases, which suggests - at a purely "let's check our arithmetic" level - the MoFe cluster contains a sum of a [4Fe-4S] cluster, a [3Fe-4S] cluster, and our Mo atom.

The Metalloprotein Database and Server (http://metallo.scripps.edu/) at Scripps (http://www.scripps.edu) might be a useful place to bookmark for further inquiries. Some of the citations are a bit on the dusty side, but the Nitrogenase review page (http://metallo.scripps.edu/PROMISE/NITROGENASE_REV.html) has some citations that may make for useful background if a more substantial piece is being planned.

[Principia]

Thanks and keep 'em coming, MikeH!

For completeness, the following is a recently proposed mechanism for the FeMoco catalysis of N2, based on numerical simulations.
http://pubs.acs.org/isubscribe/journals/bichaw/41/i47/figures/bi025623zh00002.gif

Nitrogenases are enzymes which catalyze the reduction of dinitrogen to ammonia. The most extensively characterized nitrogenase contains both Mo and Fe and is referred to as Mo nitrogenase; in addition, some organisms have alternative nitrogenases containing Fe and V, or Fe only (1). These alternative nitrogenases are inferred to have much in common with Mo nitrogenase in terms of structure, but appear to be less efficient than the latter and are generally only expressed when Mo nitrogenase is unavailable to the organism. Mo nitrogenase consists of two component proteins. The Fe protein (component 2) is a homodimer of ~60 kDa, contains an Fe4S4 cubane and acts as a specific electron donor to the MoFe protein (component 1) (2). When dithionite is used as the primary reductant in vitro, electron transfer involves association and dissociation of the two nitrogenase proteins and is accompanied by the hydrolysis of ATP. The MoFe protein is an [alpha2beta2] tetramer of ~230 kDa which contains two unique metal-sulfur clusters. The P-clusters (stoichiometry Fe8S7) mediate the supply of electrons to the iron-molybdenum cofactor (FeMoco,1 stoichiometry MoFe7S9·homocitrate, Figure 1) (2, 3). A considerable weight of evidence implicates the FeMoco as the site of reduction of N2 and other substrates, including H+ (3). Detailed spectroscopic studies of 57Fe-enriched FeMoco led to formal resting state valency assignments of [MoIVFeIIIFeII6] (4) or [MoIVFeIII3FeII4] (5) and suggested that MoIII is accessed on reduction, while recent theoretical studies have provided additional evidence that [MoIVFeIIIFeII6] is the correct alternative (6). The production of N2 by Mo nitrogenase is believed (1) to have the limiting stoichiometry shown in reaction 1:
http://pubs.acs.org/isubscribe/journals/bichaw/41/i47/eqn/bi025623ze00001.gif
Recently, some doubts have been raised over the stoichiometry of the ATP component of reaction 1 following the observation that the Fe protein can be reduced to a stable all-ferrous state, whose crystal structure has been reported (7). This form of the Fe protein, if biologically accessible, might be able to transfer two electrons to the MoFe protein for each protein association-dissociation event, halving the catalytic ATP requirement. Recent experimental studies suggest that this is indeed the case (8). The stoichiometry of reaction 1 with respect to H2 follows from the observation that very high pressures of N2 still result in approximately one H2 produced per N2 reduced (9). It is not yet clear, however, whether this obligatory hydrogen evolution (OHE) (10) is an essential feature of the enzyme's mechanism or merely the consequence of an unavoidable leakage of reducing equivalents during turnover (3). Such leakage effects certainly appear to be significant for the alternative nitrogenases; for example, the limiting H2/N2 ratio for V nitrogenase is 3.5 (1).

Marcus C. Durrant, An Atomic-Level Mechanism for Molybdenum Nitrogenase. Part 1. Reduction of Dinitrogen Biochemistry, 41 (47), 13934 -13945, 2002

[Nic Tamzek]

The difficulty is always in connecting:

****The world of peer-reviewed lit up here****
...which is always technical and usually very piecemeal (small parts of the system discussed individually, homology cited but the "big picture" of implications not given, usually model organisms)...


...and...

****The world of generally well-educated people down here****
...who know what proteins, etc. are but may not have the first clue about nitrogen fixation, and who really just want the "big picture" of evolutionary origins...

A tough thing to do.

However, I've been meaning to create an EvoWiki (http://www.evowiki.org) page discussing literature on the evolution of complex systems. Heck, I shall:

EvoWiki Irreducible complexity and the scientific literature (http://www.evowiki.org/wiki.phtml?title=Irreducible_complexity_and_the_scientific_literature)

[Nic Tamzek]

I kicked off a page on Nitrogen fixation:

http://www.evowiki.org/wiki.phtml?title=Nitrogen_fixation

...I encourage you to add and edit!

[Principia]

For those of you :
down here ... who know what proteins, etc. are but may not have the first clue about nitrogen fixation, and who really just want the "big picture" of evolutionary origins
I have to say that I am probably in the same boat as you :). Sometimes, though, the researchers who do much of the work are kind enough to provide some succinct overviews of the current models and the remaining unanswered questions. In addition to the ones above, I cite:Nature 412, 26 - 27 (2001);

Biogeochemistry: The nitrogen fix

JAMES F. KASTING AND JANET L. SIEFERT

To what extent is biological evolution driven by environmental evolution? This is one of the big questions for both evolutionary biologists and geoscientists. On page 61 of this issue, Navarro-González and colleagues1 describe their investigation into a central innovation in the evolution of life. This was the invention of biological nitrogen fixation, by which life could create biologically usable forms of nitrogen instead of depending on abiotic sources. The authors propose that it was triggered by a change in environmental circumstances — a steep fall in the rate of abiotic nitrogen fixation by lightning at some point during the Archaean era, the first half of Earth's history.

[...]

The authors' thesis is as follows. On the early Earth, concentrations of CO2 in the atmosphere were high — because of oxidation of CO produced by impacts of extraterrestrial bodies5 and only slow removal of CO2 by weathering (the continents were smaller at this time6, meaning that a smaller area of minerals was exposed for weathering). With these CO2 conditions, the authors estimate that the initial production rate of NO was about 3 10^11 g N yr^-1. Atmospheric CO2 levels declined with time, however, as the impact rate dropped and the continents grew. A rise in atmospheric CH4 produced by methanogenic — methane-generating — bacteria may have warmed the Archaean Earth and speeded the removal of CO2 by silicate weathering7. As this happened, the production rate of NO by lightning dropped to below 3 10^9 g N yr^-1 because of the reduced availability of oxygen atoms from the splitting of CO2 and H2O. The resulting crisis in the availability of fixed nitrogen for organisms triggered the evolution of biological nitrogen fixation about 2.2 billion years ago.

This hypothesis is attractive but, like many good ideas, difficult to confirm. As the authors point out, genetic studies of the enzyme involved (nitrogenase) indicate that biological nitrogen fixation is an ancient metabolic pathway. If anything, one might imagine that it was invented during the early Archaean, before 3 billion years ago, rather than later on. But then, the crisis in abiotic nitrogen fixation could have occurred very early as well. One model8, which emphasizes rapid weathering of fragments from impacts and efficient sequestration of carbon in the Earth's mantle, proposes that concentrations of atmospheric CO2 were low from the very beginning of Earth's history and that the Archaean climate was warmed almost exclusively by CH4. In that case, the availability of fixed nitrogen could have been a problem almost as soon as life originated, around 3.5 billion years ago.

[..]

The issues might be clarified by comparing the evolutionary histories of biological nitrogen fixation and methanogenesis. The Archaean climate was probably warm, so the atmosphere must have contained large amounts of either CO2 or CH4. Navarro-González and colleagues' argument requires that methanogenesis preceded biological nitrogen fixation. On the face of it, this seems unlikely because the ability to fix nitrogen is widespread among prokaryotes (Bacteria and Archaea), whereas methanogenesis is restricted to a single group within the Archaea.

Parsimony would dictate that the more ubiquitous metabolic process came first. However, if the ability to fix nitrogen was spread by rampant gene transfer between organisms, then methanogenesis could conceivably have evolved first and the premise of Navarro-González et al. could hold. This question might be resolved by comparing the phylogenies of various nitrogenase-related (nif) genes with those derived from ribosomal RNA. If the phylogenies of nif genes are in accord with those from ribosomal RNA, then nitrogen fixation can be assumed to have been vertically inherited, and so be an anciently derived character. The phylogeny of one component of nitrogenase (nifH gene)11 is in partial agreement with that of ribosomal RNA, but still does not constitute conclusive evidence for either vertical or horizontal descent of nitrogen-fixing genes. Analyses of other nif genes might allow us to distinguish between these alternatives.
Incidentally, earlier I wanted to quote from an article by Line who briefly discussed (though sympathetically) the notions of panspermia and early evolution. I think I'll provide some snippets of the full (and free) article (http://mic.sgmjournals.org/cgi/content/full/148/1/21?view=full&pmid=11782495) for balance: Microbiology (2002), 148, 21-27.
The enigma of the origin of life and its timing
Martin A. Line

The first 3·5 billion years of evolution on Earth was dominated by unicellular life, significant multicellularity being independently evolved on numerous occasions from about 1200 million years ago in Eukarya (Carroll, 2001 ) or earlier in Bacteria. Rather than being novel, much of recent evolution in terms of biochemistry is derived, being attributable largely to ‘tinkering with the available equipment, adapting existing organs to new purposes’ (Nisbet & Sleep, 2001 ). An example of the manner of working of ‘modern’ evolution is seen in the origin of some forms of fermentation in Bacteria, which was derived from respiration, which was derived from photosynthesis, which were derived from nitrogen fixation (Burke et al., 1993 ). Nitrogen fixation was probably present prior to the divergence of the three domains of life, and because of its complexity, was almost certainly derived from an earlier function, perhaps as a detoxyase responsible for detoxifying cyanides or other chemicals present in the early atmosphere (Fani et al., 2000 ). It has been suggested that nitrogen fixation arose late in the Archaean, 2·2–2·3 gigayears ago (Gya), due to decreasing abiotic production of fixed nitrogen in the atmosphere (Navarro-González et al., 2001 ), but phylogenetic evidence strongly indicates an origin earlier than 3·5 Gya.

[...]

Recent evidence has lent support to the claim of Fenchel & Finlay (1995) that ‘all basic types of bioenergetic processes probably existed 3·5 billion years ago and the biogeochemical cycling of carbon, nitrogen and sulfur was established as we know it today...’. The canonical genetic code was undoubtedly already highly evolved prior to the divergence of the LCC and has changed little since, being at, or very close to, the optimum for error minimization (Freeland et al., 2000 ). There is good evidence, provided by extant relatives lying deep in the Bacteria and Archaea, that the LCC was thermophilic to extremely thermophilic and included in its repertoire the machinery for nitrogen fixation and possibly aerobic respiration (Saraste, 1994 ), although the utility of aerobic respiration prior to the emergence of oxygenic cyanobacteria is questionable. The presence of molybdenum, iron and sulfur in the nitrogenase enzyme complex suggests a hydrothermal heritage (Nisbet & Sleep, 2001 ), and one that was almost certainly anoxic since the complex is rapidly and irreversibly inactivated in the presence of oxygen.

The ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme 13/12C signature, which is indicative of carbon dioxide fixation and possibly oxygenic photosynthesis, appears in sedimentary carbonates dating from 3·5 Gya and perhaps even before 3·7–3·8 Gya (Rosing, 1999 ; Nisbet & Sleep, 2001 ). This isotopic evidence is supported by fossil records (although controversial) of possible cyanobacteria dating to 3·3–3·5 Gya (Des Marais, 2000 ), with firm palaeontological evidence of cyanobacteria dating from 2·7 Gya (Buick, 1992 ). Characteristic biomarkers of both cyanobacteria and eukaryotes appear in 2·7-gigayear-old shales (Brocks et al., 1999 ), and since the major lineages of anaerobic photosynthetic bacteria arose before the development of (cyanobacterial) oxygenic photosynthesis (Xiong et al.,2000), their evolution by about 2·8–3·0 Gya, or perhaps much earlier, is suggested (Des Marais, 2000 ). The deepest calibration of a node in Bacteria is provided by the isotopic signature (depletion in 34S) for sulfate reduction in sedimentary sulfides dated to about 3·47 Gya (Shen et al., 2001 ). This report provided evidence that the original precipitates were formed at temperatures below 60 °C, which is indicative of mesophiles to moderate thermophiles, suggesting that divergence of the responsible biota occurred later than that of the hyperthermophiles which constitute deeper levels of the phylogeny.

The combined evidence, particularly derived from isotopic data, places the advent of the LCC earlier than 3·5 Gya. Synthesis of isotopic evidence with phylogenetic data (16S rDNA sequencing), extends this to probably earlier than 3·8 Gya. In view of the complexity of the LCC relative to that resulting from all subsequent evolution, it is difficult to avoid the conclusion that at least as great a timespan preceded the LCC as that which followed it. If so, we must search beyond Earth for the cradle of life.

[...]

Evidence is accumulating that life originated at high temperature and pressure, probably in a hydrothermal vent habitat. The finding of Ribó et al. (2001) that vortex motion can lead to chiral selection should stimulate research on this aspect in relation to the formation of key amino acids and sugars in the hydrothermal context, where sustained vortexes are readily envisaged. Work on synthetic ribozymes is at an exciting stage: at the present pace of progress we might soon be able to simulate all essential facets of the RNA world. That the ribosome is a ribozyme hints of its deeper heritage; the discovery of an RNA polymerase potential within the core sequence of rRNA would provide an important ‘missing link’ between the RNA world and the DNA world which followed, and possibly a window to the original replicator molecule.

If Earth was the cradle for life, the time interval between its origin and the existence of the LCC ["last common community"] appears incomprehensibly short. In view of the apparent complexity of the LCC, particularly in terms of biochemistry, it would be reasonable to allow perhaps 4 gigayears for its evolution from the primordial cell. Acceptance of such an extended period of evolution must however lead to the conclusion of an extra-terrestrial origin for life on Earth. Unfortunately, examination of other planets or moons of our solar system as potential cradles for life provides very little comfort since the time extension thereby obtained is minimal. The remaining possibility for consideration is an origin beyond our solar system via directed or undirected panspermia. The following significant constraints would tend to minimize the likelihood of directed (relative to undirected) panspermia being the option that applied to Earth.

1. Our failure to detect non-natural radio signals indicates intelligent life to be at most rare in the galaxy. This contrasts with the potential for lithopanspermia, since planetary systems are demonstrably common and clearly undergo a lot of splashing.

2. If Earth was deliberately seeded with extra-terrestrial bacteria it took 4 gigayears of further evolution to produce a lifeform capable of sending a spacecraft to another star. Adding a ‘comfortable’ 8 gigayears for the evolution from a primordial cell to the human equivalent elsewhere, takes us to the first generation of stars in our galaxy. Therefore only a very small fraction of the habitable galaxy would be seeded in this manner to the present time, again contrasting with undirected panspermia which in the scenario described would have had a 4 gigayear lead-time for galactic colonization.

3. If Earth was deliberately seeded, it is odd that it was only with microbes. Although the transport of macro-organisms such as ourselves may never be possible over interstellar distances, we foresee the time when robust, intelligent, self-replicating machines will be capable of a trip taking a million years or more. The time-span between our capacity for achieving directed panspermia using microbes (now) to that when we might provide the seed for both life and machine-intelligence is probably less than 200 years, a very small time-span in galactic terms.

Against undirected panspermia is the very low density of stars in our region of the galaxy, militating against a random serendipitous seeding event at the very time when Earth became habitable. This apparent difficulty may however relate to our poor comprehension of the capacity of meteoritic splashing to propel large numbers of organisms into space. What number of carbon/silicon-coated microbes splattered randomly into space is needed to provide a good probability of seeding another planetary system in our part of the galaxy, and why should this number not be achievable?

Further work is needed to support contentions of the survival of bacteria for a million years or more, since this has a direct bearing on the viability of panspermia hypotheses. Particularly important is the confirmation of estimates of the survivability of ensheathed bacterial spores (and their nucleic acid) following exposure to high-energy radiation equivalent to that existing in the interstellar medium over prolonged periods. While asserting that the total energy input of energetic particles is generally orders of magnitude lower than that of UV light, Weber & Mayo Greenberg (1985) did not consider survival of cocooned cells protected from this source.

Future expeditions to the outer solar system should shed some light on the reality or otherwise of extra-terrestrial origins of life. If the conclusions outlined here are correct, Mars and the habitable moons of other planets would have been seeded from beyond our solar system in the same event that seeded Earth. Tidal or other forces on ice crusts of some of the outer moon systems would ensure that microbes seeded at the surface would reach a warm ocean beneath. Consequently all such life would be related to that on Earth, linking to the ‘base’ of the universal phylogeny, quite possibly at a common node (point in time) regardless of its planetary or satellite origin. In that case we would certainly obtain a fascinating perspective of independent evolution from about 4 Gya, but would gain no deeper insight as to what came before.

Note added in proof

The prospects of interstellar panspermia have been dealt a blow at a recent conference on Lunar and Planetary Science (Johnson Space Centre), from calculations by planetary physicist, Jay Melosh. Simulations of the orbits of Martian rocks ejected from the solare system by Jupiter indicated a very low probability of rock capture by another stellar system, of about one meteorite ejected from a planet belonging to out solar system each 100 million years. Further calculation of the chances of rocks captured by another star hitting a terrestrial planet reduced the odds even further. A similar conclusion, that the chances of life getting to our solar system in this way were ‘essentially nil’, was expressed by geophysicist Norman Sleep (Kerr, 2001 ).

The concept of interstellar panspermia has been a philosophical luxury; it may soon become a necessity if constraints of evolutionary theory continue to conspire against an origin of life in our solar system. Perhaps fresh calculations are in order, to consider possibilities other than those provided by a Mars/Jupiter slingshot. Pluto may once have hosted a stable ocean beneath an ice cover, to therefore be a contender as a cradle for life. Would it be possible for splashed material from Pluto (by Oort cloud debris) to be expelled from the solar system by its moon Charon if the obliquity of the Pluto/Charon system was better aligned in the plane of the solar system? What of a mud/micorbial ‘fog’ created in a splash event being accelerated to escape velocity by a red giant? What optimal system might we construe to improve the odds of interstellar transport?
Thanks Nic for setting up that EvoWiki page. Since you have some experience compiling literature for Web archives, could you send some pointers my way about how to distill all this information down? I think EvoWiki might be the best next step.

[Mike H]

In terms of a somewhat more recent review-oriented paper on nitrogenase, you might want to check out this paper (http://www.pnas.org/cgi/content/full/100/7/3595), particularly on understanding biological function with the assistance of synthetic analogues. Some of you may also want to check out that issue of PNAS (http://www.pnas.org) in any case, it's a rather broad overview of bioinorganic chemistry as it stands today. It also mentions the rather recently discovered result that there is a light element ligand bound to the MoFe cofactor, most likely a nitride.

For the paper which originally put forth this idea based on refinement of crystallographic data, you will have to either have online or library access to Science (http://www.sciencemag.org). The citation itself is
Oliver Einsle, F. Akif Tezcan, Susana L. A. Andrade, Benedikt Schmid, Mika Yoshida, James B. Howard, and Douglas C. Rees. "Nitrogenase MoFe-Protein at 1.16 Å Resolution: A Central Ligand in the FeMo-Cofactor." Science 2002 September 6; 297: 1696-1700.
Mainly just in case anyone bothers you with the "When I learned about nitrogenase, there was no mention of this ligand!" question.

Of a more broad-based nature is the review by Doug Rees (citation is D.C. Rees (2002) "Great Metalloclusters in Proteins." Ann. Rev. Biochem; 71:221-46.) where he discusses nitrogenase (as well as a few other proteins with large metal clusters) in some detail. The interesting thing - from an evolutionary standpoint - are the common features that are shared between the proteins he discusses. I would also note that if you are interested in nitrogenase above and beyond laying the smack down on dubious creationist claims, a PubMed search with his name will lead you to many interesting structural and biochemical studies that should be of interest.

I apologize for the outburst of technical material, but metalloproteins are what I do. Just...can't...help...myself.

[lpetrich]

On the subject of metalloproteins, here is an article that identifies one of them as very ancient:
Molecular evolution before the origin of species (http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12225777&dopt=Abstract), Brian Davis, 2002

Ferredoxin is an iron-sulfur enzyme which can be traced back to the Last Universal Common Ancestor, where it had undergone a tandem repeat. This repeat allows for further extrapolation to a 23-amino-acid ancestral sequence. This sequence has not only the appropriate cysteines for making an iron-sulfur complex, but also a negatively-charged "tail" that can stick to a positively-charged mineral substrate. Which is the sort of environment often considered the most plausible for abiogenesis. Which also means that it could have existed in some pre-cellular phase, something like Ernst Haeckel's Urschleim.

It is so remarkable to be able to look back all that way and get so very close to the origin of life.

Here are all 10 proteins that BD examined, in order of inferred age:

Ferredoxin (Fe-S protein; does redox reactions)
Proteolipid h1 (lives in cell membranes; part of ATPase complex)
FtsZ (involved in cell division)
FEN-1 (flap exonuclease)
RNA polymerase beta'
Reverse transcriptase (RNA -> DNA)
DNA topoisomerase I (alters DNA topology)
Ribonucleotide reductase (Fe) (RNR's make DNA nucleotides from RNA ones)

Age was inferred by the number of biosynthesis steps needed to produce the proteins' amino acids from the Krebs cycle. The "easier" amino acids were likely prebiotic, the "harder" ones were possibly metabolic leakage.

It would be interesting to see how ferredoxin relates to other metal-sulfur proteins; they may be the result of gene duplication and specialization in a different direction.

There is an illustration of that ancestral ferredoxin from that article that I've clipped and included in this message.

(I've thought it over, and I've decided that the illustration from the article will not be a copyright violation. Mods, if you think otherwise, please feel free to remove it.)

[Nic Tamzek]

I just got around to reading the long quote Principia provided from this article:

Microbiology (2002), 148, 21-27.
The enigma of the origin of life and its timing (http://mic.sgmjournals.org/cgi/content/full/148/1/21?view=full&pmid=11782495)
Martin A. Line

...does it strike anyone else as a rather flaky article, a surprising thing to find in Microbiology? I mean, where does Line get his estimates that 4 billion years would be required for the natural origin of life?

PS: I just went and looked at the rest of the article, it's a bit better.

[Guts]

I wonder when Principia is actually going to discuss these papers instead of just posting them. I really don't see the relevance to what was stated in the OP.

For example, the OP states:


No. You either have nitrogenase as we know it or have nothing. There are no simpler versions of nitrogenase and there is no reason to think it would function in a biologically significant manner in a much sloppier state.


I was wondering which of these papers shows a simpler version of nitrogenase?

[Principia]

Guts: I really don't see the relevance to what was stated in the OP. No, I don't expect you to.
I was wondering which of these papers shows a simpler version of nitrogenase? Let me know when you figure it out, will ya?

[Principia]

More about nifV- phenotypes:
J Bacteriol. 1988 Apr;170(4):1978-9

Homocitrate cures the NifV- phenotype in Klebsiella pneumoniae.

Hoover TR, Imperial J, Ludden PW, Shah VK.

Dinitrogenase was isolated from a culture of a Klebsiella pneumoniae NifV- strain derepressed for nitrogenase in the presence of homocitrate. The enzyme isolated from this culture was identical to the wild-type dinitrogenase. These data provide in vivo evidence that the absence of homocitrate is responsible for the NifV- phenotype.
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6517-21

Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.

Madden MS, Kindon ND, Ludden PW, Shah VK.

In vitro synthesis of the iron-molybdenum cofactor (FeMo-co) of dinitrogenase using homocitrate and its analogs allows the formation of modified forms of FeMo-co that show altered substrate specificities (N2, acetylene, cyanide, or proton reduction) of nitrogenase [reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolyzing), EC 1.18.6.1]. The (1R,2S)-threo- and (1S,2S)-erythro-fluorinated diastereomers of homocitrate have been incorporated in vitro into dinitrogenase in place of homocitrate. Dinitrogenase activated with FeMo-co synthesized using threo-fluorohomocitrate reduces protons, cyanide, and acetylene but cannot reduce N2. In addition, proton reduction is inhibited by carbon monoxide (CO), a characteristic of dinitrogenase from NifV- mutants. Dinitrogenase activated with FeMo-co synthesized using erythro-fluorohomocitrate reduces protons, cyanide, acetylene, and N2. In this case proton reduction is not inhibited by CO, a characteristic of the wild-type enzyme. Cyanide reduction properties of dinitrogenase activated with FeMo-co containing either fluorohomocitrate diastereomer are similar, and CO strongly inhibits cyanide reduction. Dinitrogenases activated with FeMo-co containing homocitrate analogs with a hydroxyl group on the C-1 position are much less susceptible to CO inhibition of cyanide reduction. However, proton and cyanide reduction by dinitrogenase containing FeMo-co activated with (1R,2S) threo-isocitrate is only one-third that of dinitrogenase activated with the racemic mixture of -isocitrate and shows strong CO inhibition of substrate reduction. These results suggest that CO inhibition of proton and cyanide reduction occurs when the hydroxyl group on the C-1 position of analogs is "trans" to the C-2 carboxyl group (i.e., in the threo conformation). When racemic mixtures of these analogs are used in the system, it seems that the erythro form is preferentially incorporated into dinitrogenase. Finally, carbonyl sulfide inhibition of substrate reduction by dinitrogenase is dependent on the homocitrate analog incorporated into FeMo-co.
Several organic acids (Fig. 1) were tested for their ability to replace homocitrate in the FeMo-co syntehsis system (11, 13). The resulting variant forms of FeMo-co exhibited altered substrate specificty and inhibitor susceptibility. Use of citrate in place of homocitrate in the in vitro FeMo-co synthesis system resulted in the formation of holodinitrogenase with effective proton and acetylene reduction activites but with poor N2 reduction and no 1H2H formation. In addition, proton reduction is inhibited by carbon monoxide (CO). These results are analogous to dinitrogenase from nifV mutants (14, 15), and recently citrate was found in the dinitrogenase of such mutants (16). Dinitrogenase activated with FeMo-co containing homoisocitrate, isocitrate, or 1-OH citrate effectively catalyzed the reduction of protons but not the reduction of acetylene or N2 (13). The highest stringency pertaining to substrate specificty was found for N2 reduction, a six-electron process. 15N enrichment observed in experimnts where apodinitrogenase was activated with homocitrate/FeMo-co was 80 times that of background levels, compared with citrate/FeMo-co with 6.3 times background levels (13). These data suggest that none of the FeMo-co analogs tested produce a dinitrogenase capable of supporting significant dizaotrophic growth.

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=54567&action=stream&blobtype=pdf
Kind of interesting in light of the proposed detoxyase models.

EDIT: Proposed enzymatic functions of NifV --
J Bacteriol. 1997 Sep;179(18):5963-6

Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase.

Zheng L, White RH, Dean DR.

The nifV gene product (NifV) from Azotobacter vinelandii was recombinantly expressed at high levels in Escherichia coli and purified. NifV is a homodimer that catalyzes the condensation of acetyl coenzyme A (acetyl-CoA) and alpha-ketoglutarate. Although alpha-ketoglutarate supports the highest level of activity, NifV will also catalyze the condensation of acetyl-CoA and certain other keto acids. E. coli cells in which a high level of nifV expression is induced excrete homocitrate into the growth medium.

[Principia]

Beautfiul:
J Biol Chem. 1997 Oct 17;272(42):26627-33

N2 fixation by Streptomyces thermoautotrophicus involves a molybdenum-dinitrogenase and a manganese-superoxide oxidoreductase that couple N2 reduction to the oxidation of superoxide produced from O2 by a molybdenum-CO dehydrogenase.

Ribbe M, Gadkari D, Meyer O.

N2 fixation by Streptomyces thermoautotrophicus follows the equation N2 + 4-12MgATP + 8H+ + 8e- --> 2NH3 + H2 + 4-12MgADP + 4-12Pi and exhibits features which are not obvious in the diazotrophic bacteria studied so far. The reaction is coupled to the oxidation of carbon monoxide (CO) by a molybdenum-containing CO dehydrogenase that transfers the electrons derived from CO oxidation to O2, thereby producing superoxide anion radicals (O-2). A manganese-containing superoxide oxidoreductase reoxidizes the O-2 anions to O2 and transfers the electrons to a MoFeS-dinitrogenase for the reduction of N2 to ammonium. Among the most striking properties of the S. thermoautotrophicus nitrogenase system are the dependence on O2 and O-2, the complete insensitivity of all components involved toward O2 and H2O2, the inability to reduce ethine or ethene, and a low MgATP requirement. In addition, the subunit structure of the S. thermoautotrophicus nitrogenase components and the polypeptides involved seem to be dissimilar from the known nitrogenases.
N2 Fixation Is Linked to the Oxidation of Superoxide

The CO dehydrogenases from Oligotropha carboxidovorans3 and S. thermoautotrophicus can transfer the electrons formed upon CO oxidation (CO + H2O CO2 + 2e + 2H+) to O2, thereby producing superoxide anion radicals (O2) and hydrogen peroxide (H2O2). In assays containing purified CO dehydrogenase, St1 and St2 protein, and MgATP under an atmosphere of (v/v) 50% CO and 50% air, N2 was reduced to ammonium, indicating that N2 reduction was coupled to the oxidation of CO. Ammonium was not formed in the absence of CO. The requirement of O2 for N2 reduction in cytoplasmic fractions (Table IV) is explained by the formation of O2 by CO dehydrogenase and the subsequent reoxidation of O2 to O2 by the St2 protein. The functioning of O2 as an electron donor for N2 reduction is further substantiated by the sensitivity of the reaction to added SODs, the ability to replace CO dehydrogenase for xanthine oxidase, plus a suitable substrate or to provide O2 chemically from riboflavin plus light (Table IV). MnSODs from Escherichia coli and B. stearothermophilus could not substitute for the St2 protein (Table IV). Oxidation of H2 by CO dehydrogenase in the presence of O2 also leads to O2 formation (Table IV). During chemolithoautotrophic utilization of H2, S. thermoautotrophicus employs a membrane-bound hydrogenase for energy generation; this hydrogenase cannot produce O2. Under these conditions the hydrogenase activity of CO dehydrogenase (32, 33) is used for the generation of O2 as an electron donor for N2 fixation.

[...]
When cytoplasmic fractions of S. thermoautotrophicus were kept for 24 h at room temperature under N2, helium, air, or O2, nitrogenase activities ranged from 20.2 to 29.6 nmol of NH4+/mg of protein/h. Shaking of the purified proteins St1 and St2 for 24 h in air also did not affect nitrogenase activity. Under oxic conditions St1 was purified 11-fold with a yield of 1% and a specific activity of 139 nmol of NH4+/mg of protein/h, and St2 was purified 21-fold, with a yield of 31% and a specific activity of 233 nmol of NH4+/mg of protein/h. The results characterize the N2-fixing system of S. thermoautotrophicus as absolutely O2-insensitive.

Discussion

N2 fixation by S. thermoautotrophicus involves CO dehydrogenase, free O2, and O2 as electron carriers and the proteins St2 and St1 (Fig. 7). CO dehydrogenase generates superoxide anion radicals (O2) from O2, which makes N2 fixation in S. thermoautotrophicus obligately O2-dependent and establishes a molecular coupling via O2, which is in contrast to the electronic coupling in the known nitrogenase systems. The O2 anions are free intermediates and can be trapped by SOD (Table IV). Consequently, interaction of CO dehydrogenase and the St2 protein for O2 transfer is not required. Generally, O2 is considered a highly reactive and destructive metabolic by-product that requires detoxification, e.g. by superoxide dismutases. N2 fixation in S. thermoautotrophicus shows that the redox couple O2/O2 (E0 = 160 mV) operates as an electron carrier with similar efficiency as ferredoxins, flavodoxins, or hydroquinones in the known nitrogenase systems (34, 35). In addition, the use of O2 in N2 fixation by S. thermoautotrophicus seems to be a powerful mechanism to scavange O2 radicals.

The dinitrogenase reductases known so far are 2 dimeric iron proteins with molecular mass ~63 kDa and contain 4Fe and 4S2 atoms per dimer (36). In contrast, the St2 protein of S. thermoautotrophicus has been identified as a manganese-superoxide oxidoreductase with molecular mass ~48 kDa and does not contain Fe or S2 (Table II). In contrast to SOD, St2 is unable to disproportionate O2 into O2 and H2O2. The function of St2 is rather to generate electrons through the reoxidation of O2 ions to O2 and to deliver them to the St1 protein (Fig. 7). Like the known nitrogenases, the S. thermoautotrophicus system also has a requirement for MgATP. With S. thermoautotrophicus nitrogenase the most efficient MgATP/N2 ratio is 4, instead of 16 reported for nitrogenases from other sources, indicating its superb efficiency. Future work must unravel how MgATP interacts with the nitrogenase proteins.

http://www.jbc.org/content/vol272/issue42/images/medium/bc4270490007.gif

It is likely that the reduction of N2 takes place at the St1 protein of S. thermoautotrophicus. The known MoFeS-dinitrogenases are complex 22 tetrameric proteins with Mr ~230 kDa containing 2 molybdenum atoms and 30-34 iron atoms and an approximately equivalent number of acid-labile sulfide atoms (36). The St1 protein was also identified as a MoFeS-protein but with molecular mass ~144 kDa and a differing LMS heterotrimeric subunit structure (Figs. 1 and 7). It revealed per molybdenum atom, 13.8-21.7 iron atoms and 8.8-15 acid-labile sulfide atoms (Table II). A substoichiometric amount of acid-labile sulfide compared with iron is frequently found with nitrogenases (37). There was a moderate sequence similarity between the N-terminal sequences of the subunits St1-L and Kp1- or St1-M and Kp1-, although we are aware of the limitations of a comparison of subunit N termini. The overall reaction catalyzed by S. thermoautotrophicus nitrogenase compares to that of known nitrogenases, including the concomitant formation of H2 (Fig. 4).
It will be interesting to see if these critters have deleted or nonfunctional nifDK/NE since the St2 (superoxide dismutase) and St3 (CO dehydrogenase) are the primary electron donors in this system.

EDIT to add: The N termini of the St1 polypeptides had the sequences: ALPQTELRPMGKPILRKXDP (St1-L), MFPNAFKYEAPASVDEAVRLLAEYGYDGKV (St1-M), and MKIRVKVNGTLYEADVEP (St1-S). The N termini of St1-L and the dinitrogenase beta-subunit of Anabaena 7120 or Klebsiella pneumoniae showed 42.1 or 44.4% sequence similarity and 26.3 or 16.7% identity, respectively. The N termini of St1-M and the dinitrogenase alpha-subunit of Anabaena 7120 or K. pneumoniae showed 55.2 or 52.4% sequence similarity and 38.1 or 20.7% identity, respectively. The N terminus of St1-S revealed no significant sequence similarity to the alpha- or beta-subunits of other nitrogenases or to the delta-subunit of alternative dinitrogenases.

Upon nondenaturating PAGE, the St2 protein revealed a single 40-kDa band at pH 6.5 or pH 7.5 (Fig. 1), or a single 98-kDa band at pH 8.5. Other methods revealed masses of 49 kDa (sucrose density gradient centrifugation) or 41.5 kDa (gel filtration). Denaturating PAGE showed a single noncovalently bound 24-kDa subunit, designated D (Fig. 1), suggesting a homodimeric subunit structure. The N terminus2 MFELPPLPYPYDALEPYFDAKKMEIHYYGGHGA of the St2-D polypeptide revealed no significant sequence similarity to sequenced nitrogenase polypeptides. Instead, St2-D showed 96.0% sequence similarity and 72.0% identity to the N terminus of the manganese-containing SODs of Bacillus stearothermophilus (22) or Bacillus caldotenax (23). In assays containing xanthine oxidase, xanthine, and O2 for the production of superoxide anion radicals (O2) the St2 protein revealed O2 oxidizing activity of 126 units/mg of protein (1 unit equals 50% inhibition of NBT reduction). The reaction was insensitive to 3.5 mM potassium cyanide, 60 mM sodium azide, or 10 mM H2O2. Although the St2 protein displays many properties similar to a manganese SOD (24) it actually functions as a superoxide oxidoreductase transferring the electrons to the St1 protein (see below). In the presence of CO and O2, CO dehydrogenase produced H2O2 and O2. The addition of commercial E. coli SOD effected an approximately 15% increase in H2O2 formation. When SOD was replaced by St2 protein, H2O2 formation decreased by 13%, indicating the inability of the St2 protein to convert O2 to H2O2. Dithionite reduced St2 protein could transfer 2.3 ± 0.7 electrons to oxidized phenazine methosulfate with an activity of 77 nmol/mg of protein/min. Another suitable electron acceptor was 2,6-dichlorophenolindophenol (112 nmol/mg of protein/min), whereas viologen dyes, methylene blue, ferricyanide, INT, nitro blue tetrazolium, NAD(P), FAD, FMN, and riboflavin were ineffective.

[Guts]

So Principia, I guess you're not gonna discuss any of those papers eh?

I actually have some cool information, using these papers, relevant to what was stated in the OP. Unfortunately I'm on my way to New Years party. So I'll see you next year. :D

[Principia]

J Bacteriol. 1992 Nov;174(21):6840-3

Chemolithoautotrophic assimilation of dinitrogen by Streptomyces thermoautotrophicus UBT1: identification of an unusual N2-fixing system.

Gadkari D, Morsdorf G, Meyer O.

Streptomyces thermoautotrophicus UBT1, which was isolated previously from a burning charcoal pile, was shown to utilize N2 as a sole nitrogen source when growing chemolithoautotrophically with CO or H2 plus CO2 under aerobic conditions at 65 degrees C. Doubling times under diazotrophic conditions were 10 h. S. thermoautotrophicus is a new CO- or H2-oxidizing, obligately chemolithoautotrophic, thermophilic, free-living, aerobic, N2-fixing streptomycete. Its ability to fix N2 was also evident from (i) the incorporation of substantial amounts of 15N2 (about 13%) into cell material, (ii) the formation of H2 during diazotrophic growth, (iii) the repression of 15N2 assimilation and H2 formation by ammonia, and (iv) culture growth yields with N2 as a nitrogen source that were significantly higher than those without any added nitrogen compounds (ca. 2.4 versus < 0.1 mg [dry weight]). The N2-fixing system of S. thermoautotrophicus exhibited several properties not apparent in the diazotrophic bacteria studied so far, since it was (i) incapable of reducing acetylene to ethylene or ethane and (ii) resistant to inhibition by acetylene or ethylene (5% [vol/vol] each), CO (40 to 70% [vol/vol]), or H2 (40% [vol/vol]). Under stringent conditions, nifH and nifDK gene probes from Klebsiella pneumoniae did not hybridize with total DNA from S. thermoautotrophicus.

http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=207360&action=stream&blobtype=pdf Well, that's good enough for me at the moment. Let me see if I can dig up something more definitive...

[Principia]

Here is another oldie:
Proc Natl Acad Sci U S A. 1993 August 1; 90 (15): 7134–7138

Early Evolution of Photosynthesis: Clues from Nitrogenase and Chlorophyll Iron Proteins

DH Burke, JE Hearst, and A Sidow

Chlorophyll (Chl) is often viewed as having preceded bacteriochlorophyll (BChl) as the primary photoreceptor pigment in early photosynthetic systems because synthesis of Chl requires one fewer enzymatic reduction than does synthesis of BChl. We have conducted statistical DNA sequence analyses of the two reductases involved in Chl and BChl synthesis, protochlorophyllide reductase and chlorin reductase. Both are three-subunit enzymes in which each subunit from one reductase shares significant amino acid identity with a subunit of the other, indicating that the two enzymes are derived from a common three-subunit ancestral reductase. The "chlorophyll iron protein" subunits, encoded by the bchL and bchX genes in the purple bacterium Rhodobacter capsulatus, also share amino acid sequence identity with the nitrogenase iron protein, encoded by nifH. When nitrogenase iron proteins are used as outgroups, the chlorophyll iron protein tree is rooted on the chlorin reductase lineage. This rooting suggests that the last common ancestor of all extant photosynthetic eubacteria contained BChl, not Chl, in its reaction center, and implies that Chl-containing reaction centers were a late invention unique to the cyanobacteria/chloroplast lineage.
Each of the three subunits from the chlorine reductase can be aligned with one from the PChlide reductase. All three PChlide reductase genes from Rhodobacter capsulatus are more similar to those in cyanobacteria and chloroplasts than they are to the respective subunits of the chlorin reductase (Table 2). The strongest conservation between PChlide and chlorin reductases is among bchX, bchL, and chlL (Fig. 2; ref. 8). These also share notable sequence identity with the nitrogenase iron proteins. The greatest conservation is in sites known to be important for binding the gamma-phosphate of MgATP (23,24), for binding a [4Fe-4S] cluster (25-28), and in those postulated to have a role in catalyzing ATP hydrolysis (Asp-39 and Asp-43; ref.28). All of these imply mechanistic similarities between the chlorophyll and nitrogenase iron proteins (8, 11, 29-31).

There is also evidence for structural similarities. First, when conserved regions I-IV (Fig. 2) are mapped onto the nifH crystal structure (28), a number of the matches are located within the subunit interiors adn at at the subunit-subunit interface of the homodimer (Fig. 3). [...] Since this region has been shown to be involved in ionic interactions of nifH with the beta subunit of nitrogenase, nifK (32-35), similar ionic interactions may bind the chlorophyll iron proteins to one or both of their respective ancillary proteins bchYZ, bchNB, or chlNB. Third, in all nitrogenase iron proteins and in bchX, position 100 is occupied by an arginine residue. In both bchL and chlL, it is tyrosine. In site-directed mutations of [i]Azotobacter vinelandii nifH, tyrosin is the only other amino acid ath this position that still allows a significant amount of electron transfer activity (35). Finally, the structural compatibility method of Bowie et al. (36), which assesses the likelihood of burying and exposing residues in a given sequence, predicts all chlorophyll iron proteins to be compatible with adopting a nitrogenase iron protein-like structure (D. Eisenberg, personal communication).

[...]

Proposed History of the Iron Protein Family The ancestral iron protein duplicated and diverged into nitrogenase type I and type III prior to the speciation event that separated the eubacteria from the methanogenic archaebacteria. Subsequent to the this speciation, there was a duplication of the nitrogenase iron protein gene in the eubacterial line, possibly accompanied by a duplication of nifK and nifD. One of the copies divferged sufficiently to enable it to interact with a PChlide-binding protein (or even directly with the PChlide), thereby forming the first PChlide reductase. This enzyme reduced its substrate twice to form a bacteriochlorin. Subsequent duplication of each of the three subunits of this reductase allowed the two copies to specialize toward reduction of PChlides on one hand and chlorins on the other. Chl apeared later during the early evolution of cyanobacteria. Reduced reaction center pigments and the modern oxygen-rich atmosphere are therefore partly consequences of ancient duplications of the type I nitrogenase iron protein.

Transcribed from http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=47090&action=stream&blobtype=pdf

[Principia]

An interesting, through speculative, hypothesis:
Angew Chem Int Ed Engl. 2003 Apr 4;42(13):1540-3

A possible prebiotic formation of ammonia from dinitrogen on iron sulfide surfaces.

Dorr M, Kassbohrer J, Grunert R, Kreisel G, Brand WA, Werner RA, Geilmann H, Apfel C, Robl C, Weigand W

Herein we describe a method for the synthesis of NH3 from N2 using H2S as a reductant and freshly precipitated iron sulfide as a mediator, which could have served as a primordial inorganic substitute for the enzyme nitrogenase. The reductant as well as the reaction conditions (atmospheric nitrogen pressure and temperatures of the order of 70-80 °C) are rather mild and comparable to biological processes. The driving forces of the overall reaction are the oxidation of iron sulfide to iron disulfide and the formation of hydrogen from H2S.

http://www3.interscience.wiley.com/cgi-bin/fulltext/104519974/HTMLSTART

[Principia]

The story gets more interesting:
Mol Gen Genet. 1992 Feb;231(3):494-8

The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii.

Kennedy C, Dean D.

Strains with mutations in 23 of the 30 genes and open reading frames in the major nif gene cluster of A. vinelandii were tested for ability to grow on N-free medium with molybdenum (Nif phenotype), with vanadium (Vnf phenotype), or with neither metal present (Anf phenotype). As reported previously, nifE, nifN, nifU, nifS and nifV mutants were Nif- (failed to grow on molybdenum) while nifM mutants were Nif-, Vnf- and Anf-. nifV, nifS, and nifU mutants were found to be unable to grow on medium with or without vanadium, i.e. were Vnf- Anf-. Therefore neither vnf nor anf analogoues of nifU, nifS, nifV or nifM are expected to be present in A. vinelandii.
In this post, let's focus on nifU (though nifS is not irrelevant):
Proc Natl Acad Sci U S A. 2000 January 18; 97 (2): 599–604

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein

Pramvadee Yuvaniyama,* Jeffrey N. Agar,† Valerie L. Cash,* Michael K. Johnson,†‡ and Dennis R. Dean*‡

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S]2+,+ cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=10639125#B3
Genes encoding homologs to NifU and NifS are also located within the genomes of a wide variety of non-nitrogen fixing organisms (14). We have designated these as isc genes to indicate the proposed role of their products in the housekeeping function of general Fe-S cluster assembly. In line with this proposal the isc genes have been found to be essential for A. vinelandii viability (14). There is also mounting biochemical and genetic evidence from other laboratories that the isc gene products are involved in Fe-S cluster assembly in both prokaryotic (15) and eukaryotic (16–19) organisms. A comparison of the organization of the NifU protein and its proposed housekeeping counterpart, designated IscU, is relevant to the work described here. For example, the IscU protein is considerably truncated when compared with NifU, bearing sequence identity only to the N-terminal third of NifU. This portion of NifU corresponds to the NifU-1 fragment described in the present work. The NifU Cys35, Cys62, and Cys106 residues contained within this segment are also strictly conserved in all iscU gene products identified so far. In fact, the IscU primary sequence is among the most conserved sequence motifs in nature (20). The IscU protein does not contain a sequence corresponding to the permanent [2Fe-2S]2+,+ cluster-binding domain present in NifU. However, there is another gene contained within the isc gene cluster whose product does harbor weak primary sequence identity when compared with the [2Fe-2S]2+,+ cluster-binding region of NifU. This small ferredoxin has been purified and shown to contain a [2Fe-2S]2+,+ cluster that is nearly identical in its spectroscopic and electronic properties when compared with the [2Fe-2S] clusters contained within as-isolated NifU (21, 22). Thus, a function analogous to that provided by the NifU permanent cluster might also be duplicated by this ferredoxin. It is interesting that a bacterial ferritin-associated [2Fe-2S]2+,+ ferredoxin having the same spatial arrangement of cluster-coordinating cysteines, as well as the same spectroscopic and electronic properties as the isc-specific ferredoxin, has also been identified in E. coli (23, 24). This observation has led to speculation that a function of the bacterial ferritin-associated ferredoxin could involve the release of iron from ferritin for Fe-S cluster assembly, a suggestion also in line with a possible role for the permanent clusters contained within NifU.
Hell, might as well throw in another review:
Biochem. Soc. Trans. (2001) 30, (680–685)

Biosynthesis of iron–sulphur clusters is a complex and highly conserved process

J. Frazzon*, J. R. Fick† and D. R. Dean

Iron–sulphur ([Fe–S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe–S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe–S] clusters and their insertion into various protein partners. Among the [Fe–S] cluster biosynthetic proteins are included a pyridoxal phosphate- dependent enzyme (NifS) that is involved in the activation of sulphur from L-cysteine, and a molecular scaffold protein (NifU) upon which [Fe–S] cluster precursors are formed. The formation or transfer of [Fe–S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe–S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe–S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe–S] cluster formation that were discovered in the eubacteria.

http://www.biochemsoctrans.org/bst/030/0680/bst0300680.htm

[Principia]

On MikeH's recommendation, I looked up some of Doug Rees's recent review articles on metalloenzymes. Here's one that I found particularly insightful:
Science. 2003 May 9;300(5621):929-31.

The interface between the biological and inorganic worlds: iron-sulfur metalloclusters.

Rees DC, Howard JB.

Complex iron-sulfur metalloclusters form the active sites of the enzymes that catalyze redox transformations of N2, CO, and H2, which are likely components of Earth's primordial atmosphere. Although these centers reflect the organizational principles of simpler iron-sulfur clusters, they exhibit extensive elaborations that confer specific ligand-binding and catalytic properties. These changes were probably achieved through evolutionary processes, including the fusion of small clusters, the addition of new metals, and the development of cluster assembly pathways, driven by selective pressures resulting from changes in the chemical composition of the biosphere.
The article concludes with a recapitulation of Fani et al.'s detoxyase model, and the following observation:
How these more complicated structures might have arisen from the simpler Fe:S clusters is a matter of conjecture (11). It is apparent that in many cases (perhaps most notably for the nitrogenase FeMo-cofactor), the cluster requires additional assembly processes before incorporation by the enzyme (28). Hence, the evolution of an enzyme such as nitrogenase is, in fact, the coevolution of multiple components, some of which are only indirectly expressed as the enhanced activity of the more complex clusters (29). One clear indication that the cluster assembly process has accommodated the change from a reductive to an oxidative environment is that cells living in aerobic niches must actively seek and recover the poorly soluble ferric iron from their surroundings, and further cluster assembly entails the use of a protected sulfide in the form of the sulfur donation from cysteine.

As shown in Fig. 1, a progression in sophistication from simple Fe:S clusters with broad nonspecific properties to clusters with highly specific and efficient catalytic properties could be envisioned. Hence, when considering how an early Archaean metabolic function was acquired, one may need to consider initially less elegant, simpler structures with lower efficiencies (8), which evolved under selective pressures such as changes in atmospheric composition. Such an evolutionary process must include the gain of function by the fusion of small clusters under the influence of altered host protein structures, the addition of new metals, and the action of chaperone-like proteins.
Incidentally, ref. 8 points to the Dorr et al. article about prebiotic nitrogenase pathways linked on a few posts above.

[Principia]

NafY is a non-nif gene, but shares similarities with nifX, nifY, and nifB which are all (including NafY) involved in nitrogenase maturation of A. vinelandii. J. Biol. Chem., Vol. 277, Issue 16, 14299-14305, April 19, 2002

Cloning and Mutational Analysis of the Gene from Azotobacter vinelandii Defines a New Family of Proteins Capable of Metallocluster Binding and Protein Stabilization*

Luis M. Rubio, Priya Rangaraj, Mary J. Homer§¶, Gary P. Roberts§, and Paul W. Ludden

Dinitrogenase is a heterotetrameric (22) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition 222, which can be activated in vitro by the addition of FeMo-co. The protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now . In vitro FeMo-co insertion experiments presented in this work demonstrate that stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.
Interestingly, the protein fold of nafY belongs to the RNase H superfamily, as observed by the authors here: J. Biol. Chem., Vol. 278, Issue 34, 32150-32156, August 22, 2003

The Three-dimensional Structure of the Core Domain of Naf Y from Azotobacter vinelandii determined at 1.8-Å Resolution

David H. Dyer , Luis M. Rubio , James B. Thoden, Hazel M. Holden, Paul W. Ludden and Ivan Rayment

The Azotobacter vinelandii NafY protein (nitrogenase accessory factor Y) is able to bind either to the iron molybdenum cofactor (FeMo-co) or to apodinitrogenase and is believed to facilitate the transfer of FeMo-co into apodinitrogenase. The NafY protein has two domains: an N-terminal domain (residues Met1–Leu98) and a C-terminal domain (residues Glu99–Ser232), referred here to as the "core domain." The core domain of NafY is shown here to be capable of binding the FeMo cofactor of nitrogenase but unable to bind to apodinitrogenase in the absence of the first domain. The three-dimensional molecular structure of the core domain of NafY has been solved to 1.8-Å resolution, revealing that the protein consists of a mixed five-stranded beta-sheet flanked by five -helices that belongs to the ribonuclease H superfamily. As such, this represents a new fold capable of binding FeMo-co, where the only previous example was that seen in dinitrogenase.

http://www.jbc.org/cgi/content/full/278/34/32150
In Mo limiting situations, NafY is thought to bind the dinitrogenase and stabilize it while awaiting a cofactor. However: The primary role of NafY appears to be that of a molecular chaperone that maintains apodinitrogenase in a conformation that facilitates receipt of FeMo-co, thereby acting as a metallo-insertase (8). In addition, NafY can bind in vitro to FeMo-co, suggesting that this protein has two distinct activities. The presence of multiple domains within NafY, as suggested by the facile generation and stability of the core domain and by amino acid sequence alignments with NafY homologs, raises the question of whether the molecular chaperone and FeMo-co binding functions of NafY are mediated by different modules within the polypeptide chain.

[...]

The present study establishes the modular nature of NafY from A. vinelandii. It shows that the C-terminal region of NafY (Glu99–Ser232; referred to here as the core domain) folds autonomously and is sufficient for FeMo-co binding, whereas the N-terminal region of the protein (Met1–Leu98) is required for interaction with apodinitrogenase. The determination of the three-dimensional structure of NafY is of interest because it is the first FeMo-co-binding protein, other than dinitrogenase, that has been solved, and it represents a new fold for FeMo-co binding. The core domain of NafY has a beta1-beta2-beta3-alpha1-alpha2-beta4-alpha3-beta5-alpha4-alpha5 fold and a polarized surface charge distribution that are surprisingly similar to those of the ribonuclease H family. NafY is part of a conserved family of proteins, whose members are involved at different steps in the biosynthesis of FeMo-co in A. vinelandii and K. pneumoniae, and we predict that the structure presented here represents a general protein fold for the carriage of FeMo-co or FeMo-co precursors. Yet, NafY does not appear to bind DNA or RNA. But as the authors observe, the nif homologs (nifX and nifY) apparently do: Although there is unequivocal evidence that NafY interacts with dinitrogenase and with FeMo-co, there is no experimental evidence to suggest that NafY interacts with DNA or RNA molecules. However, NafY belongs to a family of proteins whose members are involved at different steps in the biosynthesis of FeMo-co, including NifX and NifY proteins, and it has been reported that both NifX and NifY from K. pneumoniae can influence the stability of the nifHDK mRNA, which encodes the structural components of dinitrogenase and dinitrogenase reductase (29, 30). The deletion of nifY or nifX genes in K. pneumoniae increases the stability of the nifHDK mRNA, which accumulates to higher levels than in the wild-type strain under conditions of nitrogenase expression. Conversely, when nifY or nifX are overexpressed, the stability of the nifHDK mRNA is reduced, and it accumulates at lower levels than in the wild type. Given their amino acid sequence similarity to NafY, it is likely that NifX and the C-terminal domain of NifY (Fig. 1) have similar structures to that of the NafY core domain and, in turn, to ribonuclease H. This extrapolation would give structural credence to the reports by Roberts and collaborators (29, 30) assigning a role for NifX and NifY as negative regulators of nitrogenase expression. What a versatile fold. Here is another article reporting a conserved hypothetical protein MTH1175 with unknown function from Methanobacterium thermoautotrophicum: J Struct Funct Genomics. 2000;1(1):15-25

NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum.

Cort JR, Yee A, Edwards AM, Arrowsmith CH, Kennedy MA.

Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA.

The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy. MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima. Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX. MTH1175 adopts an alpha/beta topology with a single mixed beta-sheet, and contains two flexible loops and an unstructured C-terminal tail. The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity.

[RufusAtticus]

Okay, I think we get the point.

Now your mission if you chose to accept it is to turn all of these leads into a suitable FAQ. Barring that a reference to a suitable review article would be appreciated.

This message will self destruct in 5. . . .

[Principia]

RA, I believe that there are people working on a compiling the available research.

Let's explore another claim from this CreatoID: The duplication explanations also come with several theoretical problems. First, we must begin with one gene product that was similar to the gene products of nifE,N,D, and K (let's call it DENK'). It would then duplicate to give nifDE and nifJK [sic] which in turn duplicate to give us nif D, E, J [sic], and K. But throughout all this, there must have been some selection pressure that maintained the tertiary structure and shared amino acid sequences that are necessary for nitrogenase function today. What was it? From what we know of nifE,N,D, and K, there is simply no good reason to think DENK' was fixing nitrogen as the four "duplicates" exist in an IC relationship that appears to be required for this function. In other words, the IC nature of the relationship between these four genes argues against an original reductase function that was simply improved step-by-step. Fani et al. (article cited earlier in this thread) do in fact posit these 4 genes as having evolved from 2 in tandem duplication events. Coupled with the observations by Blankenship and others that homologs to these proteins have other functions besides nitrogenase, I think a good evolutionary case is crystallizing. Suffice it to say, the "design" argument here is a bit bizarre. Here, the nifNE heterotetramer works with other nif gene products (notable nifH, nifQ, nifB, and possibly nifS/U) to synthesize a cofactor, that must then be transported from nifNE to its homologous heterotetramer made by nifDK. The whole process is like having, say, a Mac that needs an operating system, which must first be made on a PC, using PC based tools, before it is installed into the Mac. :confused: Someone is bound to explain this one to me. ;)

In any case, let's indulge in a little evidentiary reasoning by analogy: J Mol Biol. 2003 Aug 22;331(4):829-60

Catalysing new reactions during evolution: economy of residues and mechanism.

The diversity of function in some enzyme superfamilies shows that during evolution, enzymes have evolved to catalyse different reactions on the same structure scaffold. In this analysis, we examine in detail how enzymes can modify their chemistry, through a comparison of the catalytic residues and mechanisms in 27 pairs of homologous enzymes of totally different functions. We find that evolution is very economical. Enzymes retain structurally conserved residues to aid catalysis, including residues that bind catalytic metal ions and modulate cofactor chemistry. We examine the conservation of residue type and residue function in these structurally conserved residue pairs. Additionally, enzymes often retain common mechanistic steps catalyzed by structurally conserved residues. We have examined these steps in the context of their overall reactions. This is a very pretty article.

[lpetrich]

I hope that some of you people can put together some account of the overall trends of nitrogenase evolution, because I've gotten lost in all the bewildering detail of those abstracts.

[Principia]

lp,

From what I know, there are something like 10 different structural genes (off the top of my head, nifHDKENBQVSU) involved in making Mo-dependent nitrogenase or its FeMo-cofactor. Then there are some more involved in its regulation or enhancing its activity (off of the top of my head, nifALXY, nafY), or whose function is not well determined or not essential (e.g. nifCFJOW).

Only recently, the evolutionary relationship of the structural genes has been considered to some extent. It has long been known that nifDK (two genes required to make the heterotetrameric aponitrogenase, component I) share significant similarity with nifNE (two genes require to make a scaffold for the biosynthesis of the FeMo-cofactor to be inserted later into nifDK). Fani et al. make a good case that the 4 genes, nifNE and nifDK, may in fact share a common ancestor that existed before the divergence of Archaea and Bacteria and underwent 2 duplication events. Blankenship and Bauer (which are cited on evowiki) suggest that the nif reductases in fact had other roles to play in the Archaean earth by linking showing plausible relationships between nif (specifically nifHDK) and bch (BchBLN?) genes. These bch genes are involved in the reduction of protochlorophyllide to chlorophyllide in a key step of the biosynthesis of bacteriochlorophyll.

But as has been demonstrated, nifHDK and nifNE are neither sufficient nor necessary for the nitrogenase activity. For instance, nifV, a homocitrate synthase, is required to alter the substrate specificity of the nitrogenase cofactor in Azotobacter. In its absence, citrate takes its place and nitrogenase activity is diminished or lost. It remains to be seen if nifV was invented at the same time as nifHDK. Clearly, the biosynthesis genes are required, and their evolutionary past remains to be elucidated. Perhaps these products evolved from molybdenum/iron/sulfur chaperones, whose dimerization tended to form novel clusters.

A slew of alternative nitrogenases are also being discovered. Two well known ones use V and Fe instead of Mo, and these systems are switched on in Mo limiting situations. This indicates to me that the nitrogenase subunits are not tailor made for the FeMo-co cluster. In fact, other nif genes such as nifB are required to make these alternative systems functional, though vnf and anf structural genes tend to group together phylogenetically apart from nif genes. Another most interesting alternative nitrogenase is the Meyer nitrogenase which is found in a carboxydotrophic bacteria (S. thermoautotrophicus). These guys apparently solved the oxygen sensitivity dilemma of the "classical" nitrogenase by using superoxide radicals as electron donors in reducing nitrogen. What this system illustrates is that the 2 distinct functional components of nitrogenase systems (a reductase coupled to some energy source, and an enzyme for channeling binding nitrogen and channeling the electrons of the reductase to N2) can in fact have other cellular functions.

Well, that's all I can summarize off the top of my head, but the gist is there.

[Nic Tamzek]

Well, in a fit of inspiration I EvoWikified Principia's summary (making minor edits to remove "conversational" wording, changed from gene to protein nomenclature to avoid italicizing all the genes). I also added a link to Berg et al.'s Biochemistry textbook section on Nitrogen Fixation (free at NCBI), and included a quote of an image/caption giving the very basic process.

The page looks pretty good now, check it out:

http://www.evowiki.org/wiki.phtml?title=Nitrogen_fixation

Of course Principia or others may see fit to modify the page or the summary further...

[Guts]

Mike H wrote:


It is so remarkable to be able to look back all that way and get so very close to the origin of life.

Here are all 10 proteins that BD examined, in order of inferred age:

Ferredoxin (Fe-S protein; does redox reactions)
Proteolipid h1 (lives in cell membranes; part of ATPase complex)
FtsZ (involved in cell division)
FEN-1 (flap exonuclease)
RNA polymerase beta'
Reverse transcriptase (RNA -> DNA)
DNA topoisomerase I (alters DNA topology)
Ribonucleotide reductase (Fe) (RNR's make DNA nucleotides from RNA ones)


Going to be correcting the misinfo here by chronological order. Actually this is incorrect. Ribonucleotide reductase sequencs show that it comes earlier than reverse transcriptase and topoisomerase I.

Here are some more interesting comments on nitrogenase:


The iron-molybdenum (Fe-Mo) cofactor of nitrogenase forms an integral part of the active site of dinitrogenase. The Fe-Mo cofactor consists of Mo, Fe, S and homocitrate in a 1:7:9:1 ratio. At least six proteins are necessary for its biosynthesis; these are the gene products of nifQ, V, B, H, N and E. The synthesis process also requires MgATP. The NifE and NifN proteins form a scaffold similar to nitrogenase component I. The Fe-Mo cofactor is assembled there, then is transferred to nitrogenase component I.

http://biocyc.org:1555/META/new-image?type=ENZYME&object=CPLX-544

[Guts]

The evo-wiki page contains some errors which I will continue to comment on as I read the relevant papers. For starters it states:


For example: Here a creationist is quoted as asserting:
"Is there evidence of an evolutionary continuum with nitrogenase indicating sloppy simplicity?

No. You either have nitrogenase as we know it or have nothing. There are no simpler versions of nitrogenase and there is no reason to think it would function in a biologically significant manner in a much sloppier state


The site claims that this was stated by a "creationist" and falls under the heading of antievolution comments. In fact, this was stated by Julie Thomas in talk.origins, who is neither a creationist nor an anti-evolutionist (ID does not necessarily mean antievolution). Here is a link to the entire article:

http://makeashorterlink.com/?H34923607

At any rate, none of the comments have been responded to, with respect to a "much sloppier state". All the parts that have been discussed exist as system-dependant parts.

[Principia]

The site claims that this was stated by a "creationist" and falls under the heading of antievolution comments. In fact, this was stated by Julie Thomas in talk.origins, who is neither a creationist nor an anti-evolutionist (ID does not necessarily mean antievolution). This is merely a matter of your spin, which no doubt you have difficulty supporting. For instance, how do you know that Julie Thomas is not a creationist or an antievolutionist? Where is the evidence? In fact, I wholeheartedly invite people to read the thread that Guts linked, and let themselves decide if Julie Thomas (whom we know better as Mike Gene) was making antievolutionary commentary.

At any rate, none of the comments have been responded to, with respect to a "much sloppier state". Tell me how one is supposed to respond to "much sloppier state." Is the Azotobacter system "sloppier" than the cyanobacterial systems? Or perhaps the Streptomyces system is not "sloppy" enough? Vague terminology befits a creationist rant, and merely serves to pose rhetorical questions which are not the focus of any scientific research. In any case, I don't see where the evowiki article claimed to address specifically the quote above. Your knee-jerk reaction to potential attacks on your idols are premature. All the parts that have been discussed exist as system-dependant parts. I have no idea what "system-dependent parts" mean. Nor do I know what "all the parts that have been discussed" refer to. You apparently are a disciple of Julie Thomas's writing. Why don't you start a thread defining these non-standard terminology like "sloppy state" or "system-dependent parts"? I'm sure that people will be glad to be so enlightened.

[Guts]

Principia:


This is merely a matter of your spin, which no doubt you have difficulty supporting. For instance, how do you know that Julie Thomas is not a creationist or an antievolutionist? Where is the evidence? In fact, I wholeheartedly invite people to read the thread that Guts linked, and let themselves decide if Julie Thomas (whom we know better as Mike Gene) was making antievolutionary commentary.


All you had to do was read her writings, where she makes statements like this.


1. The stranglehold of stereotype, where instead of
an ability to understand novel opinions, there was only
a need to view my opinions as just another form of
creationism.


I don't think you even read the article that you quoted from.



Principia:


Tell me how one is supposed to respond to "much sloppier state."


I thought you said you were laying her comments to rest. Now you're telling me you don't even know how.

Principia:


Is the Azotobacter system "sloppier" than the cyanobacterial
Or perhaps the Streptomyces system is not "sloppy" enough? Vague terminology befits a creationist rant, and merely serves to pose rhetorical questions which are not the focus of any scientific research.


There was no vague terminology, the IC system was laid out in the article you quote mined from:


From a biosynthetic pathway perspective, it appears to be at least a 7 part system entailing nif H,D,K,E,N,B, and Q. From each one of these perspectives, it remains true that if one player is removed, nitrogenase ceases to function.


One would simply need to show a significant reduction of parts, but where the nitrogenase function is retained. Instead, this thing seems pretty complex. So far it seems the biosynthetic pathway still requires seven parts.


Principia:


In any case, I don't see where the evowiki article claimed to address specifically the quote above. Your knee-jerk reaction to potential attacks on your idols are premature.


Right, it attributes the quote to a "creationist", calls it an "assertion", never bothers actually attributing the quote, then quotes an irrelevant rant to make it look like there is a response to the quote. If you were truly intellectually honest, you would note that you were not responding to that quote, or simply remove it, I don't see any reason for it being there other than give the image of a response.

Principia:


I have no idea what "system-dependent parts" mean. Nor do I know what "all the parts that have been discussed" refer to.


It shows.

Principa:


You apparently are a disciple of Julie Thomas's writing. Why don't you start a thread defining these non-standard terminology like "sloppy state" or "system-dependent parts"? I'm sure that people will be glad to be so enlightened.


But I thought you were responding to her comments.

[Principia]

All you had to do was read her writings, where she makes statements like this: 1. The stranglehold of stereotype, whe