Nitrogen fixation

From EvoWiki

Contents 1 Introduction 1.1 Nitrogen Fixing Organisms 1.2 Nitrogenases 2 Example antievolutionist claims 3 Summary of what is known about the evolution of this system 4 External links to helpful resources 5 Literature

Introduction

Nitrogen fixation is a biological process whereby atmospheric nitrogen (N₂), which is chemically rather inert, is converted into biologically available forms ("fixed"), initially as ammonia. Nitrogen in these forms can then be used in the manufacture of nitrogen-requiring molecules such as the amino acids that make up proteins.

Nitrogen Fixing Organisms

Living organisms capable of fixing nitrogen are known as diazotrophs. All diazotrophs identified so far are prokaryotes distributed widely in the archaeal and bacterial domains. Well-characterized nitrogen-fixing systems have been found in some free-living species of cyanobacteria (e.g. Trichodesmium), methanogens (e.g. Methanococcus), obligate aerobes (e.g. Azotobacter), facultative anaerobes (e.g. Klebsiella), and obligate anaerobes (e.g. Clostridium). Diazotrophs may also reside in symbiotic relationships with plants or (rarely) lichens. Nitrogen fixing bacteria belonging to genera such as Rhizobium, Bradyrhizobium, etc. are commonly found in root nodules on plants (mostly legumes), and some crops are grown only because the high levels of nitrogen compounds produced in the roots. Diazotrophs are important in the maintenance of the biogeochemical nitrogen cycle. Recently, the legume-rhizobium symbiosis has also proved useful as an experimental model to study the evolution of cooperation within and between species.

Nitrogenases

The enzymes that carry out the reduction of nitrogen are known in the literature as nitrogenases. All known members of nitrogenases are members of metalloenzymes in which the catalytic sites are bioinorganic compounds. Specifically, iron-sulfur (Fe:S) and iron-molybdenum (Fe:Mo) clusters play vital roles.

http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/stryer/ch24f2.gif

Figure 24.2. [from Berg et al., Biochemistry, quoted as fair use]: "Nitrogen Fixation. Electrons flow from ferredoxin to the reductase (iron protein, or Fe protein) to nitrogenase (molybdenum-iron protein, or MoFe protein) to reduce nitrogen to ammonia. ATP hydrolysis within the reductase drives conformational changes necessary for the efficient transfer of electrons."

Enzymes involved in Mo-dependent nitrogen reduction:

• nifA, nifL
  Function: regulation of nif; nifA is a transcriptional activator, and nifL a negative regulator that modulates nifA by sensing redox states of its FAD cofactor (Schmitz 2002)

• nifB
  Function: synthesizes nifB-co, a precursor to FeMo-cofactor (Thiel 1995)

• nifD, nifK (dinitrogenase, MoFe protein, component I)
  Function: site of actual dinitrogen reduction
  Structure: nifD and nifK form an alpha₂beta₂ heterotetramer. At the interface of the alpha and beta subunits are two P clusters which faciliate e^- transfer from the dinitrogenase reductase to the FeMo-cofactor core

• nifE, nifN
  Function: scaffold for assembling FeMo-cofactor

• nifH (dinitrogenase reductase, iron protein, component II)
  Function: hydrolyzes ATP and reduces dinitrogenase
  Structure: homodimer containing two ATP binding sites and a single [Fe₄S₄] cluster

• nifQ
  Function: incorporates Mo during biosynthesis of FeMo-cofactor (Imperial 1984)

• nifS, nifU
  Function: S and Fe donors, respectively, for building Fe-S clusters (Thiel 1995, Kennedy 1992)

• nifV
  Function: homocitrate syntase, incorporates homocitrate into FeMo-cofactor (Zheng 1997, Madden 1990)

• nifX, nifY, nafY
  Function: FeMo-co binding proteins (Rubio 2002, Shah 1999); nifX and nifY may be negative regulators via destabiliizing nifHDK mRNAs(Gosink 1990)

Alternative nitrogenases replace Mo in the FeMo-cofactor with either iron (synthesized by Anf genes) or vanadium (synethesized by Vnf genes). Anf and Vnf systems are invariably found with Nif genes, and then only expressed under Mo limiting situations, producing less nitrogen-reducing activity than the Mo-dependent nitrogenase. Recently a nif-independent nitrogenase system was discovered in Streptomyces thermoautotrophicus (Ribbe 1997).

Example antievolutionist claims

• 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."

Summary of what is known about the evolution of this system

About 10 different structural proteins (e.g. NifHDKENBQVSU) are involved in making Mo-dependent nitrogenase or its FeMo-cofactor. Several more are involved in regulation or enhancing activity (NifALXY, NafY), or whose function is not well determined or not essential (e.g. NifCFJOW).

Only recently, the evolutionary relationship of the structural proteins has been considered to some extent. It has long been known that NifDK (two proteins required to make the heterotetrameric aponitrogenase, component I) share significant similarity with NifNE (two proteins require to make a scaffold for the biosynthesis of the FeMo-cofactor to be inserted later into NifDK). Fani et al. (2000) make a good case that the 4 proteins, NifNE and NifDK, may in fact share a common ancestor that existed before the divergence of Archaea and Bacteria and underwent two duplication events. Blankenship and Bauer (coauthors on references below) suggest that the nif reductases in fact had other roles to play in the Archaean earth by showing plausible relationships between Nif (specifically NifHDK) and Nch (BchBLN?) proteins. These Bch proteins 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 that the nitrogenase subunits are not tailor-made for the FeMo-co cluster. In fact, other Nif proteins such as NifB are required to make these alternative systems functional, though Vnf and Anf structural proteins tend to group together phylogenetically apart from Nif proteins. 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.

• Summary EvoWikified by oplo from Principia's summary post on IIDB. Further modifications/additions are of course welcome.

External links to helpful resources

• IIEC thread Nitrogen fixation and shadowing AE thread "The origins of nitrogen fixation systems"

• Prosthetic groups and Metal Ions in Protein Active Sites Database -- Nitrogenases are listed under Iron-Sulphur proteins

• Nitrogen fixation discussed in free online textbooks at the NCBI

• Lecture notes on nitrogenases from Gary Roberts

Literature

1. Dos Santos PC, Dean DR, Hu Y, Ribbe MW. Formation and insertion of the nitrogenase iron-molybdenum cofactor
   Chem Rev 2004 Feb;104(2):1159-73 PubMed

2. Schmitz RA, Klopprogge K, Grabbe R. Regulation of nitrogen fixation in Klebsiella pneumoniae and Azotobacter vinelandii: NifL, transducing two environmental signals to the nif transcriptional activator NifA
   J Mol Microbiol Biotechnol 2002 May;4(3):235-42 PubMed

3. Imperial J, Ugalde RA, Shah VK, Brill WJ Role of the nifQ gene product in the incorporation of molybdenum into nitrogenase in Klebsiella pneumoniae
   J Bacteriol 1984 Apr;158(1):187-94 PubMed

4. Zehr JP, Jenkins BD, Short SM, Steward GF. Nitrogenase gene diversity and microbial community structure: a cross-system comparison
   Environ Microbiol 2003 Jul;5(7):539-54 PubMed

5. Rubio LM, Rangaraj P, Homer MJ, Roberts GP, Ludden PW. Cloning and mutational analysis of the gamma gene from Azotobacter vinelandii defines a new family of proteins capable of metallocluster binding and protein stabilization.
   J Biol Chem 2002 Apr 19;277(16):14299-305. PubMed

6. Shah VK, Rangaraj P, Chatterjee R, Allen RM, Roll JT, Roberts GP, Ludden PW. Requirement of NifX and other nif proteins for in vitro biosynthesis of the iron-molybdenum cofactor of nitrogenase
   J Bacteriol 1999 May;181(9):2797-801 PubMed

7. Gosink MM, Franklin NM, Roberts GP. The product of the Klebsiella pneumoniae nifX gene is a negative regulator of the nitrogen fixation (nif) regulon.
   J Bacteriol 1990 Mar;172(3):1441-7. PubMed

8. Berman-Frank I, Lundgren P, Falkowski P. Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria
   Res Microbiol 2003 Apr;154(3):157-64 pdfPubMed

9. Lyons EM, Thiel T. Characterization of nifB, nifS, and nifU genes in the cyanobacterium Anabaena variabilis: NifB is required for the vanadium-dependent nitrogenase.
   J Bacteriol 1995 Mar;177(6):1570-5. PubMed

10. Kennedy C, Dean D. The nifU, nifS and nifV gene products are required for activity of all three nitrogenases of Azotobacter vinelandii
    Mol Gen Genet 1992 Feb;231(3):494-8 PubMed

11. Zheng L, White RH, Dean DR. Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase.
    J Bacteriol 1997 Sep; 179(18):5963-6 PubMed

12. Madden MS, Kindon ND, Ludden PW, Shah VK. Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor.
    PNAS 1990 Sep;87(17):6517-21 PubMed

13. Raymond J, Siefert JL, Staples CR, Blankenship RE. The Natural History of Nitrogen Fixation
    Mol Biol Evol 2003 Dec 23 [Epub ahead of print]. pdf PubMed
    
    Abstract: In recent years our understanding of biological nitrogen fixation has been bolstered by a diverse array of scientific techniques. Still, the origin and extant distribution of nitrogen fixation has been perplexing from a phylogenetic perspective, largely 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.

14. Fani R, Gallo R, Lio P. Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes
    J Mol Evol 2000 Jul;51(1):1-11. PubMed
    
    Abstract: 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.

15. Ribbe M, Gadkari D, Meyer O. 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
    J Biol Chem 1997 Oct 17;272(42):26627-33. PubMed
    
    Abstract: N₂ fixation by Streptomyces thermoautotrophicus follows the equation N₂ + 4-12MgATP + 8H⁺ + 8e^- --> 2NH₃ + H₂ + 4-12MgADP + 4-12P_i 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.

16. Fujita Y, Bauer CE, Reconstitution of light-independent protochlorophyllide reductase from purified bchl and BchN-BchB subunits. In vitro confirmation of nitrogenase-like features of a bacteriochlorophyll biosynthesis enzyme
    J Biol Chem 2000 Aug 4;275(31):23583-8. PubMed
    
    Abstract: Protochlorophyllide reductase catalyzes the reductive formation of chlorophyllide from protochlorophyllide during biosynthesis of chlorophylls and bacteriochlorophylls. The light-independent (dark) form of protochlorophyllide reductase plays a key role in the ability of gymnosperms, algae, and photosynthetic bacteria to green (form chlorophyll) in the dark. Genetic and sequence analyses have indicated that dark protochlorophyllide reductase consists of three protein subunits that exhibit significant sequence similarity to the three subunits of nitrogenase, which catalyzes the reductive formation of ammonia from dinitrogen. However, unlike the well characterized features of nitrogenase, there has been no previous biochemical characterization of dark protochlorophyllide reductase. In this study, we report the first reproducible demonstration of dark protochlorophyllide reductase activity from purified protein subunits that were isolated from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. Two of the three subunits (Bchl and BchN) were expressed in R. capsulatus as S tag fusion proteins that facilitated affinity purification. The third subunit (BchB) was co-purified with the BchN protein indicating that BchN and BchB proteins form a tight complex. Dark protochlorophyllide reductase activity was shown to be dependent on the presence of all three subunits, ATP, and the reductant dithionite. The similarity of dark protochlorophyllide reductase to nitrogenase is discussed.

17. Cort JR, Yee A, Edwards AM, Arrowsmith CH, Kennedy MA. NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum.
    J Struct Funct Genomics 2000;1(1):15-25. PubMed
    
    Abstract: 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.

18. Denison RF. Legume sanctions and the evolution of symbiotic cooperation by rhizobia.
    American Naturalist 2000; 156:567-76. PDF

19. Kiers ET, Rousseau RA, West SA, Denison RF. Host sanctions and the legume-rhizobium mutualism.
    Nature 2003; 425:78-81