Nitrogen fixation

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 of 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.
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 cofactorSchmitz 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
*nifB Function: synthesizes nifB-co, a precursor to FeMo-cofactorLyons 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
*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-cofactorImperial 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
*nifS, nifU Function: S and Fe donors, respectively, for building Fe-S clustersKennedy 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
*nifV Function: homocitrate syntase, incorporates homocitrate into FeMo-cofactorZheng L, White RH, Dean DR. Purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase. J. Bacteriol. 1997 Sep; 179(18):5963-6.PubMedMadden 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
*nifX, nifY, nafY Function: FeMo-co binding proteins;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.PubMedShah 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 nifX and nifY may be negative regulators via destabiliizing nifHDK mRNAsGosink 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
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 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
== 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)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 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.
==External links==
*Archive of 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
* Archived lecture notes on nitrogenases from Gary Roberts
==Sources==
*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
*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
*Berman-Frank I, Lundgren P, Falkowski P. Nitrogen fixation and photosynthetic oxygen evolution in cyanobacteria. Res. Microbiol. 2003 Apr;154(3):157-64.pdfPubMed
*Raymond J, Siefert JL, Staples CR, Blankenship RE. The Natural History of Nitrogen Fixation. Mol. Biol. Evol. 2003 Dec 23.pdf PubMed
*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
*Kiers ET, Rousseau RA, West SA, Denison RF. Host sanctions and the legume-rhizobium mutualism. Nature 2003; 425:78-81.
==References==
Category:Biology
Category:Evolution