Nitrogen fixation

Live, reproduce, die Biology

Life as we know it

Genetics Evolution Fundamental unit of life: The cell Zoology Botany

Divide and multiply

Species Extinction Chlorella Homo ergaster Microbial growth

Great Apes

Bruce Lipton Darren Naish Maurice Hilleman Alexander Fleming Frank Lewis Marsh v - t - e

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

"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."

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 cofactor

• nifB
  Function: synthesizes nifB-co, a precursor to FeMo-cofactor

• 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

• nifS, nifU
  Function: S and Fe donors, respectively, for building Fe-S clusters

• nifV
  Function: homocitrate syntase, incorporates homocitrate into FeMo-cofactor

• nifX, nifY, nafY
  Function: FeMo-co binding proteins; nifX and nifY may be negative regulators via destabiliizing nifHDK mRNAs

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.

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.

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.