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 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.