Though complex microbial relationships regulate these processes, we can assume that fixation, mineralization, nitrification, and denitrification are carried out almost exclusively by bacteria, whereas algae play a main active role only in nitrogen fixation and assimilation. Greatly simplifying the overall nitrogen cycle, and from an algal point of view, atmospheric molecular nitrogen is converted by prokaryotic algae (cyanobacteria) to compounds such as ammonia (fixation), which are in part directly converted into amino acids, proteins, and other nitrogen-containing cell constituents of the fixators, and in part excreted into the open environment. Eukaryotic algae, unable to perform fixation, incorporate fixed nitrogen, either ammonium or nitrate, into organic N compounds by assimilation. When organic matter is degraded, organic compounds are broken down into inorganic compounds such as NH3 or NH+4 and CO2 through the mineralization process. The resultant ammonium can be nitrified by aerobic chemoautotrophic bacteria that use it as electron donor in the respiration process. The cycle is completed by denitrification carried out usually by facultative anaerobic bacteria that reduce nitrate used as electron acceptor in respiration to nitrogen gas. We must stress that a realistic depiction of the N cycle would have an almost infinite number of intermediary steps along the circumference of a circle, connected by a spider web of internal, crisscrossing complex connections.
It is one of nature’s great ironies that though all life forms require nitrogen compounds the most abundant portion of it (98%) is buried in the rocks, therefore deep and unavailable, and the rest of nitrogen, the N2 gas (2%), can be utilized only by very few organisms. This gas cannot be used by most organisms because the triple bond between the two nitrogen atoms makes the molecule almost inert. In order for N2 to be used for growth this gas must be “fixed” in the forms directly accessible to most organisms, that is, ammonia and nitrate ions.
A relatively small amount of fixed nitrogen is produced by atmospheric fixation (5–8%) by means of the high temperature and pressure associated with lightning. The enormous energy of this phenomenon breaks nitrogen molecules and enables their atoms to combine with oxygen in the air forming nitrogen oxides. These molecules dissolve in rain, forming nitrates that are carried to the Earth. Another relatively small amount of fixed nitrogen is produced industrially (industrial fixation) by the Haber-Bosch process, in which atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH3) using an iron-based catalyst, very high pressures and a temperature of about 6008C. Ammonia can be used directly as fertilizer, but most of it is further processed to urea (NH2)2CO and ammonium nitrate NH4NO3.
The major conversion of N2 into ammonium, and then into proteins, is a biotic process achieved by microorganisms, which represents one of the most metabolically expensive processes in biology. Biological nitrogen fixation can be represented by the following equation, in which two moles of ammonia (but in solution ammonia exists only as ammonium ion, that is, NH3 + H2O ↔ NH+4 + OH1) are produced from one mole of nitrogen gas, at the expense of 16 moles of ATP and a supply of electrons and protons (hydrogen ions):
N2 + 8H+ + 8e- + 16ATP → 2NH3 + H2 + 16ADP + 16Pi (4.2)
All known nitrogen-fixing organisms (diazotrophs) are prokaryotes, and the ability to fix nitrogen is widely, though paraphyletically, distributed across both the bacterial and archaeal domains. In cyanobacteria nitrogen-fixation is an inducible process, triggered by low environmental levels of fixed nitrogen.
The capacity of nitrogen fixation in diazotrophs relies solely upon an ATP-hydrolyzing, redox active enzyme complex termed nitrogenase. In many of these organisms nitrogenase comprises about 10% of total cellular proteins and consists of two highly conserved components, an iron protein (Fe-protein) and a molybdenum-iron (MoFe-protein). The Fe-protein is a g2 homodimer composed of a single Fe4S4 cluster bound between identical 32–40 kDa subunits. The Fe4S4 cluster is redox-active and is similar to those found in small molecular weight electron carrier proteins such as ferredoxins. It is the only known active agent capable of obtaining more than two oxidative states and transfers electrons to the MoFe-protein. The MoFe-protein is a a2b2 heterotetramer; the ensemble is approximately 250 kDa. The a subunit contains the active site for dinitrogen reduction, typically a MoFe7S9 metal cluster (termed FeMo-cofactor), although some organisms contain nitrogenases wherein Mo is replaced by either Fe or V. These so-called alternative nitrogenases are found only in a limited subset of diazotrophs and, in all cases studied so far, are present secondary to the MoFe-nitrogenase. The MoFe-nitrogenase has been found to be more specific for and more efficient in binding N2 and reducing it to ammonia than either of the alternative nitrogenases. The catalytic efficiency of these alternative nitrogenases is lower than that of the MoFe-nitrogenase. In addition to variations in metal cofactors, the nitrogenase complex is non-specific and reduces triple and double bond molecules other than N2. These include hydrogen azide, nitrous oxide, acetylene, and hydrogen cyanide. The non-specificity of this enzyme and the alternative nitrogenases containing other metal co-factors implicate the role of varying environmental pressures on the evolutionary history of nitrogenase that could have selected for different functions of the ancestral enzyme. The nitrogenase enzyme system is extremely O2 sensitive, because oxygen not only affects the protein structure but also inhibits the synthesis of nitrogenase in many diazothrops. The repression is both transient (lasting only a few hours) and permanent. The reactions occur while N2 is bound to the nitrogenase enzyme complex. The Fe-protein is first reduced by electrons donated by ferredoxin. Then the reduced Fe-protein binds ATP and reduces the MoFe-protein, which donates electrons to N2, producing diimide (HN=NH). In two further cycles of this process (each requiring electrons donated by ferredoxin) HN=NH is reduced to imide (H2N-NH2), and this in turn is reduced to 2NH3. Depending on the type of microorganism, the reduced ferredoxin that supplies electrons for this process is generated by fermentation or photosynthesis and respiration.
As already stated, nitrogenase is highly sensitive to molecular oxygen (in vitro it is irreversibly inhibited by exposure to O2). Therefore, during the course of planetary evolution, cyanobacteria have co-evolved with the changing oxidation state of the ocean and atmosphere to accommodate the machinery of oxygenic photosynthesis and oxygen-sensitive N2 fixation within the same cell or colony of cells. As nitrogen fixation occurs in a varied metabolic context in both anaerobic and aerobic environments, strategies have followed a very complex pattern of biochemical and physiological mechanisms for segregation that can be simplified in some spatial and/or temporal separation of the two pathways.
Nitrogenase is an ancient enzyme that almost certainly arose in the Archean ocean before the oxidation of the atmosphere by oxygenic photoautotrophs. An attractive hypothesis of the development of biological nitrogen fixation is that it arouse in response to changes in atmospheric composition that resulted in the reduction in the production of abiotically fixed nitrogen. On the early Earth, concentrations of CO2 in the atmosphere were high, because of the oxidation of CO produced by impacts of extraterrestrial bodies and only slow removal of CO2 by weathering (the continents were smaller at this time, meaning that a smaller area of minerals was exposed for weathering). With these CO2 conditions, the initial production rate of NO was estimated to be about 3 x 1011 g 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 weathering. As this happened, the production rate of NO by lightning dropped to below 3 x 109 g 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. Under the prevailing anaerobic conditions of that period in Earth’s history anaerobic heterotrophs, such as Clostridium, developed. With the evolution of cyanobacteria and the subsequent generation of molecular oxygen, oxygen-protective mechanisms would be essential. A semitemporal separation of nitrogen fixation and oxygenic photosynthesis combined with spatial heterogeneity was the first oxygenprotective mechanism developed by marine cyanobacteria such as Trichodesmium sp. and Katagnymene sp. A full temporal separation, in which nitrogen is only fixed at night, then developed in unicellular cyanobateria diazotrophs and in some non-heterocystous filamentous diazotrophs (e.g., Oscillatoria limosa and Plectonema boryanum). Finally, in yet other filamentous organisms, complete segregation of N2 fixation and photosynthesis was achieved with the cellular differentiation and evolution of heterocystous cyanobacteria (e.g., Nostoc and Anabaena).
The non-heterocystous filamentous cyanobacteria Trichodesmium sp. and Katagnymene sp., unlike all other non-heterocystous species fix nitrogen only during the day. Nitrogenase is compartimentalized in 15–20% of the cells in Trichodesmium sp., and 7% of the cells in Katagnymene sp. often arranged consecutively along the trichome, but active photosynthetic components (PSI, PSII, RuBisCo, and carboxysomes) are found in all cells, even those harboring nitrogenase. A combined spatial and temporal segregation of nitrogen fixation from photosynthesis, and a sequential progression of photosynthesis, respiration, and nitrogen fixation over a diel cycle are the strategies used by these cyanobacteria. These pathways are entrained in a circadian pattern that is ultimately controlled by the requirement for an anaerobic environment around nitrogenase. Light initiates photosynthesis, providing energy and reductants for carbohydrate synthesis and storage, stimulating electron cycling through PSI, and poising the plastoquinone (PQ) pool at reduced levels. High respiration rates early in the photoperiod supply carbon skeletons for amino acid synthesis (the primary sink for fixed nitrogen) but simultaneously reduce the PQ pool further. Linear electron flow to PSI is never abolished. The reduced PQ pool leads to a downregulation of PSII, which opens a window for N2 fixation during the photoperiod, when oxygen consumption exceeds oxygen production. As the carbohydrate pool is consumed, respiratory electron flow through the PQ pool diminishes, intracellular oxygen concentrations rise, the PQ pool becomes increasingly oxidized, and net oxygenic production exceeds consumption. Nitrogenase activity is lost until the following day.
A full temporal separation between oxygenic photosynthesis and nitrogen fixation occurs in P. boryanum and O. limosa. Transcription of nitrogenase and photosynthetic genes are temporally separated within the photoperiod, that is, nitrogenase is expressed primarily during the night. Nitrogenase is contained in all cells in equal amounts. The onset of nitrogen fixation is preceded by a depression in photosynthesis that establishes a sufficiently low level of dissolved oxygen in the environment. Plectonema sp. has a versatile physiology that allows it to reversibly modulate uncoupling of the activity of the two photosystems in response to intracellular nitrogen status. Oscillatoria sp. initiates nitrogen fixation in the dark and performs it primarily in the absence of light.
In non-heterocystous cyanobacteria such as the filamentous Symploca sp. and Lyngbya maiuscola, and the unicellular Gloeothece sp. and Cyanothece sp., the temporal separation does not need a microaerobic environment. Phormidium sp. and Pseudoanabaena sp. are other examples of cyanobacteria fixing only under microanaerobic conditions. Under contemporary oxygen levels, all of these organisms are relegated to narrow environmental niches.
In heterocystous cyanobacteria, such as Anabaena sp. and Nostoc sp., a highly refined specialization spatially separates oxygenic photosynthesis from N2 fixation. Here, nitrogenase is confined to a microanaerobic cell, the heterocyst, characterized by a thick membrane that slows the diffusion of O2, high PSI activity, loss of division capacity, absence of PSII (that splits the water forming O2). This cell differentiates completely and irreversibly 12–20 h after combined nitrogen sources are removed from the medium. The development of these cells, formed at intervals between vegetative cells, is a primitive form of cell differentiation. In this process, all PSII activities are gradually lost, and the proteins involved in oxygenic photosynthesis are degraded, whereas PSI activity is maintained. Simultaneously, the production of active nitrogenase is triggered. Nitrogen fixation is localized specifically in heterocysts, and light is used for cyclic electron flow around PSI to maintain a supply of ATP for the process. The primary organic nitrogen product (glutamate) is exported to adjacent vegetative and photooxygenic cells, while carbon skeletons, formed by the respiratory and photosynthetic processes in the latter cells, are translocated to the heterocysts. In some heterocystous cyanobacteria such as Anabaena variabilis, under anaerobic conditions, a different Mo-dependent nitrogenase can be synthesized inside vegetative cells. This nitrogenase expresses shortly after nitrogen depletion, but prior to heterocysts formation, and can support the fixed N needs of the filaments independent of the nitrogenase in the heterocysts.
The biotic nitrogen fixation is estimated to produce about 1.7 x 108 tons of ammonia per year, whereas atmospheric and industrial nitrogen fixation produce about 1.7 x 107 tons of ammonia per year.
For all eukaryotic algae, the only forms of inorganic nitrogen that are directly assimilable are nitrate (NO-3), nitrite (NO-2), and ammonium (NH+4). The more highly oxidized form, nitrate, is the most thermodynamically stable form in oxidized aquatic environments, and hence is the predominant form of fixed nitrogen in aquatic ecosystems, though not necessarily the most readily available form. Following translocation across the plasmalemma (which is an energy-dependent process), the assimilation of NO-3 requires chemical reduction to NH+4. This process is mediated by two enzymes, namely, nitrate reductase and nitrite reductase. Nitrate reductase is located in the cytosol and uses NADPH to catalyze the two-electron transfer:
NO-3 + 2e- + 2H+ → NO-2 + H2O (4.3)
In cyanobacteria nitrate reductase is coupled to the oxidation of ferredoxin rather than a pyridine nucleotide as in eukaryotic algae. The nitrite formed by nitrate reductase is reduced in a six-electron transfer reaction:
NO-2 + 6e- + 8H+ → NH+4 + 2H2O (4.4)
Nitrite reductase utilizes ferredoxin in both cyanobacteria and eukaryotic algae; in the latter, the enzyme is localized in the chloroplasts. In both cyanobacteria and eukaryotic algae photosynthetic electron flow is an important source of reduced ferredoxin for nitrite reduction. The overall stoichiometry for the reduction of nitrate to ammonium can be written as:
NO-3 + 8e- + 10H+ → NH+4 + 3H2O (4:5)
The incorporation of ammonium into ammino acids is primarily brought about by the sequential action of glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT). Ammonium assimilation by GS requires glutamate as substrate and ATP, and catalyzes the irreversible reaction:
Glutamate + NH+4 + ATP → Glutamine + ADP + Pi (4.6)
The amino nitrogen of glutamine is subsequently transferred to 2-oxoglutarate, and reduced, forming two moles of glutamate:
2-Oxoglutarate + Glutamine + NADPH → 2[Glutamate] + NADP+
Both GS and GOGAT are found in chloroplasts, although isoenzymes (multiple forms of an enzyme with the same substrate specificity, but genetic differences in their primary structures) of both enzymes may also be localized in the cytosol. Whatever the location of the enzymes, however, glutamate must be exported from the chloroplast to the cytosol where transamination reaction (the reversible transfer of an amino group of a specific amino acid to a specific keto acid, forming a new keto acid and a new amino acid) can proceed, thereby facilitating the syntheses of other amino acids.
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