The Nitrogen Cycle: Nitrogen Fixation

The conditions for the reaction are extreme: 500^oC and 200 atm. of pressure are required. Surprisingly, the reaction is spontaneous at high temperature and pressure (delta G = -53 kJ). The harsh reaction conditions provide the activation energy necessary for the reaction to occur.

2. Biological Nitrogen Fixation
Early in studies of nitrogen fixation, it was expected that biological extracts could produce ammonia if provided with nitrogen gas and a strong biological reductant:

However, without further supplements, the reaction does not occur. It was discovered that ATP hydrolysis was necessary to drive the reaction. In addition, hydrogen gas is produced. The biological mechanism of nitrogen fixation requires eight electrons rather than six electrons, the extra two being used to produce the hydrogen gas:

Two points about the energetics of biological nitrogen fixation:

(i) The mechanism is somewhat wasteful - reducing power is lost in the formation of hydrogen gas. Although some microbes recycle the evolved hydrogen, many others do not. Four more electrons are required to recycle the hydrogen gas, for the final reaction:

Overall Reaction

• Step 1. N₂ + 2e^- + 4ATP---->HN = NH (diimide)
• Step 2. HN = NH + 2e^- + 4ATP----> H₂N - NH₂ (imide)
• Step 3. H₂N - NH₂ + 2e^-----> 2NH₄⁺

Limitations

• 12 ATP for reduction of dinitrogen to ammonia
• 4 ATP for evolution of competitive inhibitor, hydrogen
• 2 ATP to remove ammonia, a repressor of enzyme synthesis, oxygen will denature enzymes
• Carbon derived from photosynthesis/Calvin (C₃) cycle

(ii) Despite this, biological nitrogen fixation is still favorable - the overall free energy change is negative. However, it is not as favorable as the energetics in the process of chemical fixation.

3. Nitrogenase Enzymes
Nitrogenase is the diagnostic enzyme for nitrogen-fixing organisms; it consists of two proteins:

(i) Dinitrogenase - This component reduces nitrogen (N₂) to ammonia (NH₃). It contains an iron-molybdenum cofactor (abbreviated FeMoCo) that accepts electrons from dinitrogenase reductase.

(ii) Dinitrogenase reductase - This component transfers electrons to dinitrogenase. It contains an iron atom that is involved in the redox chemistry. A noteworthy limitation: dinitrogenase reductase is irreversibly inactivated by oxygen. As a result, nitrogen fixation cannot be done in the presence of oxygen.

(iii) Assay for nitrogenase activity - The alternate substrate acetylene (HC=CH) can be reduced by nitrogenase, forming ethylene (2HC=CH₂).These two gases can be conveniently resolved and quantitated by gas chromatography.

Characteristics of the Nitrogenase Complex

• Consists of two enzymes: Dinitrogen reductase and Dinitrogenase
• Enzymes are denatured by oxygen
• Dinitrogen reductase - contains the transitional metal iron...Dinitrogenase contains the transition metal atoms -- iron and molybdenum...
• Needs Mg ions to activate the ATP...
• Requires 12 ATP to reduce N₂ to 2NH₄⁺
• Hydrogen is a competitive inhibitor of Dinitrogenase...
• NH₄⁺ represses the synthesis of the enzymes...
• Reduces other triple bonded compunds in addition to N₂

4. Nitrogen Fixers (see Tables N8 and N9)
Despite the extreme oxygen-sensitivity of nitrogenase, some microbes that live in aerobic environments can fix nitrogen. Organisms that fix nitrogen include:

(i) Azotobacter spp. - soil bacteria; occur in oxic environments (fix ~ 0.26 lbs N₂ per agricultural acre per year);

(ii) Klebsiella spp. - soil bacteria, can live in the presence or absence of oxygen;

(iii) Cyanobacteria (e.g. Anabeana spp., Nostoc) - water bacteria; occur in oxic environments (fix ~ 22 lbs N₂ per agricultural acre per year);

Table N8. Nitrogen fixing organisms: representative free-living bacteria.

Nitrogen Fixing Bacteria	Azotobacter vinelandii	Clostridium pasteurianum	Klebsiella pneumoniae	Rhodospirillum rubrum
Associated Organism	None	None	Various	None
Natural Habitat	Aerobic Soils	Anaerobic Soils	Aerobic Soils and Anaerobic Soils; Water;Also in association with plants, man	Surface of polluted ponds (a photosynthetic bacterium)

(iv) Rhizobium spp. - plant symbionts; responsible for significant fixation (fix approx. 220 lbs N₂ per agricultural acre per year).

Table N9. Nitrogen fixing organisms: representative symbiotic bacteria.

	Nonlegume	Nonlegume	Nonlegume	Legume	Legume	Legume
Nitrogen Fixing Organism	Frankia alni	Nostoc muscorum	Anabaena azollae	Bradyrhizobium japonicum	Rhizobium trifolii	Rhizobium meliloti
Associated Organism	Alder	Gunnera macrophylla (tropical herb)	Azolla (aquatic fern)	Soybean	Clover	Alfalfa
Natural Habitat	Root nodules in the Alder tree	In stems; a cyanobacterium	In leaf pores; a cyanobacterium	Root nodules of the soybean	Root nodules of clover	Root nodules of alfalfa

The image to the left shows nodules on red alder roots formed by Frankia alni, and the image on the right shows nodules on soybean roots formed by Bradyrhizobium japonicum. Check out the excellent NITROGEN FIXATION website at Reed College (Portland, OR) for an outstanding collection of images and descriptions of legumes and non-legumes that form symbiotic associations with different bacteria and fix atmospheric nitrogen. As well as common North American legumes (peas, beans, alfalfa, clovers, vetches, lupines, trefoils) there are many North American non-legumes (such as alder, buffalo berry, snowbrush, wax myrtle, and bitter-brush) that also fix nitrogen in symbiosis with some procaryotic microorganism.

5. Nitrogenase Protection
How do organisms living in oxic environments protect their nitrogenase from oxygen?
(i) Azotobacter has an unusually high rate of respiration. It consumes oxygen so fast that the intracellular concentration of oxygen is kept low enough that nitrogenase is not inhibited.

(ii) The facultative aerobe Klebsiella fixes nitrogen only when it is in an oxygen-free environment.

(iii) Filamentous cyanobacteria have specialized cells called heterocysts that fix nitrogen. Diffusion of oxygen into these cells is limited.

(iv) Rhizobia - see discussion of root nodules below.

6. Genetics and Regulation
Most of the research on genetics and regulation of nitrogen fixation has been done with Rhizobium meliloti, the fast-growing bacterium that forms root nodules on alfalfa:

The products of the genes fixLJ encode a sensor and a transcription factor that induces the expression of the gene nifA under anaerobic conditions. The product of nifA is a transcriptional activator protein that in turn activates expression of many nif and fix genes. Among the genes induced by NifA are two operons - nifNOQP and nifHDK, the latter containing the genes encoding dinitrogenase reductase (nifH) and the two subunits of dinitrogenase (nifDK).

An important point is that this regulatory cascade that results in the expression of genes involved in nitrogen fixation will not occur in the presence of oxygen. This makes sense - since nitrogenase is not functional under oxic conditions, there is no point in expressing these genes when oxygen is present.

(i) How was this pathway mapped? Transposon insertion mutagenesis. Complementation of mutants to isolate nitrogen fixation genes using gene libraries.

(ii) Why is it called a cascade of gene expression?
Because one transcriptional activator (phospho-FixJ) activates the expression of yet another transcriptional activator (NifA) that then activates the expression of other genes. The net effect is a large amplification of gene expression.

(iii) How is an "anaerobic" environment achieved in nodules?
Nodules are structures on plants where nitrogen fixation is done by symbiotic bacteria. The plant provides the bacteria with fixed carbon produced by photosynthesis, while the bacteria supply fixed nitrogen to the plant. An anaerobic environment is maintained in the nodule via the collection of free oxygen by a heme protein, leghemoglobin (LHb):

The equilibrium dissociation constant (Kd) for the interaction between LHb and oxygen is 4 x 10^-8 M. This means that when the concentration of oxygen is 4 x 10^-8 M (which is extremely low) half of the protein is oxygen-bound and half of it is free. This means that even trace amounts of oxygen will be bound. In other words, LHb has a very high affinity for oxygen. The concentration of LHb in nodules is maintained at very high levels. This helps to ensure an anaerobic environment. In addition, oxygen does not diffuse into the nodules easily - a thick layer of cells acts as a barrier to oxygen diffusion. As a result of these phenomena, the final concentration of free oxygen in nodules is 10uM O₂, a value so small that the environment is essentially anaerobic. However, it should be remembered that there actually is a great deal oxygen present, but it is bound to LHb. This oxygen is still available as a terminal electron acceptor in energy-generating reactions.

(iv) How is the cascade turned on and off based on the concentration of oxygen?
A sensor-kinase system is employed. The oxygen sensor, FixL, is a heme protein that exists in two states - oxy-FixL when oxygen is bound and deoxy-FixL when no oxygen is bound. Deoxy-FixL (but not oxy-FixL) can phosphorylate itself using ATP as a substrate, producing phospho-FixL and ADP. The phosphate can subsequently be transferred from phospho-FixL to an inactive transcription factor, FixJ, producing phospho-FixJ. Phosphorylation activates FixJ, continuing the cascade. Thus, covalent modification by the phosphate group induces conformational changes in FixJ that makes it a competent transcription factor.

Dephosphorylation of phospho-FixJ occurs continuously, even when oxygen is not present. It must therefore be regenerated constantly. This mechanism ensures that the system does not get "stuck" in the "on" position; a good biological "switch" must be readily reversible so that the organism can quickly adapt to a rapidly changing environment.

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