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Lecture Twenty Two
Nitrogen Metabolism:  Source, Nomenclature, N₂ Fixation

Goal: Introduction to the sources of nitrogen available to plants, their chemistry and fixation into forms usable by plants. A good understanding of nitrogen fixation in plants. 

Outline:

1. Nitrogen -- General introduction and chemistry
2. Sources of nitrogen
3. N₂ fixation

Background Readings for the discussion on N₂ fixation:

Next to C, H and O, N is the most abundant element in plant tissues.  In what form is most of the N on planet earth found?

The abundance (relative to 1000 atoms of C) of major elements in plants and animals is illustrated in the following table:

As mentioned in the class text, Pliny the Elder ~ 80 A.D.  stated "...It is however universally agreed that no manure is more beneficial than a crop of lupine turned in by the plough..."

Why is this?

Unlike O₂ or CO₂, plants (or any other eukaryote) cannot directly utilize N₂ in biosynthetic reactions.  The N₂ must 1^st be "fixed".  Most fixed N in the biosphere is derived by biological N₂ fixation by certain prokaryotes often in association with plants.  Electrical energy from lightning can cause a small, but significant amount of N₂ fixation, ~ 5 kg N ha^-1 year^-1.  Until ~50 years ago agriculture depended on natural N₂ fixation and recycling of previously fixed N.

1. Nitrogen
• Nitrogen is a vital macronutrient for all life (2 to 6% plant DW).
• Classes of N molecules
1. amino acids
• building blocks of proteins
• precursors of other secondary metabolites
2. Proteins
• involved in metabolic process
• serve regulatory roles
• structural and storage roles
3. Nucleotides
• purines, pyrimidines
• genetic information
• energy carriers (e.g. ATP)
4. Other essential compounds
• alkaloids
• glycosides
• polyamines
• chlorophyll, etc.
• N occurs in various oxidation state from -3 to +5
•  

	+3	+1	0	-1
NO3-1	NO2-1	N2O2-2	N2	NH2OH	NH3
nitrate	nitrite	hyponitrite	nitrogen gas	hydroxylamine	ammonia

 

2. Nitrogen cycles and source of N for plant (and animals)
• In soil N can be present in the form of NH₃, NO₂^-, NO₃^- or N₂.
1. N₂
• Although nitrogen is readily available in the air, only certain bacteria and cyanobacteria have the ability to carry out N₂ fixation and synthesize ammonia.
• In the root nodule of legumes, this is a particularly important process.

2. NH₃
• Ammonia is present as NH₄⁺ in certain acidic, anaerobic soils and may be taken up directly but normally it is rapidly oxidized to nitrate by nitrifying bacteria, so NH₃ does not normally accumulate.
• What is this process and how does it occur?

 

 

 

3. In the majority of situations, nitrate is the sole source of nitrogen and is the form of N taken up from the soil by the roots.

Santoro (2016) described a do-it-all nitrifier.

The standard free energy, DG^°' of this reaction is -33.5 kJ/mol indicating that this reaction is exergonic.  This suggests that in the presence of a suitable catalyst this reaction would proceed spontaneously at room temperature.  However, no catalyst is known for this to occur.  Instead the Haber-Bosch process needs high temperatures (400-650°C) and pressures (100-400 atm) requiring considerable energy and expense. The synthetic nitrogen fixation consumes ~ 1/3 of the total energy for corn production (see Box 16.1 class text).

Assignment 2

N₂ + 10H⁺ + 8e^- 2NH₄⁺ + H₂

is also exergonic but in practice requires 16 molecules of ATP per molecule of N₂ reduced.  A source of e^- is required and hydrogen gas is an additional product.  12-17 g of carbohydrate are consumed per g of N fixed.

The most efficient prokaryotic N₂ fixation systems involve a symbiotic partnership with plants. There are at least 4 groups of such symbiotic interactions.  Two involve soil bacteria and two involve cyanobacteria. 

Various rhizobia genera and species are in the legume symbioses and Frankia bacteria in the actinorhizal symbioses.  With the rhizobia and Frankia bacteria new structures are formed in the plant partners upon infection by the bacterial symbiont wherein the N₂ fixation takes place. 

The Nostoc cyanobacterium fix N in preexisting stem glands in Gunnera.  Nitrogen can be supplied to rice paddies by the symbiosis of the water fern, Azolla, with the cyanobacterium, Anabaena.

In at least the first 3 cases the prokaryotes fix N as they reside inside their host cells.  The symbiotic bacteria are released from infection threads into host cells in membrane-bound vacuoles termed symbiosomes.  They are separated from their host cell cytoplasm by a peribacteroid membrane (PMB) derived from the plant host membranes.  

Burkholderia...

The formation of N₂ fixing root nodules is known as nodulation and Rhizobia produce signal molecules known as Nod factors that play a key role in the initiation of nodulation.  The host plant roots release specific flavonoid inducers of nod factors in the appropriate Rhizobial symbiont.  The nod factors are lipochito-oligosaccharides with 3-5 ß-1,4-linked N-acetylglucosamine residues and a fatty acid (16:0-3, 18:0-1 or 18:4) on the non-reducing sugar monomer (Fig. 2; Mylona et al. 1995, The Plant Cell 7:869-885 or 16.24 of the class text).

Nitrogenase catalyzes the reaction:

N₂ + 8H⁺ + 8e^- 2NH₃ + H₂

Each e^- transfer requires 2 ATP molecules so the reaction includes:

16 ATP 16 ADP + 16 Pi  

Nitrogenase enzymes contain an iron protein (dinitrogenase reductase) and a MoFe protein (dinitrogenase).  The iron protein is composed of two ~65 kD O₂ sensitive subunits containing 4 Fe and 4 S^2- atoms per dimer. The MoFe protein is an a₂b₂ heterotetramer.  The iron-molybdenum cofactor is a large redox center containing Fe₄S₃ and Fe₃MoS₃, which are linked to each other via 3 inorganic sulfide groups (see Fig. 11.7 of the Heldt text and 16.9 of the class text):  

A model proposed by Seefeldt et al. (2004) for creation of a binding site for N2 at Mo by dissociation of the carboxylate ligand of (R)-homocitrate and hydrogen bonding of the longer arm of (R)-homocitrate to -442His (also see Weare et al., 2006).  

The iron-sulfur center has a midpoint potential of -350 mV that increases to -450 mV when MgATP binds to the Fe protein and causes a conformational change.  Upon reduction by NADH and binding MgATP, the Fe protein donates e^- to the MoFe protein with concomitant hydrolysis of MgATP.  It is the MoFe protein that carries out the N₂ reduction.  Thus the nitrogenase reaction can be divided into 3 steps:

1. reduction of the Fe protein by an external e^- donor;
2. reduction of the MoFe protein by the Fe protein;
3. and reduction of N₂ by the MoFe protein.

The overall nitrogenase reaction is:

This involves transfer of 8 e^- from 4 NADH to N₂ producing 2NH₃ as illustrated in Fig. 11.6 of the Heldt text:

• Conformational changes in the nitrogenase complex during ATP turnover (Tezcan et al., 2006).

Nitrogenase can catalyze the reduction of other substrates with structures similar to dinitrogen.  Acetylene reduction provides a convenient measure of nitrogenase activity:

HCCH + 2e^- + 2H⁺ H₂CCH₂

¹⁵N₂ ¹⁵NH₃ ¹⁵N amino acids

How is the ¹⁵N detected?

Nodule function

When bacteria are released from the infection threads into the infected cells, nodule primordia differentiate into N₂ fixing nodules.  Some nodules develop indeterminately in which a developmental gradient from the meristem to a senescence zone exists with the central tissue divided into specific zones (Fig. 4; Mylona et al. 1995, The Plant Cell 7:869-885).  The nodule parenchyma (green) (red in Mylona et al. 1995). surrounding the nodule vascular bundle (black) provides an O₂ barrier that reduces access of O₂ to the central tissue. Since this is interrupted at the meristem, an O₂ gradient is created dropping off toward the senescence zone.  In the 1^st interzone cell layer, the low O₂ level activates the trans-membrane O₂ sensor protein FixL.  FixL activates the transcriptional factor, FixJ, by phosphorylation(*).  FixJ* induces transcription of the nifA and fixK genes whose products in turn induce transcription of genes encoding the various proteins involved in the N₂ fixation process.  The NifA protein is also O₂ sensitive providing an additional level of control of the process.

Van de Velde et al. (2010) report that plant peptides can govern terminal differentiation manipulating the cell fate of endosymbiosisbacteria. See Fig. 1. In some cases this symbiosis can result in "reverse parasitism"!

O₂ is transported to sites of respiration by leghemoglobin (Lb) encoded by the host plant.  This can accumulate to 25% of the soluble protein of infected cells and confers a pink/red color characteristic of nodules active in N₂ fixation.  Like hemoglobin, leghemoglobin has a high affinity for O₂ and controls delivery of O₂ to respiratory enzymes while keeping O₂ low in the vicinity of nitrogenase.

 
The ammonium produced via N₂ fixation is assimilated into other nitrogenous compounds as illustrated for a generalized legume in the following figure. Drag the intermediate compounds and enzymes into the appropriate place in the figure from the table at the right:

α-Ketoglutarate Phosphoenolpyruvate Glutamine Malate Glutamine (2) Glutamate Aspartate Oxaloacetate Glucose Glutamine Malate NH+4 Asparagine Oxaloacetate Sucrose

The overall nitrogenase reaction is:

N₂ + 4NADH+ H⁺ + 16MgATP 2NH₃ + H₂ + 4NAD⁺ + 16MgADP + 16Pi

Nitrogen assimilation and photoautotrophic nitrification in legumes, Fig. 1 Hipkin et al. (2004).

Background Readings for the discussion on nitrate reduction and ammonia assimilation: