Biological Nitrogen Fixation

Only Certain Prokaryotes Can Fix Nitrogen

Very few organisms are able to use nitrogen directly from its abundant atmospheric N2 form. The ability to reduce N2 to ammonia — biological nitrogen fixation — depends entirely on the enzyme nitrogenase, which occurs only in certain prokaryotes. Organisms capable of this are called N2-fixers, and they fall into two broad groups:

• Free-living N2-fixers — including the aerobic bacteria Azotobacter and Beijerinckia, the anaerobic bacterium Rhodospirillum, the bacterium Bacillus, and free-living cyanobacteria such as Anabaena and Nostoc.
• Symbiotic N2-fixers — the best-known example is the partnership between legume plants and the rod-shaped bacterium Rhizobium, which colonises the roots of legumes such as alfalfa, sweet clover, sweet pea, lentil, garden pea, broad bean and clover. This partnership is visible as root nodules — small outgrowths on the root. The bacterium Frankia forms similar nitrogen-fixing nodules on non-legume plants such as Alnus. Both Rhizobium and Frankia can live freely in the soil, but they only fix atmospheric nitrogen once they are established as symbionts inside a nodule. Cutting a nodule open reveals a red or pink centre, coloured by leghaemoglobin.

How a Root Nodule Forms

Nodule formation (Figure 12.4) proceeds through a defined sequence of stages:

1. Rhizobium bacteria multiply in the soil around the root and attach themselves to epidermal and root-hair cells.
2. The root hair responds by curling up into a hook shape as the bacteria begin to invade it.
3. An infection thread forms and carries the bacteria inward into the root's inner cortex, where the cortical and pericycle cells start dividing and nodule formation begins.
4. The bacteria are released from the infection thread into these dividing cells and change shape, differentiating into rod-shaped, nitrogen-fixing bacteroids.
5. Continued division and growth of the cortical and pericycle cells builds up the mature nodule, which ends up connected to the root's own vascular system, allowing nutrients and fixed nitrogen to be exchanged between the nodule and the rest of the plant.

The Nitrogen-Fixing Reaction

The mature nodule contains both nitrogenase (a molybdenum-iron protein) and leghaemoglobin. Nitrogenase catalyses the conversion of atmospheric nitrogen to ammonia, the first stable product of nitrogen fixation:

N₂ + 8e⁻ + 8H⁺ + 16ATP → 2NH₃ + H₂ + 16ADP + 16Pᵢ

Figure 12.5 shows this happening step by step on the enzyme itself: the N2 molecule binds to nitrogenase and is then progressively reduced through repeated additions of two hydrogen atoms at a time, until the fully hydrogenated product — ammonia — is released and the enzyme is freed to bind another N2 molecule.

Protecting Nitrogenase From Oxygen

Nitrogenase is extremely sensitive to oxygen and only works under anaerobic conditions, so the nodule protects it using leghaemoglobin as an oxygen scavenger, mopping up oxygen before it can reach and disable the enzyme. Interestingly, the nitrogen-fixing microbes behave as ordinary aerobes when they are free-living, but switch to anaerobic metabolism specifically during nitrogen fixation, to keep nitrogenase safe. Making ammonia this way is also energetically expensive, costing 8 ATP molecules for every molecule of ammonia produced — ATP that comes from the host plant cell's own respiration.

What Happens to the Ammonia

At the plant's normal internal pH, ammonia is protonated into the ammonium ion (NH4+). Plants can use both nitrate and ammonium as nitrogen sources, but ammonium itself is fairly toxic and cannot be allowed to accumulate, so it is converted into amino acids quickly, through one of two routes:

• Reductive amination: ammonia reacts with alpha-ketoglutaric acid, using NADPH and the enzyme glutamate dehydrogenase, to form the amino acid glutamic acid. …