Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Biosynthesis of Amino Acids and Nucleotides
Nitrogen fixation is a complex enzymatic process

In Nitrogen Fixation, The Role of catalyst is played by an enzyme complex known as the Nitrogenase system, the exact mechanism of which is not yet fully understood. Because the nitrogenase system is unstable and rapidly inactivated upon exposure to atmospheric oxygen, it is difficult to isolate in an active form and proves rather refractory to purification. Ammonia (NH₃) is considered the first stable product of nitrogen fixation. Thus, the overall process is generally believed to reduce molecular nitrogen (N₂) to two molecules of ammonia:

Class="center">N₂ + 3Н₂ → 2NH₃

∆G⁰' = -8,0 ккал/моль.

Since The change in Standard Free energy for this reaction is a large negative value, the reaction should proceed spontaneously from left to right under standard conditions. However, molecular nitrogen is a fairly inert gas, and its two atoms are held together by a very strong bond; consequently, its reduction to ammonia requires a very high activation energy. The nitrogenase system overcomes this activation barrier (Section 9.4) by a mechanism that remains unknown to us.

The ultimate hydrogen donor for the nitrogenase complex is NADPH. Reducing equivalents are first transferred from NADPH to ferredoxin. This iron-sulfur protein serves as the immediate donor of reducing equivalents during nitrogen reduction. Its molecule contains seven iron atoms and an equal number of acid-labile sulfur atoms, and the Molecular Weight of ferredoxin is 6,000. From ferredoxin, the reducing equivalents are passed on to the nitrogenase complex, which consists of two metalloenzymes: one containing iron, and the other containing both iron and molybdenum (Fig. 22-28). Nitrogen fixation also requires ATP, which is hydrolyzed to ADP and phosphate. The exact role of this ATP is unknown, as no phosphorylated intermediates have been detected. It is hypothesized that the Free energy of ATP Hydrolysis helps overcome the high activation barrier. It is very likely that for every molecule of N₂ reduced to two NH⁺₄ ions, 12 molecules of ATP are hydrolyzed to ADP and P_i. If so, the overall equation for nitrogen fixation can be written as:

N₂ + 3NADPH + 5Н⁺ +

+ 12АТР + 12Н₂О →

→ 2NH⁺₄ + 3NADP⁺ + 12ADP + 12Р_і.

Fig. 22-28. Hypothetical pathway of the nitrogenase reaction. It is assumed that a series of intermediate steps takes place at the active sites of these Enzymes.

The nitrogenase system possesses one interesting property that enables the quantitative study of nitrogen fixation in intact plants. Nitrogenase catalyzes not only the reduction of N₂, but also the reduction of acetylene (HC=CH) to Ethylene (H₂C=CH₂). Since The ratio of ethylene to acetylene in the atmosphere can be measured by physical Methods, it is possible to determine nitrogenase activity in any plant-soil system within an experimental greenhouse by introducing acetylene into the gas phase and measuring The rate of ethylene formation.

An enormous number of experimental studies have been dedicated to the nitrogenase system, which is explained by its immense practical importance. Industrially, ammonia for fertilizers is produced by the catalytic reduction of atmospheric nitrogen via the Haber process. This reaction

N₂ + 3Н₂ → 2NH₃

requires very high temperatures and pressures. If the nitrogenase reaction could be replicated using a simpler set of inexpensive catalysts, it would pave the way for relatively low-cost production of fertilizers such as soluble ammonium salts—an achievement of particular importance for developing nations that cannot afford the energy-intensive Haber process.

Biological approaches to making atmospheric nitrogen more accessible are also being tested. For example, attempts have been made to determine whether nonleguminous crop plants, such as corn, could be colonized by various species of nitrogen-fixing Bacteria or mutants thereof, thereby establishing new beneficial symbiotic associations. Along the way, it was discovered that the roots of several nonlegume tropical plants also harbor nitrogen-fixing bacteria. Unfortunately, these plants require very warm soil for nitrogen fixation; when grown in temperate zones, nitrogen fixation does not occur.

Another approach involves isolating the bacterial DNA that encodes the nitrogenase system and introducing it into The Genome of microorganisms that lack nitrogenase, or into plant genomes. Such Gene transfer from nitrogen-fixing bacteria to non-fixing organisms, notably E. coli, has been successfully accomplished. It is far more difficult to incorporate nitrogenase DNA into the genome of Higher Plants and compel it to function there. Nevertheless, Introduction/32.html">Genetic Engineering techniques are steadily improving, meaning that this challenge may eventually be solved over time.