Fundamentals of Biochemistry - A. A. Anisimov 1986

Protein and Amino Acid Metabolism
Fixation of Atmospheric Molecular Nitrogen

It is estimated that the Earth contains about 3.8×10¹⁵ t of nitrogen (calculated as elemental nitrogen). The bulk of it is atmospheric molecular nitrogen—N₂. However, humans, animals, and higher plants are incapable of assimilating such nitrogen on their own. Nevertheless, for normal functioning, All living organisms must consume nitrogen in large quantities; nitrogen-containing compounds such as Proteins AND AMINO acids are vital Components of the Human and Animal diet, just as mineral nitrogen salts are essential for the Nutrition of higher plants.

Providing the population with dietary protein is one of the most pressing challenges currently facing humanity, especially in developing and former colonial countries. Today, this issue receives so much attention that a special committee has been established under the United Nations to comprehensively study protein resources and develop practical recommendations for preventing Protein deficiency. D. I. Mendeleev, assessing this problem, wrote that the question of how to convert atmospheric nitrogen into soil nitrogen compounds or into assimilable nitrogen—capable of being absorbed by plants and transformed into complex (protein) substances—is one of those issues of immense theoretical and practical importance. According to Academician S. P. Kostychev, nitrogen starvation is the primary factor limiting The Development of life on Earth and hindering the reproduction of organisms.

In The production of mineral nitrogen fertilizers, atmospheric nitrogen is converted industrially into ammonia or nitric acid. However, this nitrogen accounts for only 2–3% of the nitrogen contained in agricultural crop yields worldwide. Practically all the nitrogen present in the living organisms of our planet originates from atmospheric nitrogen fixed by microorganisms, as only they are capable of independently assimilating molecular nitrogen.

Some higher plants fix atmospheric nitrogen by establishing symbiotic relationships with Bacteria in their ROOT nodules, thereby making a significant contribution to the overall assimilation of molecular nitrogen. It is believed that out of the 13,000 known species of legumes, the majority are capable of symbiotically fixing nitrogen in substantial amounts, particularly cultivated legumes (peas, soybeans, etc.). Approximately 250 species from other, non-leguminous families are also capable of symbiotic Nitrogen Fixation (e.g., alder, foxtail, sea buckthorn).

The bacteria inhabiting legume nodules belong mainly to the genus Rhizobium. Among free-living bacteria, molecular nitrogen is fixed by soil bacteria of the genera Azotobacter (aerobes), Clostridium (anaerobes), and certain facultative anaerobes, as well as all photosynthetic bacteria. Blue-green Algae account for 10–15% of the total atmospheric N₂ fixed.

The industrial production of ammonia from N₂ requires a large amount of energy; even in the presence of catalysts, temperatures up to +500°C and pressures up to 300–350 atm are required. Therefore, one cannot help but admire the ability of microorganisms to carry out practically the same process at normal atmospheric pressure and moderate temperatures. The main "secret" behind this capability is the presence of a specialized enzymatic nitrogen-fixation system in microorganisms. It is hardly surprising, then, that laboratories worldwide are actively investigating N₂ fixation, and the attempt to mimic the enzymatic processes of nitrogen-fixing microorganisms under industrial conditions remains an extremely alluring, fascinating, and vital goal. Extensive and groundbreaking research into the biochemistry of atmospheric molecular nitrogen assimilation was conducted in the Soviet Union under the leadership of V. L. Kretovich at the Bach Institute of Biochemistry of the USSR Academy of Sciences.

All diverse nitrogen-fixing microorganisms share the same enzymatic system that catalyzes The conversion of molecular nitrogen into ammonia. This system is known as Nitrogenase. In addition, N₂ fixation requires strong reducing agents (an electron flow), ATP, and Mg²⁺. The Nature of the electron Donors varies among different microorganisms. In aerobic bacteria (Azotobacter, Rhizobium), the reducing agents and ATP required for N₂ fixation are generated during Carbohydrate METABOLISM via NADP-dependent reactions. Photosynthetic bacteria and blue-green algae are capable of photochemically generating strong reducing agents.

Nitrogenase (EC 1.18.2.1) consists of two proteins: a Mo-Fe protein (molybdoferredoxin) and an Fe protein (azoferredoxin). The Molecular Weight of the former ranges from 200,000 to 250,000 depending on the microbial species; it is a tetramer containing two molybdenum atoms, non-heme iron, and labile sulfide. The molecular weight of the Fe protein ranges from 50,000 to 70,000; it is a dimer that also contains non-heme iron and labile sulfide (Fig. 5.1).

During fixation, ATP interacts with azoferredoxin. This releases ADP, while azoferredoxin undergoes a conformational shift that lowers its redox potential from –280 to –400 mV. Azoferredoxin thus becomes a potent reducing agent, transferring electrons to molybdoferredoxin, where the reduction of N₂ to NH₃ takes place. Overall, the nitrogen fixation process can be expressed by the following reaction:

Class="center">N₂ + 6e^- + 12ATP + 12H₂O → 2NH⁺₄ + 12ADP + 12H₃PO₄ + 4H⁺

Fig. 5.1. Scheme of Biological Nitrogen Fixation

Nitrogenase exhibits broad substrate Specificity; besides N₂, it can reduce cyanides, nitrous oxide, acetylene, and others. In the presence of ATP, nitrogenase also catalyzes the reduction of hydrogen ions to form molecular nitrogen. In some nitrogen fixers, this process occurs simultaneously with nitrogen fixation. It is hypothesized that this property of nitrogen-fixing microorganisms could potentially be harnessed in the future to produce clean fuel in the form of molecular hydrogen.

Molybdenum is crucial for nitrogen fixation, fulfilling multiple Functions in the process. It maintains the specific conformation of the nitrogenase molecule, participates in nitrogen binding and electron transfer, and induces nitrogenase synthesis. The molecular nitrogen gas molecule is extremely stable; breaking the triple bond holding its atoms together requires about 940 kJ/mol.

A key aspect of Nitrogen fixation is the substantial energy consumption required to break the strong intramolecular bonds of N₂ during its reduction to ammonia. For instance, Azotobacter expends 1 g of sugar through Respiration to fix 15 mg of nitrogen, while Clostridium pasteurianum consumes even more (5–6 g of sugar). In root nodule bacteria, the energy source consists of photosynthetic products transported from the leaves to The Root System. A portion of these assimilates may be processed and stored as reserves within the bacteroids in the form of poly-β-hydroxybutyric acid (PHB).

The nitrogen-fixing capacity of bacteroids is also linked to the unique properties of their redox systems, particularly the presence of a specialized hemoprotein called leghemoglobin. It is believed that, much like animal Hemoglobin, it acts as an oxygen carrier to bacteroids residing within the nodule under conditions of restricted oxygen access. The ammonia produced As a result of nitrogen fixation is subsequently assimilated primarily within the plant tissue of the nodule. The initial stage of this assimilation involves the binding of ammonia by keto acids to form Amino Acids.