N2+8e+ 16mg ATP+ 16H2O→2 NH3+H2+ 16mg ADP+ 16 pi+8H+

The enzyme that catalyzes this reaction is nitrogenase. Nitrogenase is a multifunctional oxidoreductase. Besides reducing N2, it can also reduce a variety of substrates, such as acetylene, cyanide, nitrous oxide, hydrazine, azide and H+. It is easy to determine the amount of acetylene reduced to ethylene by gas chromatograph, which provides a very simple method for studying the activity of nitrogenase. This method has played an important role in the study of biological nitrogen fixation.

Nitrogen-fixing enzyme is composed of iron-molybdenum protein (Fe-Moprotein) and ferritin (Fe-protein). These two proteins do not show nitrogenase activity when they exist alone, and only when they are polymerized to form a complex can they catalyze nitrogen reduction. Iron-molybdenum protein is a tetramer (α2β2) composed of two α subunits and two β subunits, with molecular weights of 5 1kD and 60kD, respectively, and molecular weights of about 220~245kD. Each ferromolybdenum protein molecule contains two molybdenum atoms and 28 iron atoms. The molecular weight of ferritin is between 59 and 73 KD, and it is composed of two 30kD (γ2) subunits with the same molecular weight. Ferritin contains four iron atoms. In the process of reducing nitrogen to NH4+, both Fe and Mo in nitrogenase undergo redox reaction, as shown in Figure 5-20. Bacteroides use carbohydrates to breathe and produce NADH or NADPH and ATP. It has been found that natural electron transporters (donors) for nitrogen fixation include ferredoxin, flavoxidoreductin and so on. The existence of ATP and divalent metal ions (such as Mg2+) in nitrogen-fixing organisms is an indispensable condition for nitrogen fixation. Only under the action of Mg2+ can ATP combine with Fe protein, and ATP hydrolysis can only take place with the participation of Fe-Mo protein. Ferritin transfers electrons to iron molybdenum protein, and ATP is hydrolyzed to produce ADP. Fe-Mo protein finally transfers electrons to N2 and protons, resulting in two molecules of NH3 and 1 molecule of H2.

Nitrogenase is sensitive to oxygen, and its catalytic reaction needs to be carried out under anaerobic conditions. Except obligate anaerobic organisms, oxygen can damage the nitrogenase of other nitrogen-fixing organisms, but these organisms need oxygen to produce ATP necessary for nitrogen fixation through respiration, so efficient nitrogen fixation is generally carried out under micro-oxygen. Different nitrogen-fixing organisms have different mechanisms to avoid nitrogen-fixing enzyme oxygen damage. For example, the nitrogen fixation function of cyanobacteria with heterotypic cells is mainly carried out in heterotypic cells, and there is an outer membrane composed of glycolipid to prevent oxygen from entering, and there is no PS ⅱ that can release oxygen through water photolysis. Among them, the activity of two enzymes in pentose phosphate pathway is low, while the activity of superoxide dismutase and dehydrogenase is strong, which keeps the heterogenous cells in a micro-oxygen environment. Bacteroides in leguminous plant nodules has a membrane around Bacteroides, and the cells in the tumor inner cortex are closely arranged to form gaps, which is very important to maintain the anoxic environment of Bacteroides. In addition, leguminous hemoglobin in nodule cells also partially controls the demand for bacteroid oxygen. In the nitrogen fixation system of non-leguminous plants, there are vesicles in the tumor of actinomycetes, which may have the same antioxidant function as the atypical cells of cyanobacteria. Obviously, the nodule in the * * * biological system itself is a good oxygen protection system.

NH3 (probably NH4+) synthesized in Bacteroides must be transported out of Bacteroides to participate in the metabolism of host plants. In the cytoplasm of bacteroid cells, NH4+ is converted into glutamine, glutamic acid, asparagine and urea. These substances are secreted by transferred cells to xylem and transported to other parts of plants.

Because of the importance of biological nitrogen fixation, the research on environmental and genetic factors controlling biological nitrogen fixation has been paid attention to. Studies show that all factors that can increase the photosynthetic capacity of plants, such as appropriate water, temperature, strong light and high CO2 level, can promote nitrogen fixation. Genetic factors of leguminous plants and nitrogen-fixing organisms also affect the rate and yield of nitrogen fixation. For example, one of the genetic factors is the nodulation ability of leguminous plants, which depends on the identification process controlled by heredity between rhizobia and host plants. In order to improve the nodulation ability, scientists are conducting research on transforming rhizobia genes and selecting suitable host varieties. Another genetic factor is that nitrogenase reduces N2 and H+. It can be seen from the general reaction formula that 1/4 electrons are used to reduce H+ to generate H2 in the reaction catalyzed by nitrogenase. H2 is reduced and escapes into the atmosphere, wasting energy. However, most rhizobia and autotrophic nitrogen-fixing bacteria contain hydrogenase, which oxidizes H2 into H2O and promotes ADP and Pi to synthesize ATP. Studies have shown that the yield of leguminous plants (such as soybeans) with rhizobia with high hydrogenase activity is slightly higher than that with rhizobia with low hydrogenase activity. It may be that the former reduces the waste of energy. Based on this understanding, it is possible to obtain rhizobia with higher hydrogenase activity through genetic engineering technology and improve the yield of beans. In addition, the nitrogen-fixing gene was introduced into the roots of non-leguminous plants by genetic engineering technology, which made some progress in the nitrogen-fixing work of these plants.

Different growth stages of plants will affect biological nitrogen fixation. Such as soybean, peanut and cajanus cajan, 90% of nitrogen for biological nitrogen fixation is in reproductive growth period, while 10% is in vegetative growth period. Strangely, the nitrogen provided by biological nitrogen fixation of several legumes is only 1/4 ~ 1/2 of the total nitrogen needed in their lifetime, and the rest mainly absorbs NO3- or NH4+ from the soil in the vegetative growth stage. However, no amount of nitrogen fertilizer can increase production. The reason is that the absorption of nitrogen fertilizer by plants increases but the biological nitrogen fixation ability decreases. Nitrate fertilizer has several functions: inhibiting the contact between rhizobia and root hairs and preventing the formation of infected filaments; The slow growth of nodules inhibited the nitrogen fixation of mature nodules; When NO3- and NH4+ were added, nodule senescence accelerated.