|
Mechanism of Nitrogen Fixation |
|
|
Mechanism of Nitrogen Fixation
The nitrogenase reaction has two essential steps: (1) electron activation by a suitable donor or adenosine di-phosphate (ADP) and (2) substrate reduction. These two steps of the reaction take place at different sites on the nitrogenase molecule but are interdependent.
Purified preparations of nitrogenases are highly sensitive to oxygen, specially the Fe protein part of the enzyme. However, it is believed that an undefined respiratory system exists in Azotobacter near the site of nitrogen fixation which actively 'scavenges' oxygen so as to prevent the inactivation of nitrogenase.
|
Energy requirements for nitrogenase reaction come from the cellular metabolic cycles m the form of adenosine triphosphate or A TP (roughly 12 to 20 moles of A TP per mole of molecular nitrogen reduced). Pyruvate functions both as an electron donor and an energy source. In the phosphoroclastic reaction, pyruvate forms acetyl phosphate which in the presence of adenosine diphosphate or ADP gives rise to ATP. The reductants are the strongly reducing naturally occurring electron carrier proteins, ferredoxin and flavodoxin. Dithionite (Na2S2O4) and certain dyes such as methyl viologen and benzyl viologen can also serve as artificial extracellular sources of electron donors.
|
Since all nitrogen-fixing microorganisms contain hydrogenase, this enzyme system in cells catalyzes the transfer of electrons from pyruvate or hydrogen to ferredoxin or flavodoxin.
Ferredoxins are naturally occurring iron-sulphur (Fe-S) electron carrier proteins capable of undergoing reversible oxidation and reduction.
They have been isolated from plants, blue-green algae and bacteria such as Clostridium pasteurianum, Azotobacter vinelandii, Rhizobium japonicum, Anabaena cylindrica, Bacillus polymyxa, Chromatium sp. and Desulfovibrio gigas. Ferredoxins differ in molecular weight, iron and sulphide contents and biological activity.
|
|
Such electron carrier proteins isolated from several nitrogen-fixing organisms can react not only with the nitrogenase of specific microorganisms but also be effectively interchanged with other microorganisms.
Flavodoxin is a flavoprotein first isolated from Clostridium pasteurianum in media containing low concentrations of iron and was found to replace ferredoxin as a electron carrier in a large number of reactions. Most of the nitrogen-fixing microorganisms are now known to possess flavodoxins. Subsequently, such electron carriers have been isolated from other anaerobic bacteria .like Peptostreptococcus elsdenii and Desulfovibrio spp. An electron carrier named azotoflavin has been isolated from Azotobacter vinelandii possessing biological activity similar to ferredoxins.
|
|
|
The role of pyruvate and ferredoxin in nitrogenase reactions can be illustrated as follows (from Fottrell, 1968):
|
Nitrogenase, in addition to reducing atmospheric N2 to NH3, can also reduce certain other compounds, as follows:
C2H2→C2H4; HCN →CH4 + HN3; H+→H 2;
HN3→ N2 + HN3; N2O→N2 + H2O.
According to Hardy and his associate of the Du Pont Laboratory, U.S.A., the active site of the enzyme for substrate reduction is believed to be composed of an Mo-Fe dinuclear site bridged by sulphur, having the proper size and electron characteristics to provide Mo-Fe distance. This distance is specific so as to accommodate various nitrogenase substrates including N2 and to exclude others. The first reaction in nitrogen reduction is the formation of a linear complex of N2 with the, Fe of nitrogenase.
This is followed by transfer of electron from Mo which is the end point of the electrons activating system, resulting in the formation of diimide which is stabilized by hydrogen bonding from the protein as well as the metal-nitrogen bonds. Successive addition of electrons produce hydrazine followed by cleavage of NN bond to yield 2 moles of NH3.
Top Two step reaction mediated by the enzyme nitrogenase bottom proposed intermediates and dinuclear active site for n 2 reduction by nitrogenase with enzyme bound diimide and hydrazine as intermediates
The increase in the NN bond length during reduction is accompanied by compensating changes in MNN angles so that Mo-Fe distance remains constant.
Response of Plants to Azotobacter Inoculation
Inoculation of soil or seed with Azotobacter is effective in increasing yields of crops in well-manured soil with high organic matter content.
|
|
Submit Your College, Institute, Company, Products for FREE
