Electron Donors

Electron Donors

	Electron Donors - Nitrogen fixation is a reductive process which requires a strong reducing agent (electron donor) as substrate. The electron donors vary in the different physiological groups of nitrogen fixing organisms. (1) Pyruvate, produced from the fermentation of sugars, is the electron donor for anaerobic bacteria such as clostridia. Pyruvate supports active nitrogen fixation in extracts of C. pasteurianum, Bacillus polymyxa, Klebsiella pneumoniae, Chromatium and some cyanobacteria. The first nitrogen fixing extracts were prepared from Clostridium pasteurianum with pyruvate as substrate. The fermentation of pyruvate was extremely rapid, and occurred along with nitrogen fixation. A large volume of CO₂ and H₂ accumulated in the reaction vessel. Earlier workers had described a complex enzyme system capable of converting pyruvate to acetyl phosphate with evolution of CO₂ and H₂. This reaction was termed the phosphoroclastic reaction because inorganic phosphate was the final acetyl acceptor. Strong reductant are produced during pyruvate metabolism.
Nitrogen fixation by oxidative cleavage of pyruvate involves a number of steps (i) Pyruvate is decarboxylated by pyruvate dehydrogenase containing TPP. (ii) Electrons are passed from the reduced ("bleached") enzyme to ferredoxin. This reaction yields the dark oxidized form of the enzyme and "leuco" ferredoxin. (iii) Clastic Reaction - The acetyl moiety of pyruvate is transferred to coenzyme A (CoA), yielding acetyl coenzyme A. (iv)The thiolester bond energy of this enzyme is conserved as ATP via the intermediate acetyl phosphate. (v) The phosphoroclastic reaction supplies nitrogenase with both reduced ferredoxin and ATP. Nitrogenase catalyses the reduction of H₂ to NH₄⁺ (vi) Excess reduced ferredoxin is converted to molecular hydrogen by an active hydrogenase. The reduction of a variety of substrates is catalyzed by ferredoxin linked reductases.
(2) Formate is a strong reducing agent which supports nitrogen fixations in extracts containing ferredoxin linked formate dehydrogenases. It junctions as an electron donor in C. pasteurianum, B. polymyxa and K. pneumoniae. (3) Hydrogen. Gaseous hydrogen is also a strong reducing agent. It supports nitrogen fixation in extracts containing ferredoxin linked hydrogenases. The H₂ nitrogenase electron transport chain of C.pasteurianum Can be reconstructed from pure components. Purified hydrogenase contains 4 atoms each of non haeme iron and acid labile sulphur, and is therefore an iron sulphur enzyme. Its MW is 60,000. It is composed of two identical subunits, each of MW 30,000. (4) Krebs Cycle Metabolites. In strictly aerobic bacteria such as Azotobacter, the reducing power for nitrogen fixation is developed during Krebs cycle respiration. Organic acids, alcohols and sugars are utilized. Salts of malic, succinic and lactic acids support vigorous growth and acetylene reduction in pure cultures of Spirillum and Azospirillum species.
Azotobacer paspali and A.lipoferum can utilize glucose and sucrose as the sole sources for growth and nitrogen fixation. By contrast, these two sugars are poor substrates for A.brasilense. In aerobes, the TCA cycle is the major route for generating adenosine , 5'-triphosphate. In Azospirillum brasilense, respiration generates adenosine 5'-triphosphate, which is required for the operation of nitrogenase. The TCA cycle is also important for generating reducing power. (5) Pyridine Nucleotides. In A. brasilense, electrons from TCA cycle, intermediate are transferred to oxidized NAD. Reduced NAD is one of the sources for N₂ -->NH₃ reduction by nitrogenase. In Azotobacter vinelandii, NADPH₂ is the electron donor in nitrogen fixation. The substrates most active in generating NADPH₂ in cell-free extracts are isocitrate, malate and glucose 6- phosphate. The electron carriers in these organisms are azotobacter ferredoxin and azotoflavin, a flavodoxin. NADPH₂ (or NADH ) support nitrogen fixation in extracts of aerobic facultative fermentative and photosynthetic bacteria. They are the most important carriers for linking cellular reducing power to nitrogenase.
	(6) Photosynthetic Donors. There have been several reports of the coupling of a native photosynthetic electron transport chain to nitrogen fixation in cyanobacteria and photosynthetic bacteria. Nitrogen fixation in legumes depends upon the supply of photosynthate from the host to the bacteroides inside the nodules. In the cyanobacteria, photo synthetically derived electrons drive nitrogenase activity in vivo. Electrons from carbon substrates also drive nitrogenase activity.

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