Chloroplasts nitrite reduction

Photosynthetic inhibition caused by NOx may be due to competition for NADPH between the processes of nitrite reduction and carbon assimilation in chloroplasts. N02 has been shown to cause swelling of chloroplast membranes (Wellburn et al. 1972). Biochemical and membrane injury may be caused by ammonia produced from N03, if it is not utilized soon after its formation. Plants can metabolize the dissolved NOx through their N02 assimilation pathway ... 

How the mitochondria can enhance nitrite reduction in leaves in the dark is not known as the bulk of the experimental evidence indicates that nitrite reductase is localized in the chloroplast. The reductant is ferredoxin generated by light or by the NADPH-ferredoxin reductase and an unknown NADPH-generating system in the dark. The inhibition of nitrite reduction by DNP and arsenate (Kessler and Bucker, 1960 Kessler, 1964 Hattori and Myers, 1966) has been interpreted to implicate a high energy phosphate requirement in nitrite reduction. One can speculate that the role of ATP could be to form an active nitrite required for reduction or to facilitate the entry of nitrite into the chloroplast. 

Most (Losadaet /., 1963 Ritenour er a/., 1%7 Dalling /a/., 1972a) but not all (Grant er al., 1970 Lips and Avissar, 1972) studies indicate that nitrite reductase from green leaves of higher plants is associated with isolated chloroplasts. Additional confirmation of the chloroplastic location has been published (Magalhaes et at., 1974 Miflin, 1974). These studies show that illuminated isolated intact chloroplasts without supplemental cofactors or enzymes can stoichiometrically reduce nitrite to ammonia. The rates of nitrite reduction were equivalent to in situ assimilation rates. 

Furthermore, to nitrite reduction as a source of stromal alkalinity, must be added the rate of ammonium uptake by chloroplasts in photorespiratory cycling and turnover of amino acids independent of photorespiration for reassimilation by glutamine synthetase and glutamate synthase. 

Figure 1. Coupling of nitrite reduction to photorespiration in C3 chloroplasts. For coupled export of two bicarbonate molecules to the cytosol for each nitrite reduced, a sustained cycling process is necessary, since only one bicarbonate is formed in the first cycle (RuBP, ribulose bisphosphate 3-PGA, 3-phosphoglycerate GLYCOL, glycollate 2-PG, 2-phosphoglycollate GA, glycerate 2-OG, 2-oxoglutarate).

The distribution of carbonic anhydrase is also in accord with the alkaline stress theory. This enzyme is highly active in C3 chloroplasts whereas it is absent from C4 bundle-sheath chloroplasts conducting carboxlation - nor is it needed there. Bicarbonate generated on nitrite reduction in the mesophyll chloroplasts could be transferred to the cytosol at a leisurely rate for PEP carboxylase activity there. ... 

In a plant, nitrite reductase catalyses reduction of its substrate to ammonia, which is subsequently incorporated into cell material. This type of nitrite reductase is classified as an assimilatory enzyme. The plant enzyme is found in the chloroplasts and acquires reductant from ferredoxin which is in turn reduced by the action of the photosystems that derive electrons... 

In all photoautotrophs, reduction of NOj" to NH4 is achieved in two distinct enzymatic steps (Campbell, 2001). First, assimilatory nitrate reductase (NR) catalyzes the two electron reduction from NOj" to NO2. NR is a large soluble cytoplasmic enzyme with FAD (flavin adinine dinucleotide), an iron-containing cytochrome and molybdopterin prosthetic groups, and requires NADH and/or NADPH as an electron donor (Guerrero et al, 1981). Functional NR is in the form of a homodimer and therefore requires two atoms of iron per enzyme. Following transport into the chloroplast, NO2 undergoes a 6 e reduction to NH4 via assimilatory nitrite reductase (NiR). NiR, a soluble chloroplastic enzyme, contains five iron atoms per active enzyme molecule, and requires photosynthetically reduced ferredoxin as an electron donor (Guerrero et al., 1981). 

Chloroplast ferredoxin is a small water soluble protein M W 000) containing an Fe-S center [245]. Its midpoint potential ( — 0.42 V [246]) is suitable for acting as an electron acceptor from the PSI Fe-S secondary acceptors (Centers A and B) and as a donor for a variety of functions on the thylakoid membrane surface and in the stroma. Due to its hydrophylicity and its abundance in the stromal space, ferredoxin is generally considered as a diffusable reductant not only for photosynthetic non-cyclic and cyclic electron flow, but also for such processes as nitrite and sulphite reduction, fatty acid desaturation, N2 assimilation and regulation of the Calvin cycle enzyme through the thioredoxin system [245]. Its possible role in cyclic electron flow around PSI has already been discussed. The mobility of ferredoxin along the membrane plane could be an essential feature of this electron transfer process the actual electron acceptor for this function and the pathway of electron to plastoquinone is, however, still undefined. 

In broad outline, the reduction and assimilation of inorganic sulfate and nitrate in plants have several features in common. Both processes entail 8 c reductions to inorganic forms (sulfide and ammonia, respectively) in energy-requiring reactions prior to incorporation into appropriate acceptor molecules. With the exception of the partial reduction of nitrate to nitrite in the cytoplasm, assimilation of sulfate and nitrite occurs in chloroplasts in reactions which are dependent on light for a supply of Fdred and ATP. However, the processes differ in many aspects of detail. For example ATP is required for activation of sulfate prior to reduction but in the nitrate assimilation pathway ATP is required after reduction for the incorporation of ammonia into glutamine. In addition, sulfate activation has no counterpart in nitrate reduction and, whereas sulfate remains bound to a carrier during reduction, the intermediates of nitrate remain free. 

The reduction of NADP by ferredoxiniNADP reductase (FNR, EC 1.18.1.2) is the terminal step in the photosynthetic generation of strong reductant in chloroplasts (1). FNR, an FAD containing enzyme, also catalyzes electron flow from NADPH to ferredoxin (Fd), which supplies electrons for reduction of nitrite and sulfite (2), and may facilitate the redox poising of cyclic electron transport around PSl (3). 

The next step— the reduction of nitrite to ammonia—again may take place throughout the plant, although most is known about the process as it occurs in green tissues. Nitrite reductase in photosynthetic plant parts is located within the chloroplasts and the reaction it catalyses results in the transfer of 6 electrons from the photosynthetic electron transport system via ferredoxin (eqn. 65) to each molecule of nitrite. 

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