Light and Dark Reduction of Nitrite in a Reconstituted Enzymic System.

Kessler (1) has studied the reduction of nitrite to ammonia by the green alga Ankistrodesmus both in the light in an atmosphere of nitrogen and in the dark under hydrogen. No specific conclusion could be drawn as to the mechanism of the process, although it was assumed that reduced pyridine nucleotides might serve as the immediate hydrogen donors for the reduction of nitrite and that energy-rich phosphate bonds were specifically required for the occurrence of the reaction. The production of “assimilatory power” by noncyclic photophosphorylation (2) was, therefore, considered a prerequisite for nitrite reduction. Nicholas (3), working with cell-free enzyme preparations from Neurospora, found also that phosphorylations were involved in the reduction of nitrite. Roussos and Nason (4) purified from extracts of soybean leaves a soluble “pyridine nucleotide-nitrite enzyme” which, in the presence of an unidentified, heat-stable, organic factor obtained from the same extracts, required nitrite in order to catalyze the oxidation of reduced dior triphosphopyridine nucleotide. However, they could not demonstrate any disappearance of nitrite concomitant with the enzymic oxidation of pyridine nucleotide, and the fate of nitrite remained unknown. More recently, Hageman, Creswell, and Hewitt (5) have shown that the reduction of nitrite to ammonia by marrow leaf enzymes occurs with reduced benzyl viologen alone or in catalytic amounts with DPNH, but not with TPNH or DPNH in the absence of the dye. Mortenson, Valentine, and Carnahan (6) obtained from Clostridium pasteurianum a water-soluble, nonheme, nonflavin, iron-containing protein, named ferredoxin, which linked hydrogenase with a variety of electron acceptors, among them nitrit.e. According to Valentine et al. (7), spinach ferredoxin substituted for Clostridium ferredoxin in the reduction of nitrite by extracts of this bacterium with hydrogen gas as the electron donor. Huzisige and Satoh (8) isolated from spinach leaves a soluble enzyme preparation, “photosynthetic nitrite reductase,” which was required in addition to grana for the photochemical reduction of nitrite. In previous work from our laboratory (9, lo), it has been shown, however, in spinach chloroplasts, that the reduction of nitrite itself is a dark reaction. The mechanism involved in the process was found to be similar to the one implicated in the photosynthetic reduction of TPN+ (11). To take place, the reduction of nitrite required, in addition to lightor dark-reduced