Nitrite reduction in Clostridium pasteurianum

Abstract

The mechanism of nitrite reduction in plants and bacteria has not been well defined. The identification of a new, low molecular weight, non-heme iron protein, ferredoxin, as an electron carrier in C. pasteurianum, led to the discovery of a closely associated nitrite reductase activity in this organism (Mortenson, L. E., et al., Biochem. and Biophys. Research Communs., 2, 448 (1962)). This nitrite reductase system was chosen for a detailed study as an example of a ferredoxin-dependent reaction. Studies with the cell-free extract from which the ferredoxin had been removed, called the phosphoroclastic enzyme system, substantiated the reaction sequence: H2-hydrogenase-ferredoxin-nitrite reductase-N02- NH3 proposed earlier (Valentine, R. C., et al., J. Biol. Chem. 238, 856 (1963)). Nitrite was stoichiometrically converted to ammonia. The six-electron reduction occurred as one complete' reaction, and no intermediates were detected. The Michaelis constant for the overall reaction, Km =9.1 µM. Experiments with reduced flavin and pyridine nucleotides added to reaction mixtures or generated In ~ demonstrated that these nucleotides do not participate in nitrite reduction in this organism. Ferredoxin apparently mediates the electron transfer by coupling directly with the nitrite reductase system. With sodium dithionite as the electron source, benzyl viologen mediated the direct supply of electrons to nitrite reductase, but not to hydrogenase. This system provided a means of isolating the nitrite reduction activity from other cellular systems and of studying it independently of hydrogenase and ferredoxin activities. Substitution of other artificial dyes for benzyl viologen indicated the enzyme could accept electrons only from sources that had electrode potentials similar to the hydrogen electrode (E'0 =-0.35 to -0.45 v at pH 7). Nitrite reductase was inhibited reversibly by CO. Established sulfhydryl and iron inhibitors were also effective inhibitors of this enzyme system. The enzyme is apparently a metalloprotein containing iron. The Km =25 µM for the benzyl viologen-mediated reaction. Purification studies utilizing most of the common methods of protein fractionation were unsuccessful in obtaining a purified preparation of nitrite reductase. The studies implied that the reductase activity is expressed by a multi-component enzyme complex which is highly oxygen labile. Results of a comparative study of several other clostridia indicate that nitrite reductase activities are present only in those clostridial strains which also contain an active hydrogenase and nitrogenase system.