Nitrate reductase cyanide

W. B., Glutathione S-transferases in the formation of cyanide from organic thiocyanates and as an organic nitrate reductase. Biochem. Biophys. Res. Commun. 64 (1975), p. 501—506... 

Dissimilatory nitrate reductases (Pichinoty type A) in membrane fractions from bacteria have been shown capable of utilizing a variety of respiratory Intermediates and reduced pyridine nucleotides for nitrate reduction (Cole and Wimpeny, 1968 Knook et ai, 1973 Burke and Lascelles, 1975 Enoch and Lester, 1975). Reduction of nitrate by the membrane fractions, when respiratory substrates or pyridine nucleotides serve as reduc-tant, is generally inhibited by azide, cyanide and p-chloromercuribenzoate. Nitrate reduction, mediated by respiratory substrates, could be inhibited by n-heptylhydroxyquinoline-N oxide (HONO) or dicoumoral (Ruiz-Herrera and DeMoss, 1%9 Knook et al., 1973 Burke and Lascelles, 1975). However, in Micrococcus denitrificans (Lam and Nicholas, 1969) and in Bacillus stearothermophilus (Downey, 1%6) nitrate reduction is not inhibited by... 

Dissimilatory nitrate reduction by particulate preparations from bacteria can utilize reduced viologen dyes as electron donors. With this reductant, nitrate reduction is insensitive to inhibition byp-chloromercuribenzoate and HONO, but is prevented by cyanide and azide (Burke and Lascelles, 1975 Ruiz-Herrera and DeMoss, 1969). Respiratory intermediates or reduced pyridine nucleotides cannot serve as electron donors for the nitrate reductase solubilized from membrane complexes and the assay must be performed with reduced viologen dyes. 

Electrons are transferred by the respiratory electron chain to a cytochrome b which then donates electrons to the Mo-protein (nitrate reductase). By using reduced viologens it should be possible to determine the kinetics of nitrate reduction by the nitrate reductase however, no information on this aspect is currently available. The sensitivity of nitrate reduction to inhibition by cyanide is considered to be due to association of the inhibitor with molybdenum residues in the nitrate reductase (Enoch and Lester, 1975). Cytochrome b involved in nitrate reduction does not bind cyanide. 

Pyridine nucleotide mediated nitrate reductase and NADPH cytochrome c reductase activities in the purified preparations are inhibited by p-hydroxymercuribenzoate (Garrett and Nason, 1969 McDonald and Coddington, 1974 Guerrero and Gutierrez, 1977). This inhibition could be overcome with cysteine or dithiothreitol. Cyanide and azide inhibit pyridine nucleotide and reduced viologen dye mediated nitrate reduction but do not affect NADPH cytochrome c reductase activity. 

Sulfhydryl reagents and heat will inhibit nitrate and cytochrome c reduction, but do not interfere with nitrate reduction mediated by FMNH2, etc. Azide and cyanide inhibit nitrate reduction but do not interfere with the NAD(P)H-mediated cytochrome c reduction. From work with barley (Wray and Filner, 1970) and spinach (Rucklidge et al., 1976) it can be inferred that NADH nitrate reductase is composed of a NADH-cytochrome c dehydrogenase and a Mo-protein. Nitrate induces the apoprotein (dehydrogenase) in molybdenum-deficient spinach leaves. This apoprotein mixed with an acid-dissociated product of spinach nitrate reductase can form an active nitrate reductase. 

Pistorius et al. (1978) have indicated that the time course kinetics of accumulation of cyanide inactivated enzyme observed, when cells of Chlorella are transferred from nitrate to ammonium, are too slow to account for the measured rapid decline in nitrate reduction. They suggest that cessation of nitrate utilization when this organism is transferred to ammonium is due to a termination of nitrate uptake. However, Diez et al. (1977) indicated that in Ankistrodesmus the absence of a nitrogen source (i.e., no nitrate uptake) brings about a much smaller inactivation of nitrate reductase than that produced by the transfer from nitrate to ammonium. Thus, the rapid in vivo inactivation of nitrate reduction observed in this alga when it is transferred to... 

Small amounts of cyanide reversibly inhibit nitrate reductase in plants. Furthermore, in experiments in which measurements of this inhibition was used as a bioassay, it was possible to demonstrate the presence of very small amounts of cyanide in a number of plants not previously known to have cyanide (Vennesland et al., 1981). 

Vennesland s group has concluded that the inactivation in vivo after the cells have been treated with ammonia involves the formation of a firmly bound complex of reduced enzyme and cyanide. They have speculated that the product of ammonium assimilation which inhibits nitrate reductase is cyanide. In this respect the COj requirement for inactivation by ammonia in the light could be relevant. On the other hand, COg could potentiate the stimulating effect of ammonia on the photosynthetic non-cyclic electron flow.< If this interpretation would be correct, the activation of the ADP-dependent pyruvate kinase reaction by ammonia discovered by Bassham in Chlorella might be better explained by an increase in the cellular ADP level induced by the uncoupling effect of ammonia on photophosphorylation than by a direct activation of the kinase by the ammonium cation, as it has been postulated. 

The conversion in vitro of the active form of Chlorella nitrate reductase into the inactive form depends on its reduction by NAD(P)H, and is reversible by reoxidation with ferri-cyanide. Inactivation by NADH requires the first moiety of the complex to be active and proceeds much faster at high pH or when ADP at low concentration (0 3 mM) is simultaneously present. This synergistic effect is quite specific for NADH and ADP. Nitiate, as well as several of its competitive inhibitors, completely prevents and even reverses inactivation by NADH and ADP. In fact, Vennesland and co-workers have demonstrated the presence of cyanide in the in vivo inactivated enzyme by overnight incubation with nitrate and phosphate. ... 

Lorimer, G. H., Gewitz, H., Volker, W., Solomonson, L. P. and Vennesland, B. (1974) The presence of bound cyanide in the naturally inactivated form of nitrate reductase of Chlorella vulgaris. J. Bioi. Chem. 249, 6074-6079. 

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