A. Paneque

Inactivation and repression by ammonium of the nitrate reducing system in chlorella.

Addition of ammonium to a suspension of Chlorella cells growing autotrophically in the light with nitrate causes a striking inactivation of nitrate reductase in less than 1 hour. Neither the NADH2-specific diaphorase which catalyzes the first step of the reduction of nitrate to nitrite by NADH2 nor nitrite reductase are affected by the ammonium treatment. However, all the enzymes of the nitrate reducing system, including nitrite re ductase, are fully repressed by ammonium. The in vivo and in vitro reactivation of nitrate reductase and the derepression of all the enzymes of the nitrate reducing system are also described.

Ferredoxin-nitrite reductase from spinach

Summary 1. Nitrite reductase from a crude homogenate of spinach leaves has been purified about 500-fold and freed from NADP-reductase (EC 1.6.99.4) and nitrate reductase (EC 1.6.6.2). The enzyme, which does not seem to be a flavoprotein, catalyzes the reduction of nitrite to ammonia with a variety of enzyme systems as electron donors and requires ferredoxin as electron carrier. Flavin nucleotides and menadione are not capable of replacing ferredoxin, but the artificial electron carrier, methyl viologen, is also effective in the reaction. In the absence of spinach NADP-reductase, ferredoxin-nitrite reductase cannot use NADPH2 as electron donor. 2 A method based on the reduction of nitrite by ferredoxin (of methyl viologen) chemically reduced with hydrosulfite has been successfully applied to the aerobic assay of the enzyme. Nitrite reductase itself is inhibited by cyanide but not by p-chloromercuribenzoate or azide. The affinities of the various substrates and the offect of pH have also been investigated. 3. Isolated spinach chloroplasts contain nitrite reductase. On a protein basis, the activity of nitrite reductase in the chloroplasts is higher than in the rest of the cell as a whole. By breaking the chloroplasts, it has been shown that most of the enzyme is recovered in the chloroplast extract and only a small part remains bound to the grana.

Flavin nucleotide nitrate reductase from spinach

Abstract 1. 1. Nitrate reductase from spinach has been purified 130-fold by a procedure which includes, as the main steps, adsorption on calcium phosphate gel and chromatography on hydroxylapatite column. By this procedure it has been shown that NADP reductase and nitrate reductase are different proteins. 2. 2. A method based on the reduction of NO3− to NO2− with flavin nucleotides reduced by S2O42− has been successfully applied to the assay of the enzyme. 3. 3. FMN and FAD are the natural cofactors which in their reduced form mediate the transfer of electrons to the nitrate-nitrate reductase system. Menadione and ferredoxin are not capable of replacing the flavin nucleotides, but benzyl and methyl viologen are affective electron carriers in the system. 4. 4. Nitrate reductase has a pH optinum of 7.6. The Michaelis constant for either FMN or FAD is roughly 0.02 mM and that for nitrate, 0.2 mM. 5. 5. Due to the fact that FMN and FAD are the effective electron donors for the reduction of NO3− in higher plants and that they can be reduced by a variety of enzyme systems, the enzyme until now known as NAD(P)H2: nitrate oxidoreductase (EC 1.6.6.2) has to be considered as a mixture of two different proteins, NADP reductase and nitrate reductase itself, and the latter will be systematically classified as FMNH2(FADH2): nitrate oxidoreductase.

Regulation by ammonia of nitrate reductase synthesis and activity in Chlamydomonas reinhardi

Abstract In the green alga Chlamydomonas reinhardi , all the enzymes of the nitrate-reducing system are nutritionally repressed by ammonia. Besides, ammonia promotes in vivo the reversible inactivation of nitrate reductase in an indirect way by primarily raising the cellular level of reducing power, which in turn determines the reversible reduction of the enzyme. Subsequent removal of ammonia from the medium causes reactivation of the inactive enzyme. Interconversion of the active and inactive forms into one another can also be directly achieved in vitro by reducing and oxidizing nitrate reductase with its own physiological substrates.

Mechanism of nitrate reductase reversible inactivation by ammonia in Chlamydomonas

Abstract Ammonia promotes in vivo the conversion of the active form of nitrate reductase into its inactive form by indirectly causing the reduction of the enzyme. Apparently ammonia acts as an uncoupler of noncyclic photophosphorylation, a process which in turn leads to a rise in the level of reducing power in the cell. The transformation is reversible, and, upon ammonia removal, the enzyme becomes again oxidized and active. Nitrate reductase inactivation by ammonia requires light and does not occur when the noncyclic electron flow is blocked or when the photosynthetically generated reducing power is being simultaneously oxidized. In the dark and in the absence of ammonia, the enzyme appears either active or inactive in response to the degree of aerobicity or anaerobicity of the culture.

Reversible inactivation by NADH and ADP of Chlorella fusca nitrate reductase

The active form of Chlorella fusca nitrate reductase can be reversibly converted into its inactive form by reduction with NADH in the presence of ADP. Under the experimental conditions used, no inactivation occurs when nitrate is simultaneously present or when the nucleotides act isolately, the inactivating effect being maximal at a concentration of ADP (0.3 mM) equimolecular with that of NADH. The inactive enzyme thus attained can be completely reactivated by reoxidation with ferricyanide. The redox state of the pyridine nucleotide and the phosphorylation degree of the adenine nucleotide are critical for the inactivation process to ensue, since neither NAD+ nor AMP or ATP do exert any effect. ADP is also a powerful, although rather unspecific, protector against thermal inactivation of the NADH-diaphorase moiety of the NADH-nitrate reductase complex.

Mechanism of nitrate reduction in Chlorella

Abstract In Chlorella , the reduction of nitrate to ammonia takes place in two independent enzymatic steps: 1) the reduction of nitrate to nitrite, involving 2 electrons, catalyzed by NADH-nitrate reductase and 2) the reduction of nitrite to ammonia, involving 6 electrons, catalyzed by ferredoxin-nitrite reductase. Both enzymes have been purified and characterized, and some of their properties have been investigated.

Structural and functional role of FAD in the NADH-nitrate reducing system from Chlorella

Partially-purified preparations of nitrate reductase from Chlorella cells [l] and photosynthetic tissues from higher plants [2,3] were previously found to utilize reduced FAD or FMN as electron donor for the reduction of nitrate to nitrite. NADPH was only active as reductant when the nitrate reducing system was supplemented with both flavin nucleotide and NADPH diaphorase; NADH, however, could be utilized as electron donor without the addition of other cofactors or enzymes [ 1,3,4] . In the transfer of electrons from NADH to nitrate, the reaction catalyzed by NADHnitrate reductase (M.W. about 500,000), two enzymatic activities participate sequentially, which, although not physically separable, can be easily and independently assayed: the first, a NADH diaphorase which can use a variety of oxidized compounds (such as cytochrome c or dyes) as electron acceptors; and the second, a nitrate reductase proper, or terminal nitrate reductase, which can use reduced flavin nucleotides (or viologens) as electron donors, and has been named therefore FNH;!-nitrate reductase [l-3] . A peculiar characteristic of this second enzyme is that it can exist in two metabolic interconvertible (active or inactive) forms [51. In agreement with the results from other laboratories [6-B], we found not only that the NADH-dependent reduction of nitrate did not require flavin nucleotide but that the .addition of it could result in

Molybdenum and iron as constituents of the enzymes of the nitrate reducing system from chlorella

Abstract By adding radioactive 99 Mo (as molybdate) and 59 Fe (as ferrous ion) to a culture of Chlorella cells at the moment derepression of the enzymes of the nitrate reducing system was initiated as a consequence of the removal of ammonia from the medium, it could be unequivocally shown that the two metals were incorporated into nitrate reductase and nitrite reductase respectively, remaining associated with the enzymes during purification. After a mild heat treatment of nitrate reductase, exogenous molybdate could be made to interact with the enzyme and to function as electron donor after its chemical reduction with hydrosulfite.

Characterization of the nitrate-reducing system of the yeast Torulopsis nitratophila

Abstract The assimilatory nitrate-reducing system of the yeast Torulopsis nitratophila has been characterized. Nitrate is reduced to nitrite by a FAD-dependent NADPH-nitrate reductase similar to the enzyme isolated from other fungi and green plants. This enzyme may exist in an active or inactive interconvertible form, according to its redox state. Nitrite is reduced to ammonia by a FAD-dependent NADPH-nitrite reductase similar to the enzyme isolated from bacteria and other fungi but different from green plants ferredoxin-nitrite reductase.