P.J. Aparicio

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.

Blue light photoreactivation of nitrate reductase from green algae and higher plants

Abstract The inactive form of NADH-nitrate reductase from spinach and Chlorella fusca is fully reactivated in short periods of time when the enzyme-complex is illuminated with white or blue light but not with red light. Flavin nucleotides greatly accelerate the photoreactivation process. The results suggest that blue light might act as a modulating agent in the assimilation of nitrate in green algae and higher plants.

Interconversion of the active and inactive forms of Chlorella nitrate reductase

In the green alga Chlorella, the reduction of nitrate to nitrite is catalyzed by the flavomolybdoprotein NADH-nitrate reductase, an enzyme complex of high molecular weight with two different enzymatic activities participating sequentially in the transfer of electrons from NADH to nitrate: the first is a FAD-dependent NADH-diaphorase, and the second is the molybdoprotein nitrate reductase or terminal nitrate reductase, also named FNHa-nitrate reductase for it can use exogenous reduced flavin nucleotides as electron donors [1,2]. Both activities are affected in different ways by appropriate treatments or by selective inhibitors. Thus, NADH-diaphorase is heat-labile and sensitive to -SH binding reagents, such as pCMB; FAD and NADH specifically protect against inactivation by heating and pCMB, respectively [3,4]. On the other hand, FNHznitrate reductase is inactivated by incubation with NAD(P)H, particularly when minimal amounts of cyanide are simultaneously present; nitrate prevents against this inactivation that, once it has occurred, can be reversed by ferricyanide [4]. This paper presents evidence that Chlorella nitrate reductase can exist in two interconvertible forms, active and inactive, according to the redox state of the enzyme, the conversion being affected by temperature and pH. A preliminary report of this work has appeared previously [ 51.

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

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.

Inactivation by acetylene of spinach nitrate reductase

Abstract The molybdoprotein NADH-nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1) from spinach can be inactivated by acetylene only when the enzyme is in its reduced state. Other gases such as ethylene, carbon monoxide, dinitrogen and others did not alter the enzyme activity. From the two partial activities of nitrate reductase, only the terminal nitrate reductase was impaired by acetylene while the dehydrogenase activity was rather stimulated. Functional dehydrogenase activity was required for inactivation when NADH was the reductant. Dithionite, dithionite + MV or dithionite + FMN were also able to sustain acetylene inactivation, whether or not nitrate reductase was previously depleted of its dehydrogenase activity. However, ascorbate or ascorbate + DCIP did not cooperate with acetylene for inactivating nitrate reductase. Nitrate and the competitive inhibitors with respect to nitrate of nitrate reductase, namely azide, cyanate and carbamyl phosphate, protected nitrate reductase from acetylene inactivation. Cyanide-inactivated nitrate reductase was still sensitive to acetylene, since, once the cyanide-inactivated enzyme was placed under acetylene, no ferricyanide reactivation could be attained. These results suggest that reduced nitrate reductase might bind acetylene at the nitrate active site, where molybdenum is supposed to be implicated, thus impairing the reduction of nitrate.

Photoreactivation of Spinach Nitrate Reductase: Role of Flavins

Summary Flavin adenine dinucleotide (FAD) stimulates photoreactivation of inactivated nitrate reductase complex, 10 μM or 100 μM FAD being saturating within 5 or 2 min exposure respectively. Under intermittent illumination no variation in the enzyme activity levels is observed during the dark periods. Reduced pyridine nucleotides (NADH, NADPH) produce a lag of this photoreactivation, the lag being correlated with the concentration of these nucleotides in the reduced form. Oxidized pyridine nucleotides or adenine nucleotides have no effect. Nitrate enhances photoreactivation only when added together with NADH. These results are discussed in terms of flavins being the pigments responsible for the photoreactivation of nitrate reductase.

Red-light effects sensitized by methylene blue on nitrate reductase from spinach (Spinacia oleracea L.) leaves

Abstract Nitrate reductase from spinach (Spinacia oleracea L.) leaves, which had been inactivated in vitro by incubation with NADH and cyanide, was fully reactivated in minutes when irradiated in anaerobic conditions with red light in the presence of methylene blue. Both the rate and the extent of reactivation increased with light intensity (6 to 100 W·m-2) and dye concentration (1 to 10 μM). On the contrary, photoreactivation was completely abolished when NADH or ethylenediaminetetra-acetic acid were present during irradiation. We propose that methylene blue, when photo excited, exhibits a redox potential positive enough to reoxidise the CN--re-duced molybdenum complex settled in the inactive enzyme, thus causing its reactivation. On the other hand, prolonged irradiation of nitrate reductase, under air and in the presence of methylene blue, promoted an oxygen-dependent irreversible inactivation of the two partial activities of the enzyme. This inactivation was markedly enhanced in 77% deuterated water and greatly prevented by azide, which indicates that singlet oxygen is the species primarily involved in the photooxidative inactivation of the enzyme.