Molybdenum complexes nitrate reduction

A reactivation of an inactive nitrate reductase apoenzyme extracted from molybdenum-deficient plants can be achieved by the addition of acid-treated nitrate reductase or by addition of phosphate buffer washes of nitrate reductase absorbed on AMP-Sepharose. The acid treatment or phosphate wash apparently produced a molybdenum-containing complex which was responsible for reactivation of the apoenzyme. The complex had a molecular weight of less than 3 x 10 (Notton and Hewitt, 1971 Rucklidgee/< /., 1976). These reports clearly demonstrate the requirement for molybdenum for nitrate reduction however, the role of the metal in the reduction process is not completely resolved. 

The reaction of Mo(H20)63+ and nitrate in aqueous solutions results in the formation of Mo2Oi(H20)62+ and nitrite. Mo(III) coordinated to oxygen and nitrogen donor atoms of EDTA also reduces nitrate in aqueous solutions. The reduction of nitrate by a Mo(III)-EDTA complex results in the formation of nitrite and a Mo(V)-EDTA complex, as determined by chemical and spectrophotometric techniques. These reactions serve as models for biological nitrate reduction. In addition, molybdate coordinates to naturally produced phenolates. The molybdenum-coordinating phenolates also coordinate tungstate and ferric iron. Two of these phenolates contain threonine, glycine, alanine, and 2,3-dihydroxybenzoic acid. 

Molybdenum is essential to the formation and activity of assimila-tory nitrate reductases. Cells must assimilate molybdate from the environment, metabolize molybdenum in some manner to form active molybdenum cofactor, and then incorporate it into a large molecular weight protein so that it can perform a reversible redox reaction with nitrate. Investigations on the aqueous Mo (III) model systems for nitrate reduction and the coordination of molybdate by naturally produced phenolates will hopefully lead to an understanding of the complex process of molybdenum acquisitions by and molybdenum function in nitrate reductases. 

Nitrate reductase catalyzes the first step in conversion of nitrate to ammonia, the reduction of nitrate to nitrite (N02 ). The reaction multisubunit nitrate reductase enzyme, with Mr of about 800 kilodaltons, contains bound FAD, molybdenum, and a cytochrome called cytochrome 557 (which contains an Fe4S4 complex). Nitrate reductase carries out the overall reaction ... 

Although there are reports of inhibition of nitrate reductase by chelating agents which might chelate nonheme iron, analyses of the purified enzymes from eukaryotes have failed to reveal the Fe-sulfur complexes detected in the dissimilatory nitrate reductases from prokaryotes (Garrett and Nason, 1969 Ahmed and Spiller, 1976). The extensively purified nitrate reductases should be amenable for study with epr in order to determine the involvement of molybdenum in the reduction process. 

Plants usually obtain their nitrogen by absorption of nitrate or ammonium ions from the soil (symbiotic associations between higher plants and nitrogen fixing bacteria are of course exceptions to this). Ammonium ions may be utilized directly in the synthesis of amino acids (see p. 169), but nitrate must first be reduced to ammonia. This is accomplished in two stages the reduction of nitrate to nitrite followed by the reduction of nitrite to ammonia. The first step— nitrate reduction—is catalysed by the flavo-protein enzyme complex nitrate reductase (Fig. 5.12) which contains molybdenum and FAD (flavin adenine dinucleotide) as a prosthetic group. Reduced FMN... 

The enzyme contains two heme A molecules, two heme C molecules, one molybdenum atom, and five [Fe4S4] clusters in the molecule with molecular mass of 250 kDa. Molybdenum occurs as a complex with molybdopterin guanine dinucleotide (MGD) (Fukuoka et al 1987 Yoshino, 1994 Suzuki et al., 1997). The enzyme catalyzes the reduction of N. winogradskyi ferricytochrome c-550 and horse ferricytochrome c with nitrite, i.e it catalyzes the oxidation of nitrite with ferricytochromes c as the electron acceptor. The reaction is stimulated by Mn2+ and Ca2+. Although the enzyme catalyzes actively the oxidation of nitrite around pH 8, it shows also capability to catalyze the reduction of nitrate with ferrocytochrome c at pHs less than 6 the bacterium changes itself from a nitrifier to a denitrifieT at pHs less than 6. 

Nitrate Reductase.—Reduction of nitrate by molybdenum(v) complexes of the type [M0OCI3L] and [MoOClL 2l where L and L are neutral or anionic bidentate ligands, proceeds by rate-determining loss of Cl in DMF at 25 °C ... 

The reduction of various substrates (N2 RCN, RNC, CN% N03 ) by metalloenzymes with molybdenum-sulfur sites (nitnogenase, nitrate reductase) has prompted active studies of the chemistry and electrochemistry of complexes with Mo-S assemblies which could mediate or catalyse the reduction of these substrates. 

First, ferredoxin-nitrate reductase (EC 1.7.7.2) is a molybdenum-iron-sulfur protein that converts nitrate to nitrite while undergoing oxidation. Its structure is not yet well-defined. Then, ferridoxin-nitrite reductase (EC 1.7.7.1), an iron (as heme and at least one iron sulfur complex, vide infra) can continue the reduction to ammonia (NH3). 

Metallo-Flavoproteins. As was mentioned in the case of cytochrome reductase, enzymes are known that contain metal cofactors in addition to flavin. These are called metallo-flavoproteins. The presence of metals introduces complexity into the reaction, since the metals involved, iron, molybdenum, copper, and manganese, all exist in at least two valence states and can participate in oxidation-reduction reactions. The enzymes known to be metallo-flavoproteins include xanthine oxidase, aldehyde oxidase, nitrate reductase, succinic dehydrogenase, fatty acyl CoA dehydrogenases, hydrogenase, and cytochrome reductases. Before these are discussed in detail some physical properties of flavin will be presented. 

The organization of xanthine oxidase appears to be quite complex. There is evidence that various substrates are not bound at the same site, and that the primary reaction of different substrates may occur with various ones of the cofactors. The oxidation of purines and aldehydes is inhibited by pteridyl aldehyde and by cyanide, but these reagents do not affect the oxidation of DPNH. It is possible that these inhibitors influence substrate binding sites and primary electron transport, respectively, and that the oxidation of DPNH involves a different binding site and avoids the cyanide-sensitive electron transport mechanism, which may well involve iron. Xanthine oxidase, and probably all flavoproteins, require —SH groups, but a definite function for these groups cannot be ascribed at this time. Similarly, various factors influence the reactions with oxidants differentially. Cyanide inhibits cytochrome reduction, but not the reactions with 0 or dyes. Reduction of either cytochrome c or nitrate depends upon the presence of molybdenum. These observations... 

A corollary to the assumption that the enzymes cycle between Mo(VI) and Mo(IV) is that Mo(V), the valence state with which this chapter is primarily concerned, will appear in the systems only incidentally. If intramolecular electron transfer to and from the other centers of the enzymes is fast, then Mo(V) will appear as electrons are transferred to Mo(VI) or from Mo(IV) by these centers (Section 3). This hypothesis is capable of explaining the apparent stability of the nitrate complex of Mo(V) in nitrate reductase. If nitrate can undergo only two-electron reduction at the molybdenum center, then we must regard this signal-giving species itself not as an unexpectedly stable Michaelis complex, but as no more than a dead-end complex. 

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