Nitrate reductase mechanism

For nitrate reductase, evidence on the role of molybdenum in the catalytic mechanism of the enzyme from Neurospora was first presented in 1954 by Nicholas and Nason (21) and the position seems to have changed relatively little since then. The original conclusion (23) was that molybdenum functions as an electron carrier in the sequence ... 

Blom was the first to demonstrate, in 1928, the formation of HA by an unknown mixture of bacteria which utilized nitrate as their sole nitrogen source to produce ammonia , an observation substantiated by Lindsey and Rhines who generalized this reaction to a diverse set of microorganisms capable of producing NH3 by reduction of both nitrites and nitrates. The mechanism of the 6-electron reduction of nitrite to ammonia (i.e. conversion of the [N + 02] species to by bacterial cytochrome c nitrite reductase... 

For the one-electron reduction of nitrite to NO by nitrate reductase, an O atom transfer mechanism is unlikely, inasmuch as O atom transfer is inherently a two-electron mechanism. 

Nitrate reductase from Chlorella, an assimilatory enzyme, is a homotetramer of molecular weight 360 000 and contains one each of Mo, heme and FAD per subunit. The nitrate reductase from E. coli is a dissimilatory enzyme. EXAFS data are available on the molybdenum sites in both enzymes (Table 24).1050 The environment of the molybdenum in the assimilatory enzyme is similar to that found for sulfite oxidase, with at least two sulfur ligands near the molybdenum and a shuttle between monoxo and dioxo forms with redox change in the enzyme. This allows a similar mechanism to be put forward for the assimilatory nitrate reductase,1051 shown in equation (57), where an oxo group is transferred from nitrate to MoIV with production of nitrite and MoVI. 

Both assimilatory and dissimilatory nitrate reductases are molybdoenzymes, which bind nitrate at the molybdenum. EXAFS studies1050 have shown that there are structural differences between the assimilatory nitrate reductase from Chlorella vulgaris and the dissimilatory enzyme from E. coli. The Chlorella enzyme strongly resembles sulfite oxidase1050,1053 and shuttles between mon-and di-oxo forms, suggesting an oxo-transfer mechanism for reduction of nitrate. This does not appear to be the case for the E. coli enzyme, for which an oxo-transfer mechanism seems to be unlikely. The E. coli enzyme probably involves an electron transfer and protonation mechanism for the reduction of nitrate.1056 It is noteworthy that the EXAFS study on the E. coli nitrate reductase showed a long-distance interaction with what could be an electron-transfer subunit. 

Investigation of the mechanism of nitrate reduction by Desulfovibrio desulfuricans nitrate reductase at various levels of theory has confirmed the utility of the orbital-free embedding method in the description of enzymatic processes.379... 

Given the notion of microscopic reversibility, and the similarity in the active sites of sulfite oxidase and nitrate reductase (assimilatory), determining the mechanism of action of sulfite oxidase impacts upon our understanding of reductases in the (MPT)Mo(0)2 family. A key issue in the mechanism of sulfite oxidase is whether substrate binds to the metal center during the catalytic cycle. Substrate (or product) binding to the molybdenum center, as proposed for the catalytic... 

Similar mechanisms operate in the action of nitrate reductase and nitrite reductase. Both of these substances are produced from ammonia by oxidation. Plants and soil bacteria can reduce these compounds to provide ammonia for metabolism. The common agricultural fertilizer ammonium nitrate, NH4NO3, provides reduced nitrogen for plant growth directly, and by providing a substrate for nitrate reduction. NADH or NADPH is the electron donor for nitrate reductase, depending on the organism. 

The enzymatic mechanisms of nitrate bioactivation have long been the subject of debate. The glutathione-S-transferase and cytochrome P-450 systems were thought to be involved [8], while recently a nitrate reductase that specifically catalyzes the formation of 1,2-glyceryl dinitrate from glyceryl trinitrate was purified and identified as a mitochondrial aldehyde dehydrogenase [46]. Interestingly, this... 

Plant use of iron depends on the plant s ability to respond chemically to iron stress. This response causes the roots to release H+ and deduct ants, to reduce Fe3+, and to accumulate citrate, making iron available to the plant. Reduction sites are principally in the young lateral roots. Azide, arsenate, zinc, copper, and chelating agents may interfere with use of iron. Chemical reactions induced by iron stress affect nitrate reductase activity, use of iron from Fe3+ phosphate and Fe3+ chelate, and tolerance of plants to heavy metals. The iron stress-response mechanism is adaptive and genetically controlled, making it possible to tailor plants to grow under conditions of iron stress. 

The coupled proton-electron transfer mechanism can also be applied to the molybdenum reductases. For nitrate reductase, a scheme such as Reaction 20 is possible. A Mo (IV)-Mo (VI) couple is used to illustrate this, and while such a couple is viable for some nitrate reductases, the Mo(II)-Mo(IV) or the Mo(III)-Mo(V) couple could also be accommodated... 

The above schemes work reasonably well for certain enzyme reactions, especially for substrates where oxygen addition/loss occurs at a main group element (e.g., N, S, Se, Cl, see Table I). In addition to SO and nitrate reductase, key examples are DMSOR, trimethylamine oxide reductase, chlorate reductase, and selenate reductase. In the case of enzymes catalyzing C-based redox reactions of organic molecules, notably XDH and aldehyde oxidase, a direct OAT step is unlikely and is replaced by mechanistic steps typical of hydro-xylation (2). The essential features of the mechanism are shown in Fig. 10 for xanthine dehydrogenase/oxidase. 

The molybdenum-containing oxidoreductases that catalyze Eq. (1) have been variously termed molybdenum hydroxylases (6), oxotransferases (7), and oxo-type molybdenum enzymes (8). Molybdenum hydroxylase aptly describes the conversion of xanthine to uric acid, but the name seems less appropriate for the reactions catalyzed by sulfite oxidase and nitrate reductase oxotransferase implies that the function of these enzymes is to transfer oxo groups, even though relatively little is known about their actual mechanism of action and the name oxo-type molybdenum enzyme recognizes both the apparent oxo transfer chemistry of Eq. (1) and the fact that the molybdenum atom in each of these enzymes contains at least one terminal oxo group. In this chapter, we shall refer to these enzymes as pterin-containing molybdenum enzymes because a 6-substituted pterin appears to be a common chemical feature of all of the enzymes. 

As mentioned previously, Prochlorococcus is the dominant phytoplankton group in the North Pacific trades biome. Recently, the fuU genome sequences of several representative Prochlorococcus ecotypes have been pubhshed (Dufresne et al, 2003 Rocap et al., 2003). It is important to point out that none of the three genomes sequenced contain nitrate reductase, the enzyme responsible for the reduction of NOs to N02, the hypothesized mechanism for the existence of the PNM layer. This is not to say that Prochlorococcus does not contribute to the PNM, rather that we have no evidence to date that they can utilize NOs . However, recent results suggest that a yet-to-be-isolated Prochlorococcus ecotype may contain nitrate reductase (Casey et al., 2007). Furthermore, the deep living/dark-adapted ecotype of Prochlorococcus, as weU as other microbes, can utihze N02 as a source of N for biosynthesis so the net effect of phytoplankton/microbe metabolism would be to erode, not to produce or sustain, the PNM. 

Molecular mechanisms of nitrate accumulation depend not only on the nitrate reductase system, but also on the ability of roots to take from the soil, nitrate or ammonium ions, and on the plant s capacity for their conversion by assimilation processes to higher products. Besides this, the assimilation depends on the ability of a given genotype to transport substances necessary for the synthesis. It was shown that genotype differences of the nitrate reductase level do not depend on the nitrate content in tissues [25]. Nitrates are accumulated in plant organisms at high concentrations when aU the nitrogen accepted cannot be utilized for the production of amino acids and for subsequent protein synthesis [26]. This occurs when the plant, in the course of its metabolism, is unable to reduce the accepted nitrates into the assimilable ammonia form. 

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