Dissimilatory nitrate reductase

Alefounder, P. R., and Ferguson, S. J. (1980). The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitri-ficans. Biochem.J. 192, 231-240. 

Carlson, C. A., Ferguson, L. P., and Ingraham, J. L. (1982). Properties of dissimilatory nitrate reductase purified from the denitrifier Pseudomonas aeruginosa. J. Bacteriol. 151, 162-171. 

A number of other reductases and dehydrogenases, including dissimilatory nitrate reductases of E. coli and of denitrifying bacteria (Chapter 18), belong to the DMSO reductase family. Other members are reductases for biotin S-oxide,649 trimethylamine N-oxide, and polysulfides as well as formate dehydrogenases (Eq. 16-63), formylmethanofuran dehydrogenase (Fig. 15-22,... 

Bacterial assimilatory nitrate reductases have similar properties.86/86a In addition, many bacteria, including E. coli, are able to use nitrate ions as an oxidant for nitrate respiration under anaerobic conditions (Chapter 18). Tire dissimilatory nitrate reductases involved also contain molybdenum as well as Fe-S centers.85 Tire E. coli enzyme receives electrons from reduced quinones in the plasma membrane, passing them through cytochrome b, Fe-S centers, and molybdopterin to nitrate. The three-subunit aPy enzyme contains cytochrome b in one subunit, an Fe3S4 center as well as three Fe4S4 clusters in another, and the molybdenum cofactor in the third.87 Nitrate reduction to nitrite is also on the pathway of denitrification, which can lead to release of nitrogen as NO, NzO, and N2 by the action of dissimi-latory nitrite reductases. These enzymes873 have been discussed in Chapters 16 and 18. 

Generally, the assimilatory nitrate and nitrite reductases are soluble enzymes that utilize reduced pyridine nucleotides or reduced ferrodoxin. In contrast, the dissimilatory nitrate reductases are membrane-bound terminal electron acceptors that are tightly linked to cytochrome by pigments. Such complexes allow one or more sites of energy conservation (ATP generation) coupled with electron transport. 

E. coli uses nitrate as a terminal electron acceptor through a respiratory, dissimilatory nitrate reductase whose synthesis is induced when nitrate is provided, and which is repressed by oxygen. Nitrate reductase is discussed with other molybdoenzymes in Section 62.1.9, and catalyzes the reduction of nitrate to nitrite. The enzyme is isolated from the cytoplasmic membrane of E. coli, and contains three subunits (a, j8 and y) although the y-subunit may be absent in some preparations. The -y-subunit is a b-type cytochrome, and the a-subunit is reported to be the catalytic subunit. The enzyme contains a number of iron-sulfur clusters, including a HiPIP and at least two ferredoxins.1054,1437... 

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. 

The potentials found for nitrate reductases [96] vary with the role of the particular enzyme. Assimilatory nitrate reductase, found in plants, algae, and fungi, is involved in the first step in nitrogen assimilation and has a molybdenum center that operates at around 0 mV. Respiratory (dissimilatory) nitrate reductase, utilized by bacteria in energy yielding processes, has a molybdenum center that operates at around +200 mV [97,98],... 

Figure 7 The modus operandi of nitrate reductase (a) assimilatory nitrate reductase (plants, fungi, algae) (b) respiratory (dissimilatory) nitrate reductase (.Escherichia coli, Pseudomonas).

Other members of this family that have been structurally determined by X-ray diffraction include formate dehydrogenase (FDH), trimethylamine oxidase (TMAO), dissimilatory nitrate reductase(NAP), and most recently, arsenite oxidase (AsO). Only the distinctive points of their structures will be briefly described here. 

In a further development of this research (84), the reaction of [Mo(CO)2(S2C2R2)2] with the nucleophiles R Y- (R = Ph, z-Pr, or C6FsO, Y = O R = 2-Ad Y = O, S or Se) (see Fig. 10) was shown to form the corresponding des-oxo MoIV( YR )(S2C2Me2)21 complex. These complexes have considerable potential as structural analogues for the reduced forms of the catalytic centers of the DMSOR and the TMAOR (Y = O), the dissimilatory nitrate reductase (Y = S), and the formate dehydrogenase (Y = Se) (13). Thus, the spectroscopic and electrochemical properties and reactivity of these complexes will provide useful calibrations of the corresponding behavior of the catalytic centers of the MPT enzymes. 

L = OSer for DMSOR, TMAO reductase L = SCys for dissimilatory nitrate reductase L = SeCys for formate dehydrogenase... 

The enzymes are subdivided into three families based on structural and sequence comparisons (Figure 1, Table 1). Oxotransferases isolated from prokaryotes (see Prokaryote) belong to the DMSO reductase family. These enzymes include DMSO reductase, biotin X-oxide reductase, trimethylamine A-oxide reductase, dissimilatory nitrate reductase, formate... 

Enzymes in this family include DMSO reductase, biotin 5-oxide reductase, dissimilatory nitrate reductase, trimethylamine A-oxide reductase, and formate dehydrogenase they are found exclusively in bacteria and fungi and act as terminal respiratory reductases during anaerobic growth in the presence of their respective substrates. " DMSO reductases catalyze the reaction shown in equation (5) the water-soluble enzymes from the purple phototrophic bacteria R. capsula-tus and R. sphaeroides are among the simplest Mo-MPT enzymes, being relatively small (ca. 85 kDa), single subunit... 

Two types of dissimilatory nitrate reductases, localized in either the cytoplasmic membrane or the periplasm, are widespread among bacteria. The cytoplasmic membrane-bound nitrate reductase (Nar) catalyzes the first steps of denitrification (NOs" N02 NO N2O... 

Dissimilatory nitrate reductase (periplasmic) catalytic subunit... 

Dissimilatory nitrate reductase (membrane) catalytic a subunit Cytochrome c and d containing nitrite reductase (cdl-NiR)... 

In addition to N assimilation, nitrogen compounds in nature can also be used as electron sources or sinks, especially by prokaryotes (but not exclusively, see Zvyagil skaya et al., 1996). The enzymes that mediate these dissimilatory reactions have been studied extensively in the context of inputs and losses of fixed N in the marine N budget. Many of these measurements have been based on enzyme activities (e.g., dissimilatory nitrate reductase). Since dissimilatory reactions have been reviewed elsewhere in this volume (Chapter 5 by Ward and Chapter 6 by Devol, this volume), these wiU not be discussed further and we refer the reader to these chapters. 

The nitrate reductase from Haloferax denitrificans was purified to electrophoretic homogeneity aided by the ease with which the enzyme is solubilized from membranes and the stability of the reductase in solutions of low ionic strength [142]. The enzyme is composed of two subunits that resemble the a and (3 subunits of the dissimilatory nitrate reductases found in bacteria [143]. Dissimilatory nitrate reductases have a third subunit that contains a b-type cytochrome. No such subunit is detected in the nitrate reductase from H. denitrificans. However, this observation has no significance since this subunit is often lost during purification. The most striking property of the enzyme is its response to salt concentration, both when membrane-bound [144] and following purification [142]. Nitrate reduction is most active in the absence of added salt and the enzyme is stable for weeks on end in the absence of salt. Similar nitrate reductase activities occur in... 

ESR studies on the dissimilatory nitrate reductase are also available. Two signals arising from a high pH and a low pH species of Mo have been found in the enzyme from E. the low... 

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