Nitrate reductase subunits

There are four different classes of nitrate reductases (234). The nitrate reductases from D. desulfuricans show a strong homology to the a-subunit of the class of periplasmic respiratory nitrate reductases, and also to some of the enzymes that are included on the class of cytoplasmic assimilatory nitrate reductases. Because of this fact, a proposal was made for a new class of monomeric NAP, which contains the minimal arrangement of metal centers to perform nitrate reduction one [4Fe-4S] cluster and a Mo bound to two MGD. 

Fig. 6. Representative EPR spectra displayed by trinuclear and tetranucleEir iron-sulfur centers, (a) and (b) [3Fe-4S] + center in the NarH subunit of Escherichia coli nitrate reductase and the Ni-Fe hydrogenase fromD. gigas, respectively, (c) [4Fe-4S] + center in D. desulfuricans Norway ferredoxin I. (d) [4Fe-4S] center in Thiobacillus ferrooxidans ferredoxin. Experimental conditions temperature, 15 K microwave frequency, 9.330 GHz microwave power, (a) 100 mW, (b) 0.04 mW, (c) smd (d) 0.5 mW modulation amplitude (a), (c), (d) 0.5 mT, (b) 0.1 mT.

The latter authors demonstrate in their latest paper101) that bluelight (under physiological conditions stimulating conidiation and LIAC) will depress nitrate reductase activity and increase the activity of the smaller subunit of the enzyme complex in Neurospora. It is therefore suggested to be a key enzyme for physiological bluelight action. 

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. 

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. 

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. 

Blasco, F., lobbi, C., Giordano, G., Chippaux, M., and Bonnefoy, V., 1989, Nitrate reductase from Escherichia coli completion of the nucleotide sequence of the nar operon and reassessment of the role of the a and 5 subunits in iron binding and electron transfer. Mol. Gen. Genet. 218 249n256. 

Buc, J., Santini, C.-L., Blasco, F., Giordani, R., C rdenas, M. L., Chippaux, M., Comish-Bowden, A., and Giordano, G., 1995, Kinetic studies of a soluble oP complex of nitrate reductase A from Escherichia coli. Use of various (xP mutants with altered P subunits, Eur. J. Biochem. 234 766n772. 

The [2Fe 2S], [3Fe S], and [4Fe S] clusters that are found in simple Fe S proteins are also constituents of respiratory and photosynthetic electron transport chains. Multicluster Fe S enzymes such as hydrogenase, formate dehydrogenase, NADH dehydrogenase, and succinate dehydrogenase feed electrons into respiratory chains, while others such as nitrate reductase, fhmarate reductase, DMSO reductase, and HDR catalyze the terminal step in anaerobic electron transport chains that utihze nitrate, fumarate, DMSO, and the CoB S S CoM heterodisulfide as the respiratory oxidant. All comprise membrane anchor polypeptide(s) and soluble subunits on the membrane surface that mediate electron transfer to or from Mo cofactor (Moco), NiFe, Fe-S cluster or flavin active sites. Multiple Fe-S clusters define electron transport pathways between the active site and the electron donor or... 

Total mass. Per subunit. Structural data can be obtained from the Brookhaven Protein Data Bank (PDB) at http //www.rcsb.org/pdb. Other Mo-MPT enzyme PDB codes include Hydrogenophagapseudoflava CO dehydrogenase, IFFV Desulfovibrio desulfuricans aldehyde oxidoreductase, IDGJ Rhodobacter capsulatus DMSO reductase, 4DMR R. sphaeroides nitrate reductase (dissimilatory), lOGY D. 

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... 

NapB. NapB is a subunit of the heterodimeric periplasmic nitrate reductase (NapAB) and transfers electrons to the catalytic NapA molybdoprotein. Nap systems are found in a number of bacteria, iucluding enterobacteria, aerobic denitrifiers, and nonsulfur purple photo synthetic bacteria. Their physiological function is different in these groups of bacteria and includes redox balancing using nitrate as an electron sink to dispose of excess rednctant, aerobic denitrification, and nitrate scavenging in nitrate-limited environments. 

Dissimilatory nitrate reductase (periplasmic) catalytic subunit... 

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

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... 

In the presence of nitrate, formate dehydrogenase-N (FDH-N) and nitrate reductase are induced (8). As a result, the formate is consumed preferentially by FDH-N due to its inherent high affinity for formate versus FDH-H. The Km value for formate of FDH-N is 0.12 mM, while that of FDH-H is 26 mM. The FHL system is induced by the presence of formate, however, formate consumption by an FDH-N - nitrate reductase system depresses the expression level of the FHL system. Consequently, both expression of FHL and hydrogen production are repressed by the presence of nitrate in the medium. The removal of nitrate from waste water is not a practical process therefore a bacterial strain capable of hydrogen production even in the presence of nitrate is favorable. The subunits of nitrate reductase are encoded in the fdn operon. The a-subunit of nitrate reductase is encoded in the... 

The periplasmatic vanadium-containing nitrate reductase from P. isachenkovii has a molecular mass of 220 kDa (four subunits). The pterin cofactor is again absent. In media supplemented with vanadate and nitrate, vanadate is first reduced by a membrane-bound reductase using NADH as electron donor. This dissimilatory reduction was followed by nitrate consumption.I " ]... 

Enzymes of the sulfite oxidase family coordinate a single equivalent of the pterin cofactor with an MPT-Mo 02 core in its oxidized state (54, Figure 16), and usually an additional cysteine ligand, which is provided by the polypeptide. Members of this family catalyze the transfer of an oxygen atom either to or from the substrate. Among the members of this family are sulfite oxidase, sulfite dehydrogenase, assimilatory nitrate reductases, and the YedY protein, the catalytic subunit of a sulfite oxidase homologue in E. coli So far, all members of this family contain the MPT-form of Moco without an additional dinucleotide. 

The first step, conversion of N03" to N02, is catalyzed by a large and complex enzyme called nitrate reductase. Nitrate reductase is a multi-subunit enzyme with Mr of about 800 kD. It contains bound FAD, molybdenum, and a cytochrome called cytochrome 557 (which contains an Fe4S4 complex). Nitrate reductase carries out the following reaction ... 

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