Nitrate reductase catalytic activities

The idea that the same cofactor species operated in all Mo enzymes originated from a reconstitution assay. In this assay method, the isolated Moco from one enzyme, such as XO, is inserted into a cofactor-free mutant (Nit-1) of nitrate reductase from Neuraspora crassa, where it can reactivate or reconstitute normal nitrate reductase catalytic activity. It is now recognized that the Mo at the active site has many different coordination environments, as has been illustrated for the three Mo families in Fig. 1. In this context, the mutant nitrate reductase assay experiment is interpreted as involving some reprocessing of the inserted molybdenum cofactor from foreign enzymes to obtain the correct form of the cofactor for nitrate reductase catalysis. The Moco designation, if it is to be used, must refer to the family of sites present in Moco enzymes. 

Hyde, G.E. Campbell, W.H. (1990). High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH-nitrate, reductase and its comparison to human NADH-cytochrome b5 reductase. Biochemical and Biophysical Research Communications 168, 1285-91. 

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... 

Anderson, L.J., Richardson, D.J., and Butt, J.N. (2001) Catalytic protein film voltammetry from a respiratory nitrate reductase provides evidence for complex electrochemical modulation of enzyme activity. Biochemistry, 40, 11294-11307. 

In addition to the membrane-anchored enzymes that support anaerobic respiration, bacteria express a number of functionally, and structurally, distinct nitrate reductases related by the presence of an Mo[MGD]2 containing active site. PFV has demonstrated tunnel-diode behaviour from two of these the periplasmic Rhodobacter sphaeroides NapAB and the assimilatory Synechococcus elongatus NarB. In both cases preferential binding of nitrate to the Mo , over Mo , oxidation state provides an explanation for the catalytic voltammetry and this may prove to be a conserved feature of the catalytic cycle in these enzymes. 

Coelho et al have reported the eatalytic voltammetry of NapAB from Cupri-avidus necator In the absence of mediators, no nitrate reductase electrocatalysis was found but a catalytic cathodic wave was observed upon addition of H2O2 (peroxidase activity). When MV was used as a mediator a catalytic nitrate reduetion eurrent was observed. 

SECM has been employed to detect the catalytic activity of a variety of immobilized enzymes or enzyme labels including glucose oxidase [27, 58,122-124], urease [44, 125], nitrate reductase [126], diaphorase [72,127,128], horse radish peroxidase [129], NADH-cytochrome C reductase [28], and alkaline phosphatase [54]. When the enzyme kinetics are too slow for feedback measurements, genera tion/collection mode can be used. In this section, we discuss kinetic investigations and analytical appKcations, but leave patterning of activity to Sect. 3.3.4.4 on microfabrication. 

Figure 10 shows the catalytic voltammetry of a film of nitrate reductase, a membrane-bound enzyme that contains a Mo active site and Fe-S clusters [46]. The enzyme is adsorbed on a PGF electrode ... 

However, some of these structures show unusual features and a crowded active site. EXAFS studies of Rhodobacter capsulatus DM SO reductase show the expected four Mo-S ligands and one Mo-O bond arising from a serine residue plus one Mo=0 in the reduced Mo (IV) state of the active site. This oxo ligand is removed upon oxidation of the metal ion to Mo(VI). In the oxidized structme, an aquo ligand is postulated to coordinate the molybdenum ion (Fig. 11.17c) [126-128]. An equivalent restdt has been reported for BSO that catalyzes the reduction of D-biotin D-sulfoxide to D-biotin [129]. A similar overall catalytic mechanism is expected for nitrate reductases (Fig. 11.18), which catalyze the following reaction ... 

Elliott, S.J., Hoke, K.R., Heffron, K., Palak, M., Rothery, R.A., Weiner, J.H., Armstrong, FA., 2004. Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli how nitrate reduction and inhibition depend on the oxidation state of the active site. Biochemistry 43, 799-807. 

If the substrate ion is bound to the metal and the metal accepts or donates electrons while its ligand (probably terminal sulfur or oxygen) does the same with protons, then we still have to inquire about further problems. We have no information about the metal orbital in which bonding of the substrate ion takes place (but see Garner et al, 191 A). For nitrate reductase, Vincent and Bray (1978) concluded that the low-pH form of the enzyme is the one which is catalytically active, since the pH for interconversion of the EPR-detectable forms appeared to coincide with the pK governing activity of the enzyme. 

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