Nitrite enzymatic reduction

Finally, the methemoglobin reduction test may be briefly mentioned. The test has been developed for the detection of primaquine sensitivity and depends on the function of the G-6-PDH system. Its principle consists in the oxidation of Hb to MHb by sodium nitrite and the subsequent enzymatic reduction to Hb in presence of methylene blue. The activity of this system can be followed easily by observation of alterations in color after an incubation period or by means of MHb determinations before and after this period (B18). 

Finally we note that enzymatic reduction of N02 and N03 is important in the natural nitrogen cycle. Copper-containing reductases are known and, as models, copper(I) nitrite complexes with triazacyclononane ligands have been shown to evolve NO.54... 

Indeed, it has been reported by Arciero et al. (1991) that the enzymatic reduction rate of ferricytochrome c-554 with hydroxylamine is 10 times as fast as that of the ferricytochrome with NOH. Moreover, it has been confirmed by Andersson and Hooper (1983) that the enzymatic oxidation of hydroxylamine to nitrite proceeds in two steps as mentioned above. 

In the processes of the enzymatic oxidation of nitrite, the reduction by nitrite of cytochrome c-550 hardly occurs because EmJ,0 (midpoint redox potential) of nitrite/ nitrate system (+0.40 V) is higher than that of cytochrome c-550 (+0.28 V). The reduction step of molecular oxygen catalyzed by cytochrome c oxidase proceeds rapidly, because EmJ,0 of H20/02 system is +0.82 V and is very much higher than that of cytochrome c-550. Therefore, in the bacterium, the electron flow from nitrite... 

ISO. 2000. Determination of nitrate and nitrite contents—Method by enzymatic reduction and molecular absorption spectrometry after Griess reaction. Ref. No. ISP/TC 34/SC5 N546. Delft, The Netherlands International Organization for Standardization. 

Bacteria have been Implicated in the formation of N-nitroso compounds under a wide variety of conditions representing both vitro and vivo situations Mechanisms of participation and/or catalysis Include a) decrease of the pH of the system, b) reduction of nitrate to nitrite, c) adsorption of amine onto the cell surface or cytoplasmic membrane, d) actual enzymatic formation. The literature of the field will be reviewed and experimental evidence which tests the above mechanisms will be presented ... 

Since HA is unstable in vivo , and is known to rapidly associate with the heme part of heme proteins , and possibly also with a variety of biological oxidants, such as the superoxide anion that is produced by many mammalian cells, it is difficult to demonstrate its accumulation in vivo. Already in 1932 Lindsey and Rhines discussed some analytical difficulties in the detection of HA, since when added externally, it disappeared rapidly from bacterial cultures this led to the conclusion that even if it is produced as an intermediate, its consumption is too fast to allow the accumulation of sufficient quantities for analytical demonstration. Compelling indirect evidence for the presence of HA as an intermediate in the enzymatically catalyzed reduction of nitrite (N02 ) to NH3 was provided by Einsle and colleagues , who characterized the crystal structure of the complex obtained by soaking cytochrome c-nitrite reductase with NH20H. ... 

In contrast to fresh muscle, meat has low levels of NAD (Madhavi and Carpenter, 1993). Thus, NAD-dependent enzymatic pathways for NOMb formation ate relatively unimportant in meat curing. In commercial practice, nitrite is reduced to NO by nonenzymatic means, including use of reductants such as ascorbate and erythorbate. Although meat has sufficient reducing ability to obtain a slow conversion of nitrite to NO, ascorbate or its isomer, erythorbate, is commonly added to curing brines or sausage emulsions to obtain faster NO production and thus a more rapid development of cured meat color. Care must be taken... 

In summary, a considerable body of enzymatic, genetic, and analytical data supports the view that the major, if not sole, pathway of denitrification involves NO as an obligatory intermediate and requires the action of nitric oxide reductase. On the other hand, the ability of nitrite to modify nitrosyl transfer ratios and the N isotope fractionation factor during its reduction, are consistent with the reductive scheme of Averill and Tiedje (1982). It was suggested (Goretski... 

Table III also shows the values of the equilibrium constants, KVAp for the conversion of iron nitrosyl complexes into the corresponding nitro derivatives. Keq decreases downwards, meaning that the conversions are obtained at a lower pH for the complexes at the top of the table. Thus, NP can be fully converted into the nitro complex only at pHs greater than 10. The NO+ N02 conversion, together with the release of N02 from the coordination sphere, are key features in some enzymatic reactions leading to oxidation of nitrogen hydrides to nitrite (14). The above conversion and release must occur under physiological conditions with the hydroxylaminoreductase enzyme (HAO), in which the substrate is seemingly oxidized through two electron paths involving HNO and NO+ as intermediates. Evidently, the mechanistic requirements are closely related to the structure of the heme sites in HAO (69). No direct evidence of bound nitrite intermediates has been reported, however, and this was also the case for the reductive nitrosylation processes associated with ferri-heme chemistry (Fig. 4) (25).

Biocatalytic synthetic reactions also include carbon dioxide fixation with the production of methanol in artificial multi-enzyme systems [188]. Formate dehydrogenase (FDH, EC 1.2.1.2) can catalyze the reduction of carbon dioxide to formate, and methanol dehydrogenase (MDH, EC 1.1.99.8) can catalyze the reduction of formate to methanol. Both of these enzymes require NAD+-NADE1 cofactor, and in the presence of the reduced dimethyl viologen mediator (MV+), they can drive a sequence of enzymatic reactions. The cascade of biocatalytic reactions results in the reduction of CO2 to formate catalyzed by FDEI followed by the reduction of formate to methanol catalyzed by MDH. A more complex system composed of immobilized cells of Parococcus denitrificans has been demonstrated for the reduction of nitrate and nitrite [189]. 

In all photoautotrophs, reduction of NOj" to NH4 is achieved in two distinct enzymatic steps (Campbell, 2001). First, assimilatory nitrate reductase (NR) catalyzes the two electron reduction from NOj" to NO2. NR is a large soluble cytoplasmic enzyme with FAD (flavin adinine dinucleotide), an iron-containing cytochrome and molybdopterin prosthetic groups, and requires NADH and/or NADPH as an electron donor (Guerrero et al, 1981). Functional NR is in the form of a homodimer and therefore requires two atoms of iron per enzyme. Following transport into the chloroplast, NO2 undergoes a 6 e reduction to NH4 via assimilatory nitrite reductase (NiR). NiR, a soluble chloroplastic enzyme, contains five iron atoms per active enzyme molecule, and requires photosynthetically reduced ferredoxin as an electron donor (Guerrero et al., 1981). 

Like TNT, 2,4,6-trinitrophenol (picric acid, PA) is also degraded through reduction. The hydride Meisenheimer complex is recognized as a key intermediate of denitration for the microbial degradation of PA [104,105], Lenke and Knackmuss [106] described an initial reduction of PA via the intermediate formation of a hydride-Meisenheimer complex to produce 2,4-dinitrophenol by the enzymatic elimination of nitrite. Under aerobic conditions, the mono- or dinitrophenol can then be oxygenated with subsequent ring cleavage. 

One possible rationale for these observations is related to the chemical properties of the low-spin heme-di active site, to which the enzymatic reaction product, NO, is assumed to bind with high affinity, thereby inhibiting its activity. A way to minimize product inhibition is by limiting nitrite oxidation of [Fe ]heme-oxidized heme-c in the same subunit that will prevent rapid, intramolecular reduction of the heme di Fe(lll) that has just been formed. The species that fulfill this requirement are 3, 5, and 7 (9 can react in two ways, only one of which fits the requirements). The observed pattern of intramolecular rate constants supports fliis reactivity by favoring species 5, but complete control would also have to include maintaining the enzyme at a low steady-state level of reduction so that species 8, 9, and 10 are not produced. 

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