Adaptive enzyme nitrate reductase

Yamanaka and co-workers (364-366) have crystallized a cytochrome oxidase from P. aeruginosa which oxidizes Pseudomonas ferrocytochrome c-551. It is also capable of nitrite reduction with a turnover number of 4000 moles nitrite reduced under anaerobic conditions to nitric oxide per minute at 37°. It is an adaptive enzyme, nitrate being essential for its biosynthesis. The enzyme has a molecular weight of 120,000, with two subunits of equivalent molecular weight, 2 heme c and 2 heme d groups per mole (Fig. 38) (366a). Nitrite reductase activity is 94% inhibited by 8 X 10 M KCN, but only by CO. The lack of CO inhibition appears to be related to the fact that the enzyme has a greater affinity for nitrite than for carbon monoxide. 

That hydroxylamine might not be an obligatory intermediate, or occur as a free intermediate, in the reduction of nitrite to ammonia is suggested by the properties of nitrite reductases of Azotobacter chroococcum and Escherichia coli. The former is an adaptive enzyme, the formation of which requires nitrate or nitrite in the culture (31,2). It is FAD-depen-dent and presumably contains metals and p-mercuribenzoate inhibitable... 

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. 

Figure 12. (A) The amperometric response of (a) a polypyrrole-viologen-nitrate reductase electrode and (b) an identical electrode constructed without the enzyme, in response to injections (a) increasing the nitrate concentration by 3.5 pM, and (y) of buffer. (B) Calibration curves (inset smaller concentration range) for the response to nitrate of a polypyrrole-viologen-nitrate reductase electrode at —0.7 V vs. SCE. Adapted from Ref. [106a with permission.

Figure 4-12. Catalytic voltammetry of Paracoccus pantotrophus nitrate reductase (NarGH) adsorbed as a film on a PGE electrode at pH 6. (A) Increasing the electrode rotation rate from 0 to 3000 rpm removes the mass transport limitation of the catalytic response in 50 pM NO3 . (B) The enzyme s greater rate of chlorate reduction compared to nitrate reduction is reflected in greater distortion of the waveform through dispersion of sluggish interfacial electron transfer rates (see also Fig. 4-4C). Scan rate 10 mV s. Adapted from ref. 64. with permission.

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