Nitrite reductase light

With the structure of ascorbate oxidase in hand, a new structurally based alignment of the sequences of ascorbate oxidase, laccase, and ceruloplasmin has been performed (Messerschmidt and Huber, 1990). In brief, while gene triplication for ceruloplasmin is still revelant, its sequence can be further subdivided into two domains per unit of triplicated sequence, or six domains in total. Each of these sequences bears some resemblance to each of the three domains of ascorbate oxidase, as does each of the two domains in laccase. The coppers of the trinuclear site of ceruloplasmin then are predicted to be bound between domains 1 and 6, with a type I site also lying in both domains 6 and 4 (see Huber, 1990). The relative orientation of each of these domains is not predicted by this alignment, but it turns out that the structure of nitrite reductase may shed some light on this (see Section V,C). 

Absorption spectra of oxidized and dithionite-reduced nitrite reductase from Pseudomonas aeruginosa. Solid line, oxidized enzyme dashed line, dithionite-reduced enzyme, in 40 mM potassium phosphate buffer, pH 6.9. The enzyme concentration was 8 fiM and the spectra were recorded at room temperature (20°C) under N in a Thunberg cuvette with a light-path of 1 cm. From Barber etal. (1976). 

The uptake of nitrate and subsequent conversion to reduced nitrogen in cells requires a change of five in the oxidation state and proceeds in a stepwise fashion. The initial reduction takes place via the nitrate/nitrite reductase enzyme present in phytoplankton and requires large amounts of the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and of adenosine triphosphate (ATP) and thus of harvested light energy from photosystem II. Both the nitrogenase enzyme and the nitrate reductase enzyme require iron as a cofactor and are thus sensitive to iron availability. 

How the mitochondria can enhance nitrite reduction in leaves in the dark is not known as the bulk of the experimental evidence indicates that nitrite reductase is localized in the chloroplast. The reductant is ferredoxin generated by light or by the NADPH-ferredoxin reductase and an unknown NADPH-generating system in the dark. The inhibition of nitrite reduction by DNP and arsenate (Kessler and Bucker, 1960 Kessler, 1964 Hattori and Myers, 1966) has been interpreted to implicate a high energy phosphate requirement in nitrite reduction. One can speculate that the role of ATP could be to form an active nitrite required for reduction or to facilitate the entry of nitrite into the chloroplast. 

Nitrite reductase is regulated by nitrate and light in a manner similar to NR. Nitrite reductase activity increases in response to nitrate or nitrite (Ingle et al. 

The latter inhibition is reversed by light. Urea inactivation-reaclivation studies showed parallel loss and recovery of nitrite and hyroxylamine reductase activities, and nitrite was shown to inhibit hydroxylamine reduction. These results have suggested that the enzyme has a common binding site for nitrite and hydroxylamine. The absorption spectra of the A. fischeri enzyme (oxidized, reduced, and reduced plus nitrite or hydroxylamine) are shown in Fig. 39. 

Treatment of E. coli sulfite reductase with p-mercuriphenyl sulfonate results in the specific release of FMN from the enzyme (390). FMN-depleted sulfite reductase can be prepared also by photodestruction of FMN. The enzyme-FMN dissociation constant is 10 nAf at 25°, and light irradiation can deplete the enzyme of FMN by destroying the released flavin. These treatments do not lead to removal or destruction of other components of the enzyme. The FMN-depleted enzyme is no longer capable of NADPH-dependent reduction of sulfite, nitrite, hydroxylamine. 

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