The effects of site-directed mutation on spinach nitrite reductase
Tripathy, Jatindra Nath
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Ferredoxin-nitrite oxidoreductase (NiR) catalyzes the six electron reduction of nitrite to ammonia, with reduced ferredoxin (Fd) serving as the electron donor. Chemical modification studies of spinach chloroplast nitrite reductase suggested that tryptophan at 92 (W92) position in NiR is located near the Fd-binding site of the enzyme and plays an important role in the NiR-catalyzed reaction. W92 is absolutely conserved in ferredoxin-dependent NiR's from higher plants, algae, and photosynthetic bacteria, consistent with an essential role for this tryptophan. The ferredoxin dependent nitrite reductase from spinach was expressed in Escherichia coli using the pET30b expression system. Coexpression of bacterial cysG gene with pET30b-NiR or inclusion of 6-aminolevulinic acid, a heme precursor, in the growth medium, resulted in increased amount of pure protein per liter of culture and greater specific activity. W92 was mutated to alanine (A), valine (V), and tyrosine (Y) in order to further test the role of W92 in the NiR-catalyzed reaction. The wild-type NiR and its W92 variants, all were purified to homogeneity, and all exhibited essentially identical visible-region spectra. The Fd-linked specific activity of the W92A variant was only 20% of that observed with the wild-type NiR. The W92V variant exhibited a specific activity 30% of the wild-type level and the W92Y variant exhibited a specific activity 37% of the wild-type level. A very similar pattern was observed for the methyl viologen-linked specific activity of these NiR variants (methyl viologen is a non-physiological electron donor). The binding constant (Kd) for nitrite was 15 (±5) µM for the wild-type NiR and all of its W92 variants. The Kd for Fd was 1 (±0.2) µM for wild-type NiR and ranged from 2 µM to 7 µM for the three W92 variants. Km values for nitrite and ferredoxin were measured for wild-type NiR and its W92 variants, allowing calculation of the catalytic efficiencies. The W92A variant displayed 80% lower catalytic efficiency with respect to nitrite, and 40% lower catalytic efficiency with respect to ferredoxin, compared to the wild-type enzyme. The catalytic efficiencies, with respect to nitrite, for W92V and W92Y variants decreased by approximately 25% compared to the wild-type enzyme. However, the catalytic efficiencies, with respect to ferredoxin, for W92V and W92Y variants were greater than that of wild-type NiR.