Fluorescence lifetime analysis of nitrite reductase from A. xylosoxidans at the single molecule level reveals enzyme mechanism
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The blue copper-containing nitrite reductase from Alcaligenes xylosoxidans: cloning of the nirA gene and characterization of the recombinant enzyme.
The nirA gene encoding the blue dissimilatory nitrite reductase from Alcaligenes xylosoxidans has been cloned and sequenced. To our knowledge, this is the first report of the characterization of a gene encoding a blue copper-containing nitrite reductase. The deduced amino acid sequence exhibits a high degree of similarity to other copper-containing nitrite reductases from various bacterial sour...
متن کاملpH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans.
The first quantitative characterization of the interaction of NO2(-) with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit inte...
متن کاملThe enzyme mechanism of nitrite reductase studied at single-molecule level.
A generic method is described for the fluorescence "readout" of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluoresce...
متن کاملPurification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres.
Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme h...
متن کاملX-ray structure of a blue copper nitrite reductase at high pH and in copper-free form at 1.9 A resolution.
Copper-containing nitrite reductases possess a trimeric structure where the catalytic Cu site, located at the monomer-monomer interface, resembles the catalytic sites of a number of Zn enzymes. Nitrite reductase from Alcaligenes xylosoxidans has optimum activity at pH 5.2 which decreases to a negligible level at pH 8. The structure of this nitrite reductase has previously been determined at pH ...
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