Functional Analysis of Conserved Aspartate and Histidine Residues Located Around the Type 2 Copper Site of Copper-Containing Nitri Reductase

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Abstract
A heterologous expression system of the blue coppeiv-eontaining nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombi-nant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native ArgNIR were retained in the copper-reconstituted recombi-nant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved nonco-ordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NTR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4–2% of those of the recombinant enzyme, and the apparent Km values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring

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