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

Abstract

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU 1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant 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 noncoordinate residues, Asp 98 and His 255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp 98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His 255 residue hydrogen-bonded to Asp 98 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 K_m values for nitrite were greatly increased in the Asp 98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp 98 and His 255 are involved not only in the catalytic reaction but also in the substrate anchoring.