Characterization of Nitrite Reductase from a Denitrifier, Alcaligenes Sp. NCIB 11015. A Novel Copper Protein1

Abstract

A copper-containing dissimilatory nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] was purified from a denitrifier, Alcaligenes sp. NCIB 11015, by ion-exchange chromatography on CM-cellulose, gel filtration on Sephadex G-150, and adsorption on hydroxyapatite. The preparation was homogeneous by SDS-polyacrylamide gel electrophoretic criteria, and its enzymatic activity increased considerably by freezing (at -20 degrees C) and thawing. The enzyme consists of two subunits with a molecular weight of 37,000, and the isoelectric point and redox potential are 8.4 and +260 mV (pH 7.2), respectively. The EPR spectrum and copper analysis clearly indicated that the enzyme contains two type I copper atoms per molecule but no other types of copper. This is the first blue copper protein that exhibits catalytic activity despite possessing only type I copper.