Evidence that the Type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase

Abstract

Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A. cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer. The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra. These results indicate that an earlier report that the A. cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO 2 − is reduced. Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.