Nitric oxide reductase of Achromobacter cycloclastes

Abstract

Nitric oxide reductase from Achromobacter cycloclastes was solubilized with dodecyl maltoside from membranes and substantially purified by hydroxyapatite column chromatography. Preparations of enzyme had purities estimated to be 65–72% and specific activities of about 3 μmol NO/min per mg protein when measured at 30°C and pH 5.5 using ascorbate/phenazine methosulfate as the reducing system. Preparations lacked nitrite reductase activity. The enzyme consists of two peptides of approx. 38 and 17.5 kDa, and is identified as a cytochrome bc complex for which the mol ratio of cytochrome c to cytochrome b is about 1:1. A heme c containing band of approx. 55 kDa would appear to be a 1:1 complex or compound of the 38 and 17.5 kDa peptides. These and other observations suggest that A. cycloclastes possesses a nitric oxide reductase similar to that previously purified from Pseudomonas stutzeri and Paracoccus denitrificans. In contrast to the cytochrome cd 1-type nitrite reductase of the latter two bacteria, the nitrite reductase of A. cycloclastes is a copper protein. This is the first reported characterization of nitric oxide reductase from a denitrifier with a Cu-containing nitrite reductase, and provides further evidence that the denitrification pathway in such bacteria requires nitric oxide reductase and proceeds by way of NO as an intermediate.