NITRIC-OXIDE REDUCTASE OF ACHROMOBACTER-CYCLOCLASTES

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 mumol NO/min per mg protein when measured at 30-degrees-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 cd1-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.