The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: Evolutionary implications

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba₃ and caa₃) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N₂O) under reducing anaerobic conditions. The rate of NO consumption and N₂O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 ± 0.7 mol NO/mol ba₃ x min and 32 ± 8 mol NO/mol caa₃ x min at [NO] ≃ 50 μM and 20°C) that, though considerably lower than that of bona fide NO reductases (300-4,500 mol NO/mol enzyme x min), is definitely significant. We also show that for ba₃ oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar Cu(B)/⁺ coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb₃ terminal oxidases. Our findings represent functional evidence in support of this hypothesis.