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

Abstract

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba(3) and caa(3)) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) under reducing anaerobic conditions. The rate of NO consumption and N(2)O 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 ba(3) x min and 32 +/- 8 mol NO/mol caa(3) x min at [NO] approximately 50 microM and 20 degrees 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 ba(3) 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 cbb(3) terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

Figure 1

NO reductase activity of ba₃ and caa₃. (Upper) ba₃ (1.5 μM) was degassed, reduced by 10 mM ascorbate, 100 μM TMPD, 1 μM cytochrome c₅₅₂, and, after addition of 60 μM NO, mixed in the stopped flow with 104 μM deoxy-Hb at the following incubation times: 0.9, 1.8, 3.4, 7.5, 10.8, 16, and 26.8 min. Difference spectra, collected at 100 ms after mixing, show NO disappearing from solution. Baseline, deoxy-Hb in the presence of ba₃ and reductants before addition of NO; dashed spectrum, 104 μM NO-saturated Hb. (Lower) NO consumption as a function of time after addition of NO (60 μM final concentration) to: 10 mM ascorbate + 200 μM ruthenium hexamine (●) or 10 mM ascorbate + 10 μM TMPD (*); 5 μM (aa₃) beef heart oxidase reduced by 10 mM ascorbate and 200 μM ruthenium hexamine (○); 1.5 μM ba₃ reduced by 10 mM ascorbate + 200 μM ruthenium hexamine (□) or by 10 mM ascorbate + 10 μM TMPD + 1 μM cytochrome c₅₅₂ (■); 0.3 μM caa₃ reduced by 10 mM ascorbate and 10 μM TMPD (▴); or 100 μM TMPD (▵). Contrary to beef heart oxidase, both oxidases from T. thermophilus catalyze NO consumption significantly faster than reductants alone.

Figure 2

Amperometric measurements: NO binding and catalytic consumption. (Upper) Three aliquots of NO (final concentration 15 μM) were added to degassed buffer containing 2 mM ascorbate and 0.1 mM TMPD before addition of reduced oxidases [see arrows; final concentrations: 2 μM ba₃ (a); 0.2 μM caa₃ (b); 2 μM aa₃ (c)]. NO decay before addition of each enzyme is attributed to the reaction with the reductants. Upon addition of ba₃ and caa₃, a catalytic NO consumption (after initial binding seen as a vertical drop) was observed, whereas with beef heart oxidase only stoichiometric binding was seen. (Lower) Stoichiometry of NO binding to reduced ba₃ (▵) and beef heart oxidase (□) as a function of NO concentration. For both enzymes, NO binds maximally with a 1:1 stoichiometry in the NO concentration range explored.

Figure 3

N₂O formation by the ba₃ oxidase. The rate of N₂O formation, identified by head-space analysis and gas chromatography, is shown at different enzyme concentrations in acetate buffer (pH 4.8) and ascorbate-reduced PMS. The low pH is necessary to suppress nonenzymatic N₂O formation, which increases with pH and may become significant unless specific countermeasures are being taken (25). The graph combines data of duplicate experiments, which were fitted by linear regression at 4.94 (■), 2.47 (⧫), and 1.24 (▵) μM ba₃, respectively; ●, enzyme boiled in 1% SDS for 5 min; □, assay mixture without the enzyme.

Figure 4

Time course of the oxidation of reduced ba₃ by NO. ba₃ (5 μM) was reduced anaerobically by 1 mM ascorbate in the presence of 1 mM CO, and the resulting CO-bound fully reduced enzyme was mixed with 250 μM NO at 20°C. (Upper) Absorption changes collected from 10 ms to 80 s after mixing. (Lower) Optical components deconvoluted by fitting the first two V columns of the singular value decomposition output (scaled by their relative singular values) to two exponential decays (Inset: best fit). The faster phase (solid spectrum) corresponds to CO displacement by NO, proceeding at k₁ = 9 s⁻¹, whereas cytochrome b oxidation (dashed spectrum) occurs at a much slower rate, k₂ = 0.04 s⁻¹, which is consistent with the turnover number measured for NO consumption by ba₃.