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 caa3) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N 2O) under reducing anaerobic conditions. The rate of NO consumption and N 2O 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 NO͞mol ba 3 ؋ min and 32 ؎ 8 mol NO͞mol caa 3 ؋ min at [NO] Ϸ 50 M and 20°C) that, though considerably lower than that of bona fide NO reductases (300 -4,500 mol NO͞mol enzyme ؋ 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. Heme-copper terminal oxidases and bacterial NO reductases (NOR) were suggested to have originated during evolution from a common ancestor (1-3). The common phylogeny was proposed because of structural similarities between these enzymes (see ref. 4 for a review), notably in the large catalytic subunit, which displays significant sequence homology and conservation of crucial residues (including the six metal-binding histidines). The topology of the catalytic subunit of NOR (NorB) is predicted to comprise 12 transmembrane helices, as shown for subunit I of heme-copper oxidases (5, 6). Finally, the active site is, in both cases, a bimetallic center, consisting of a heme-iron and a second metal, which is Cu in oxidases and Fe in NOR (7,8).On the basis of these structural similarities, it was presumed that the mechanisms of O 2 and NO reduction may share common features and, possibly, that O 2 and NO may be used as alternative substrates by both enzyme families. The mechanism of NO reduction by NOR is, at present, largely hypothetical, which makes any comparison with the mechanism of O 2 reduction by oxidases difficult. It is interesting, however, that a bacterial NOR with O 2 reductase activity was found in Paracoccus denitrificans ATCC 35512 (9); in contrast, there is no unequivocal experimental evidence in support of the hypothesis that heme-copper oxidases catalyze the reduction of NO to N 2 O (2NO ϩ 2e Ϫ ϩ 2H ϩ 3 N 2 O ϩ H 2 O). Brudwig et al. (10) reported that beef heart cytochrome c oxidase enhances (by a factor of 2) the reduction of NO by ascorbate and N,N,NЈ,NЈ-tetramethyl-p-phenylenediamine (TMPD), but on a time...

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

“…Evidence for only one NO steadily and with high affinity bound to the active site was found, in a configuration similar to that for the aa 3 1-NO case, in agreement with the finding that a second NO would lead to NO reduction (10). In ba 3 , the relative affinity of accommodation of a second NO after formation of the heme a 3 -NO bond is substantially lower than in aa 3 , a property that may be related to the relatively small Fe a 3 -CuB distance (23).…”

“…On the other hand, it is possible that two NO molecules are accommodated by the active site (1). This has been reported in steady-state electron paramagnetic resonance (EPR) (7) and Fourier transform infrared (FTIR) studies (8) under high NO concentrations; by contrast, NO uptake experiments at low concentrations indicate stoichiometric uptake of NO on the time scale of ∼1 min (9,10). Generally, the (temporal) copresence of two NO molecules in the reduced active site is required for any NO reductase activity (2NO + 2e -+ 2H + f N 2 O + H 2 O).…”

Observation of the Equilibrium CuB-CO Complex and Functional Implications of the Transient Hemea3 Propionates in Cytochromeba3-CO from Thermus thermophilus

“…A, step-scan TR-FTIR difference spectra of the CO-bound form of fully reduced cytochrome ba 3 (pD 8.5) at 0.15, 0.5, 1, 2, 3, 4, 5,6,7,8,9,10,15,20,21,22,23,25,30,35,40,45,50,55,60,64,70, and 78.5 ms subsequent to CO photolysis. The spectral resolution was 8 cm Ϫ1 , and the time resolution was 100 s. A 532 nm, 3-10 mJ/pulse pump beam was used for photolysis.…”

O 2 reduction by a functional heme/nonheme bis-iron NOR model complex

“…In eukaryotes CcO (aa 3 type) reduces O 2 to H 2 O and is reversibly but strongly inhibited by NO (17)(18)(19)(20). Several other CcOs (ba 3 , caa 3 , cbb 3 ) are reported to exhibit limited (Ͻ1%) NOR activity, i.e., reduce NO to N 2 O (7,21). On the other hand, NORs that reduce NO to N 2 O are reversibly but strongly inhibited by O 2 , and some of them show modest (Ϸ10%) O 2 reduction activity (12).…”