Cytochrome c oxidase is a respiratory enzyme catalysing the energy‐conserving reduction of molecular oxygen to water. The crystal structure of the ba3‐cytochrome c oxidase from Thermus thermophilus has been determined to 2.4 Å resolution using multiple anomalous dispersion (MAD) phasing and led to the discovery of a novel subunit IIa. A structure‐based sequence alignment of this phylogenetically very distant oxidase with the other structurally known cytochrome oxidases leads to the identification of sequence motifs and residues that seem to be indispensable for the function of the haem copper oxidases, e.g. a new electron transfer pathway leading directly from CuA to CuB. Specific features of the ba3‐oxidase include an extended oxygen input channel, which leads directly to the active site, the presence of only one oxygen atom (O2−, OH− or H2O) as bridging ligand at the active site and the mainly hydrophobic character of the interactions that stabilize the electron transfer complex between this oxidase and its substrate cytochrome c. New aspects of the proton pumping mechanism could be identified.

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...

The multicopper proteins, nitrous-oxide reductase (N20R) and cytochrome c oxidase (COX), were investigated by EPR spectroscopy at microwave frequencies 2.4 -35 GHz. Our results support a Cu-Cu interaction in COX and N20R. At least 10 lines in the 2.7-GHz, 12 lines in the 4.6-GHz and 14 lines in the 9.2 GHz spectra were resolved for N20R. Eight copper lines at 2.7 GHz, about ninc lines at 4.6 GHz and about six lines at 9.2 G H L were resolved for COX. Simulations of the EPR spectra were consistent with most of the resonances of the multiline spectra, including regions in the center of the spectra where overlap of the three seven-line patterns is proposed. These simulations indicated that Cu-Cu interaction, in a mixed-valence [Cu(l.5). . .Cu(l.5)], S = site is consistent with, if not proof of, the unusual spectral features observed for N 2 0 R and COX.Nitrous-oxide reductase (N20R) is the terminal electron acceptor in a respiratory chain converting N 2 0 to N2 in denitrifying bacteria: N 2 0 + 2H' + 2e-N2 + H 2 0 .