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 flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O 2 -reductase activity (>40 s ؊1 ), but very low NO-reductase activity (ϳ0.2 s ؊1 ); O 2 reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K m < 2 M), producing H 2 O. The three-dimensional structure of the oxidized protein determined at 1.9 Å resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the nonheme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O 2 , allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.The flavodiiron proteins (FDP, 2 originally named A-type flavoproteins (1)) are widespread among Bacteria and Archaea, either strict or facultative anaerobes, where they have been proposed to play a role in the response to nitrosative and/or oxidative stress (2, 3). A few prokaryotic FDPs have been characterized to date, namely those from the bacteria Desulfovibrio gigas (originally named rubredoxin:oxygen oxidoreductase, ROO (4 -7), and hereafter denoted FDP Dg ), Escherichia coli (named flavorubredoxin, FlRd, 3 Refs. 2, 8 -11), Desulfovibrio vulgaris (12), Moorella thermoacetica (FDP Mt , (13, 14)), and the homologous enzyme from the methanogenic archaeon Methanothermobacter marburgensis (FDP Mm , Refs. 15, 16). The FDPs contain two redox centers: a FMN, the electron entry site into the enzyme, and a non-heme Fe-Fe center, the active site (13). They are cyanide-insensitive enzymes able to catalyze the reduction of O 2 (to H 2 O) and/or NO (to N 2 O). Some of these enzymes are almost exclusively reactive toward NO (such as E. coli FlRd, Refs. 2, 9), 4 others toward O 2 (such as the M. marburgensis enzyme, (15)), whereas some FDPs catalyze the reduction of both gases, though with different efficiency (7,12,13). These enzymes are expected to play a protective role in anaerobic or microaerobic microorganisms that need to survive under O 2 and cope with NO produced by the host defense system to counteract infection (17,18).Surprisingly, a few years ago, genes coding for FDPs were identified also in the geno...

Cytochrome bd is a prokaryotic respiratory quinol:O2 oxidoreductase, phylogenetically unrelated to the extensively studied heme-copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O2, NO and H2O2 is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

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The cytochrome cbb 3 is an isoenzyme in the family of cytochrome c oxidases. This protein purified from Pseudomonas stutzeri displays a cyanide-sensitive nitric oxide reductase activity (V max ¼ 100^9 mol NO·mol cbb 21 3 ·min 21 and K m ¼ 12^2.5 mM), which is lost upon denaturation. This enzyme is only partially reduced by ascorbate, and readily re-oxidized by NO under anaerobic conditions at a rate consistent with the turnover number for NO consumption. As shown by transient spectroscopy experiments and singular value decomposition (SVD) analysis, these results suggest that the cbb 3 -type cytochromes, sharing structural features with bacterial nitric oxide reductases, are the enzymes retaining the highest NO reductase activity within the heme-copper oxidase superfamily.

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Experimental evidence is presented supporting a mechanism of S-nitrosothiol formation and degradation mediated by copper ions using bovine serum albumin, human hemoglobin and glutathione as models. We found that Cu 2؉ , but not Fe 3؉ , induces in the presence of NO a fast S-nitrosation of bovine serum albumin and human hemoglobin, and the reaction is prevented by thiol blocking reagents. During the reaction, Cu ؉ is accumulated and accounts for destabilization of the S-nitrosothiol formed. In contrast, glutathione rapidly dimerizes in the presence of Cu 2؉ , the reaction competing with S-nitrosation and therefore preventing the formation of S-nitrosoglutathione. We have combined the presented role of Cu 2؉ in S-nitrosothiol formation with the known destabilizing effect of Cu ؉ , providing a unique simple picture where the redox state of copper determines either the NO release from S-nitrosothiols or the NO scavenging by thiol groups. The reactions described are fast, efficient, and may occur at micromolar concentration of all reactants. We propose that the mechanism presented may provide a general method for in vitro S-nitrosation. S-Nitrosothiols (RS-NOs)1 have a variety of biological activities, which are mostly attributed to their ability to release NO (1-3). RS-NOs are not only synthesized and administered clinically (2) but are also produced endogenously. Stamler et al. (4) reported that human plasma contains ϳ7 M RS-NOs, mostly as S-nitroso-albumin, a level unexpectedly high as the basal cellular NO level is in the low nanomolar range (5, 6). Thus, RS-NOs are considered as NO pools buffering the level of NO, which may be targeted at different sites (7). RS-NOs are also reported to be involved in the trans-S-nitrosation of proteins by transferring the NO ϩ moiety (8, 9), a process suggested to be a reversible post-translational modification regulating the activity of enzymes and receptors (3, 10, 11). (21), respectively. It is worth noticing that the reaction with water efficiently competes with direct thiol nitrosation by N 2 O 3 , due to the large molar excess of water over thiols. NAD ϩ substituting oxygen for the electron acceptor can also accelerate the reaction of NO with thiols (22). Several authors also suggested that S-nitrosation of thiols occurs by reaction with nitrosonium ions (NO ϩ ) formed either via metalcatalyzed oxidation of NO or via dinitrosyl-iron-cysteine complexes (8,21,23,24); efficiency and physiological relevance of these reactions remain unclear.In this study we have examined by spectroscopic and amperometric techniques the interaction of NO and thiols in the presence of cupric and ferric ions. Experiments have been carried out using the small tripeptide GSH (low millimolar amounts in the cell), bovine serum albumin (BSA, which is the most abundant plasma protein), and human hemoglobin (Hb). BSA and GSH both bear only one reduced cysteine per molecule (Cys-34 in BSA; Refs. 25 and 26), but, as shown below, they display in the presence of Cu 2ϩ a very different reactivity with NO...

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O 2 /NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O 2 /NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a 3 -Cu B binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a 3 . Reversal of inhibition in the presence of O 2 does not involve a direct reaction of O 2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k ‫؍‬ 0.13 s ؊1 ), as determined in the absence of O 2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a 3 -Cu B center.