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...

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 reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was investigated by stopped-flow, amperometry, and EPR, using the enzyme as prepared or after "pulsing." A rapid reduction of cytochrome a is observed with the pulsed, but not with the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa 3 , whereas the enzyme as prepared reacts to a very small extent (<20%). In both cases, the reactivity is abolished by pre-incubation with cyanide. These experiments suggest that the effect of "pulsing" the enzyme, which leads to enhanced NO reactivity, arises from removing Cl ؊ bound at the oxidized cytochrome a 3 -Cu B site.

The role of nitric oxide (NO) as a signalling molecule involved in many pathophysiological processes (e.g., smooth muscle relaxation, inflammation, neurotransmission, apoptosis) has been elaborated during the last decade. Since NO has also been found to inhibit cellular respiration, we review here the available information on the interactions of NO with cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain. The effect of NO on cellular respiration is first summarized to present essential evidence for the fact that NO is a potent reversible inhibitor of in vivo O2 consumption. This information is then correlated with available experimental evidence on the reactions of NO with purified COX. Finally, since COX has been proposed to catalyze the degradation of NO into either nitrous oxide (N2O) or nitrite, we consider the putative role of this enzyme in the catabolism of NO in vivo.

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The basic principles of a novel, versatile, sensitive, and selective oxygen-sensing assay are presented in this paper. For the first time, liquid chromatography with electrochemical detection (at the hmde) has been used for the determination of oxygen. All factors concerning optimization of the chromatographic separation conditions and electrochemical detection with respect to direct determination of oxygen even in complex biological samples are discussed. Due to the combination of a chromatographic technique with amperometric detection, a high selectivity can be achieved. A direct and linear relationship between the oxygen concentration in the sample and the reduction current was verified in a large concentration range from saturation down to trace level oxygen concentrations. The novel oxygen-sensing assay provides a much higher sensitivity compared to conventional oxygen sensors. In principle, O(2) concentrations down to 4.5 × 10(-)(9) mol L(-)(1) O(2) (corresponding to a signal-to-noise ratio of 3) can be detected. Precision was determined by repeated measurements (n = 6) of air-saturated solutions (2.5 × 10(-)(4) mol L(-)(1) O(2), 20 °C, 920 mbar) which yielded relative standard deviations of lower than 0.2%.