Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis aa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)−1 min−1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)−1 min−1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.


Introduction
Nitric oxide (NO) is a gaseous free radical that, while exerting physiological functions at low concentrations, can have deleterious effects on the cell at high levels, being able itself or in combination with reactive oxygen species to damage proteins, lipids and nucleic acids. NO production by activated macrophages through the inducible form of the enzyme NO synthase (iNOS) is indeed a common host defence mechanism against infections. Mycobacterium (M.) tuberculosis, like several other microorganisms, has evolved the ability to sense this ligand and elicit defensive mechanisms [1], such as the downregulation of the host iNOS expression [2,3], the degradation of proteins damaged by NO and reactive nitrogen species [4] and the production of enzymes resisting and/or metabolizing the ligand. Two types of enzymes are mainly involved in bacterial NO detoxification, the NO-reductases (NORs) and the NO-dioxygenases (NODs). The former reductively metabolize NO to dinitrogen oxide (N 2 O) under anaerobic conditions, whereas the latter degrade NO to nitrate (NO 3 − ) using O 2 as co-substrate.
Most NODs belong to the globin superfamily, such as flavohaemoglobins (flavoHb) and truncated haemoglobins (trHb) that detoxify NO with high efficiency in many microbes. Genes encoding flavoHb and trHb have been found in mycobacteria too. In M. tuberculosis, a truncated single

Cytochrome bcc-aa 3 is Resistant to NO Inhibition
The effect of NO on the dithiothreitol (DTT)/menadione (MD)-sustained O 2 -reductase activity of bcc-aa 3 was tested oxygraphically at 25 • C by simultaneously monitoring the concentration of O 2 and NO in solution ( Figure 1). Surprisingly, NO (even at relatively high concentrations) has little or no effect on the enzyme activity. As shown in Figure 1, only a small, transient decline in the O 2 consumption is caused by NO additions, followed by quick and full restoration of the control O 2 -reductase activity of bcc-aa 3 .

Cytochrome bcc-aa3 is Resistant to NO Inhibition
The effect of NO on the dithiothreitol (DTT)/menadione (MD)-sustained O2-reductase activity of bcc-aa3 was tested oxygraphically at 25 °C by simultaneously monitoring the concentration of O2 and NO in solution ( Figure. 1). Surprisingly, NO (even at relatively high concentrations) has little or no effect on the enzyme activity. As shown in Figure 1, only a small, transient decline in the O2 consumption is caused by NO additions, followed by quick and full restoration of the control O2-reductase activity of bcc-aa3. Figure 1. Effect of NO on O2 consumption by purified mycobacterial cytochrome bcc-aa3 supercomplex. The addition of cytochrome bcc-aa3 (67 nM) to the oxygraphic chamber containing the reducing system DTT/MD results in an O2 reductase activity of 144 e − min −1 . The effect of NO on the enzymatic O2 consumption is tested by sequentially adding to the chamber increasing volumes of NO-saturated water. Following O2 depletion and sample reoxygenation, O2 consumption re-starts and the activity increases up to 600 e − min −1 . Sequential additions to the 1.5-mL reaction chamber: 9.5 μL of 800 mM DTT (5 mM); 4 μL of 100 mM MD (0.26 mM); 10 μL of 10 μM cytochrome bcc-aa3 (67 nM); 3, 10 and 25 μL of 1.8 mM NO (3.6, 12 and 30 μM, respectively).
The activity recovery occurs as NO levels decline, the NO decay in the presence of the enzyme being faster than expected. Following O2 exhaustion and subsequent sample reoxygenation, the O2-reductase activity of bcc-aa3 is enhanced (Figure 1). This phenomenon resembles the so-called 'pulsing effect' initially reported for the mammalian cytochrome c oxidase [31] and more recently documented for Escherichia coli cytochrome bd-I too [32]. It should be noted that after reoxygenation the enzyme retains its resistance to NO inhibition ( Figure 1). Figure 2A shows that bcc-aa3 in turnover with excess reductants and O2 can metabolize NO. In the presence of the supercomplex, the rate of NO decay is indeed significantly higher than measured in the absence of bcc-aa3 under otherwise identical experimental conditions (Figure 2A, red trace vs. black trace). At an initial NO concentration of 30 μM, bcc-aa3 displays a maximal NO-metabolizing activity of ≈303 mol NO (mol bcc-aa3) −1 min −1 at 25 °C, as estimated from initial rate analysis. The supercomplex can catalyze the aerobic degradation of NO only under turnover conditions. Indeed, as shown in Figure 2B, in the absence of reductants, the kinetics of NO decay with and without the enzyme in aerobic solution are virtually the same. The addition of cytochrome bcc-aa 3 (67 nM) to the oxygraphic chamber containing the reducing system DTT/MD results in an O 2 reductase activity of 144 e − min −1 . The effect of NO on the enzymatic O 2 consumption is tested by sequentially adding to the chamber increasing volumes of NO-saturated water. Following O 2 depletion and sample reoxygenation, O 2 consumption re-starts and the activity increases up to 600 e − min −1 . Sequential additions to the 1.5-mL reaction chamber: 9.5 µL of 800 mM DTT (5 mM); 4 µL of 100 mM MD (0.26 mM); 10 µL of 10 µM cytochrome bcc-aa 3 (67 nM); 3, 10 and 25 µL of 1.8 mM NO (3.6, 12 and 30 µM, respectively).

Cytochrome bcc-aa3 in Turnover Metabolizes NO
The activity recovery occurs as NO levels decline, the NO decay in the presence of the enzyme being faster than expected. Following O 2 exhaustion and subsequent sample reoxygenation, the O 2 -reductase activity of bcc-aa 3 is enhanced ( Figure 1). This phenomenon resembles the so-called 'pulsing effect' initially reported for the mammalian cytochrome c oxidase [31] and more recently documented for Escherichia coli cytochrome bd-I too [32]. It should be noted that after reoxygenation the enzyme retains its resistance to NO inhibition ( Figure 1). Figure 2A shows that bcc-aa 3 in turnover with excess reductants and O 2 can metabolize NO. In the presence of the supercomplex, the rate of NO decay is indeed significantly higher than measured in the absence of bcc-aa 3 under otherwise identical experimental conditions (Figure 2A, red trace vs. black trace). At an initial NO concentration of 30 µM, bcc-aa 3 displays a maximal NO-metabolizing activity of ≈303 mol NO (mol bcc-aa 3 ) −1 min −1 at 25 • C, as estimated from initial rate analysis. The supercomplex can catalyze the aerobic degradation of NO only under turnover conditions. Indeed, as shown in Figure 2B, in the absence of reductants, the kinetics of NO decay with and without the enzyme in aerobic solution are virtually the same. Taking advantage of the spontaneous increase in the O2-reductase activity of bcc-aa3 observed along each oxygraphic assay (Figure 1), the rate of NO consumption by bcc-aa3 could be measured at different actual rates of O2 consumption and the former was found to be proportional to the latter, according to a NO/O2 ratio of 2.65 ± 0.19 ( Figure 3).  Taking advantage of the spontaneous increase in the O 2 -reductase activity of bcc-aa 3 observed along each oxygraphic assay (Figure 1), the rate of NO consumption by bcc-aa 3 could be measured at different actual rates of O 2 consumption and the former was found to be proportional to the latter, according to a NO/O 2 ratio of 2.65 ± 0.19 ( Figure 3). Taking advantage of the spontaneous increase in the O2-reductase activity of bcc-aa3 observed along each oxygraphic assay (Figure 1), the rate of NO consumption by bcc-aa3 could be measured at different actual rates of O2 consumption and the former was found to be proportional to the latter, according to a NO/O2 ratio of 2.65 ± 0.19 ( Figure 3).

Cytochrome bcc-aa 3 Possesses NO-Reductase Activity
The ability of bcc-aa 3 to degrade NO was also tested under anaerobic reducing conditions. In this assay, an aliquot of pre-reduced supercomplex was anaerobically added to an O 2 -free solution of NO containing excess reductants (DTT and MD), and the NO concentration in solution was then monitored using a NO-selective electrode. As shown in Figure 4, prior to enzyme addition, in the presence of excess DTT and MD, a slow decay of NO is observed due to reaction of NO with the reductants. The addition of bcc-aa 3 clearly accelerates the decomposition of NO (Figure 4). The fast drop in the NO concentration observed immediately after addition of bcc-aa 3 can be explained, at least partially, by NO binding to the reduced of bcc-aa 3 . The NO decay observed subsequently is consistent with a substantial catalytic NO reductase activity of bcc-aa 3 . After NO depletion, if the gas is re-added, the NO-consuming activity of bcc-aa 3 is observed again (Figure 4). A notably slower NO consumption was observed if the same volume of aerobic buffer was added instead of the supercomplex in control experiments conducted under otherwise identical conditions (Supplementary Figure S1). The enzymatic NO reductase activity value was obtained from the slope of the trace with the enzyme after subtraction of the background non-enzymatic NO-reduction rate. Under anaerobic conditions, at [NO] = 8.4 µM, the estimated NO-reductase activity of bcc-aa 3 proved to be 2.8 ± 0.5 mol NO (mol of bcc-aa 3

Cytochrome bcc-aa3 Possesses NO-Reductase Activity
The ability of bcc-aa3 to degrade NO was also tested under anaerobic reducing conditions. In this assay, an aliquot of pre-reduced supercomplex was anaerobically added to an O2-free solution of NO containing excess reductants (DTT and MD), and the NO concentration in solution was then monitored using a NO-selective electrode. As shown in Figure 4, prior to enzyme addition, in the presence of excess DTT and MD, a slow decay of NO is observed due to reaction of NO with the reductants. The addition of bcc-aa3 clearly accelerates the decomposition of NO (Figure 4). The fast drop in the NO concentration observed immediately after addition of bcc-aa3 can be explained, at least partially, by NO binding to the reduced of bcc-aa3. The NO decay observed subsequently is consistent with a substantial catalytic NO reductase activity of bcc-aa3. After NO depletion, if the gas is re-added, the NO-consuming activity of bcc-aa3 is observed again (Figure 4). A notably slower NO consumption was observed if the same volume of aerobic buffer was added instead of the supercomplex in control experiments conducted under otherwise identical conditions (Supplementary Figure S1). The enzymatic NO reductase activity value was obtained from the slope of the trace with the enzyme after subtraction of the background non-enzymatic NO-reduction rate.   Figure 1) containing DTT (5 mM), MD (0.26 mM) as the reducing system, and glucose (5 mM) and glucose oxidase (16 units/mL) to scavenge residual O2 in the 1.5-mL reaction chamber. Then, 30 μL of 10 μM cytochrome bcc-aa3 (200 nM), pre-reduced with DTT (5 mM) and MD (0.33 mM) in the presence of catalase (520 units/mL) and SOD (60 units/mL), was added to the chamber. The NO decay observed before addition of the supercomplex is likely due to the reaction with the reductants. Addition of pre-reduced cytochrome bcc-aa3 accelerates the NO decay due to catalytic NO consumption. The initial fast drop in the NO concentration observed just after addition of the supercomplex is likely due at least partly to NO binding to cytochrome bcc-aa3. Following NO depletion from solution, two aliquots of 4.2 μM NO (3.5 μL of 1.8 mM NO each) were sequentially added and catalytic NO consumption further observed. The activity of bcc-aa3 at [NO] = 8.4 μM was 2.8 ± 0.5 mol NO (mol bcc-aa3) −1 min −1 . The calculated activity is expressed as mean ± standard deviation.

Discussion
Clinical resistance of M. tuberculosis antibiotics represents an increasing threat to public health globally, preventing effective treatments against tuberculosis, one of the top 10 causes of death worldwide [33]. Interestingly, a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility has been found in clinical strains of M. tuberculosis [34]. Understanding how this bacterial pathogen resists the host immune system attack and identifying novel drug and MD (0.33 mM) in the presence of catalase (520 units/mL) and SOD (60 units/mL), was added to the chamber. The NO decay observed before addition of the supercomplex is likely due to the reaction with the reductants. Addition of pre-reduced cytochrome bcc-aa 3 accelerates the NO decay due to catalytic NO consumption. The initial fast drop in the NO concentration observed just after addition of the supercomplex is likely due at least partly to NO binding to cytochrome bcc-aa 3 . Following NO depletion from solution, two aliquots of 4.2 µM NO (3.5 µL of 1.8 mM NO each) were sequentially added and catalytic NO consumption further observed. The activity of bcc-aa 3 at [NO] = 8.4 µM was 2.8 ± 0.5 mol NO (mol bcc-aa 3 ) −1 min −1 . The calculated activity is expressed as mean ± standard deviation.

Discussion
Clinical resistance of M. tuberculosis antibiotics represents an increasing threat to public health globally, preventing effective treatments against tuberculosis, one of the top 10 causes of death worldwide [33]. Interestingly, a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility has been found in clinical strains of M. tuberculosis [34]. Understanding how this bacterial pathogen resists the host immune system attack and identifying novel drug targets are therefore crucial for the successful cure of this infectious disease. The respiratory complexes of M. tuberculosis, including the bcc-aa 3 supercomplex, are attractive targets for the development of new antitubercular agents.
Kim et al. [30] recently succeeded in the purification of an untagged hybrid bcc-aa 3 supercomplex with M. tuberculosis cytochrome bcc and M. smegmatis cytochrome aa 3 . The supercomplex is stable and remains active during protein extraction and purification in the presence of the non-ionic detergent dodecyl-β-d-maltoside (DDM) [30]. This allowed us to use these bcc-aa 3 preparations for functional studies. Assays were meant to test whether bcc-aa 3 could contribute to mechanisms subverting, suppressing or evading the host immune response. In particular, we sought to gain insight into the reactivity of bcc-aa 3 with NO, a harmful species produced by the host as part of the immune response to fight microbial infections. To this purpose, we investigated the effect of NO on the O 2 reductase activity of bcc-aa 3 and the ability of the supercomplex to metabolize NO under different conditions. First, we assayed whether the O 2 -reductase activity of bcc-aa 3 is affected by NO, which is known to bind with high affinity to the active site of respiratory terminal oxidases, resulting in a potent inhibition (reviewed in [35][36][37][38][39][40][41]). Surprisingly, we found that the mycobacterial supercomplex is resistant to NO inhibition, even at the highest concentration of the gas tested (30 µM). Such insensitivity to NO is unprecedented for a terminal oxidase. Indeed, the oxidases thus far assayed for NO inhibition, such as mitochondrial cytochrome c oxidase [42][43][44], the aa 3 oxidase from Rhodobacter sphaeroides [45], the cbb 3 oxidases from Rhodobacter sphaeroides and Vibrio cholerae [45] and the cytochromes bd from Escherichia coli and Azotobacter vinelandii [32,41,46], were found to be strongly and reversibly inhibited by NO with IC 50 values in the nanomolar range. Figure 1 (blue trace) shows that the addition of 3.6 µM NO has no detectable effect on the O 2 consumption by bcc-aa 3 in the presence of excess respiratory substrates. At the highest concentration of NO added, there is only a small decline in the O 2 consumption followed by a relatively fast (≈1-2 min after the NO addition) and complete recovery of enzyme activity. The activity recovery occurs as NO disappears from solution ( Figure 1, red trace). Careful comparison of the NO traces acquired in the presence and absence of bcc-aa 3 shows that the enzyme drastically accelerates the kinetics of NO decay (Figure 2A). The maximum NO-consuming activity of bcc-aa 3 detected at 30 µM NO and ≈130 µM O 2 was ≈303 mol NO (mol bcc-aa 3 ) −1 min −1 . Thus, one can conclude that the apparent resistance of the supercomplex to NO inhibition results from its ability to directly metabolize NO, likely to nitrite (see below). NO consumption by the supercomplex requires turnover conditions, as bcc-aa 3 in the same aerobic buffer but without the DTT/MD reducing system proved to be unable to metabolize NO ( Figure 2B).
The rates of NO and O 2 consumption catalysed by bcc-aa 3 are proportional, yielding a NO/O 2 ratio of 2.65 ± 0.19 (Figure 3). In the bovine mitochondrial cytochrome c oxidase (mtCcOX), unlike O 2 that binds only to the fully reduced (R) active site of the enzyme, NO was previously reported to bind and react also with the catalytic intermediates O (with the fully oxidized haem a 3 -Cu B site), P (peroxy) and F (ferryl), each according to a 1:1 stoichiometry [47]. The reaction with the intermediates O, P and F is accompanied by injection of 1 electron into the enzyme from NO [47]. Donation of such an electron, while causing NO oxidation to nitrite, leads to conversion of an O 2 intermediate into the succeeding one along the catalytic cycle ( Figure 5). By reacting with NO, intermediates O, P and F are therefore respectively converted into the intermediates E (with the single electron-reduced haem a 3 -Cu B site), F and O [47]. In mtCcOX, the nitrite formed from NO binds with relatively high affinity to the oxidized haem a 3 in the active site, impairing the Fe reduction and, thus, its ability to react with O 2 . Consequently, the reaction with O 2 stops. At lower reductive pressure on the enzyme, when intermediates O, P, F are more populated at steady-state, this inhibition pathway leading to the haem a 3 Fe 3+ -NO 2 − adduct prevails over the so-called nitrosyl pathway, that is instead favoured at higher reductive pressure and occurs via NO binding to reduced haem a 3 , leading to formation of a Fe 2+ -NO adduct (reviewed in [48]). Under the assumption that the mycobacterial bcc-aa 3 catalytic intermediates share a similar reactivity with NO, the observed >1 NO/O 2 stoichiometry indicates that in the case of the mycobacterial enzyme NO can also react with more than one catalytic intermediate, but possibly with the notable difference (compared to the mitochondrial enzyme) that the newly formed nitrite does not bind to the haem a 3 moiety with high affinity and, therefore, is ejected into the bulk phase from the enzyme without impairing its catalytic O 2 -reductase activity ( Figure 5).
react with more than one catalytic intermediate, but possibly with the notable difference (compared to the mitochondrial enzyme) that the newly formed nitrite does not bind to the haem a3 moiety with high affinity and, therefore, is ejected into the bulk phase from the enzyme without impairing its catalytic O2-reductase activity ( Figure 5).

Figure 5.
Proposed mechanism for the interaction of NO with the catalytic intermediates of the mycobacterial bcc-aa3 supercomplex under aerobic conditions. As shown for mtCcOX, by reacting with the intermediates O, P and F, NO is suggested to lead to formation of nitrite. However, at variance from the mammalian enzyme, in the case of the mycobacterial supercomplex, the observed NO/O2 stoichiometry = 2.65 ± 0.19 suggests that nitrite does not bind with high affinity to oxidized haem a3 and is rapidly ejected from the enzyme, without affecting its catalytic activity.
Interestingly, bcc-aa3 was found to be capable of consuming NO also under anaerobic reducing conditions (Figure 4), with an estimated NO-reductase activity of 2.8 ± 0.5 mol NO (mol bcc-aa3) −1 min −1 (at [NO] = 8.4 μM and 25 °C), most likely leading to nitrous oxide (N2O) as the reaction product, similarly to other terminal oxidases. Indeed, the ability to catalyze the reduction of NO to N2O was previously documented under similar experimental conditions and setup (amperometric measurements under anaerobic reducing conditions at [NO] = 5-10 μM) in several haemcopper oxidases, namely in the ba3 and caa3 oxidases from Thermus thermophilus [49], the cbb3 oxidases from Pseudomonas stutzeri [50] and Rhodobacter sphaeroides [51], and the bo3 oxidase from Escherichia coli [52], but not in the mitochondrial beef heart cytochrome c oxidase [49,53] or the cytochromes bd from Escherichia coli and Azotobacter vinelandii [46]. Notably, the NO reductase activities of all terminal oxidases tested so far are significantly lower than that displayed by bacterial NORs. For instance, NOR purified from Paracoccus denitrificans shows the activity as high as ≈1400 mol NO (mol enzyme) −1 min −1 [50] (see also [54,55]).
Taken together, the novel findings reported here, particularly the high resistance to NO inhibition of the mycobacterial bcc-aa3 supercomplex and its unexpected NO-metabolizing activity, suggest a role for this enzymatic complex in the defence against NO, a major effector in the immune response to M. tuberculosis infection [56]. The resistance of bcc-aa3 to NO inhibition may help the pathogen survive acute NO stress before transcriptional and late phase responses take place [57]. All in all, this study reveals unexpected properties of the mycobacterial bcc-aa3 supercomplex, further pointing to this protein as a valuable drug target. Proposed mechanism for the interaction of NO with the catalytic intermediates of the mycobacterial bcc-aa 3 supercomplex under aerobic conditions. As shown for mtCcOX, by reacting with the intermediates O, P and F, NO is suggested to lead to formation of nitrite. However, at variance from the mammalian enzyme, in the case of the mycobacterial supercomplex, the observed NO/O 2 stoichiometry = 2.65 ± 0.19 suggests that nitrite does not bind with high affinity to oxidized haem a 3 and is rapidly ejected from the enzyme, without affecting its catalytic activity.
Interestingly, bcc-aa 3 was found to be capable of consuming NO also under anaerobic reducing conditions (Figure 4), with an estimated NO-reductase activity of 2.8 ± 0.5 mol NO (mol bcc-aa 3 ) −1 min −1 (at [NO] = 8.4 µM and 25 • C), most likely leading to nitrous oxide (N 2 O) as the reaction product, similarly to other terminal oxidases. Indeed, the ability to catalyze the reduction of NO to N 2 O was previously documented under similar experimental conditions and setup (amperometric measurements under anaerobic reducing conditions at [NO] = 5-10 µM) in several haemcopper oxidases, namely in the ba 3 and caa 3 oxidases from Thermus thermophilus [49], the cbb 3 oxidases from Pseudomonas stutzeri [50] and Rhodobacter sphaeroides [51], and the bo 3 oxidase from Escherichia coli [52], but not in the mitochondrial beef heart cytochrome c oxidase [49,53] or the cytochromes bd from Escherichia coli and Azotobacter vinelandii [46]. Notably, the NO reductase activities of all terminal oxidases tested so far are significantly lower than that displayed by bacterial NORs. For instance, NOR purified from Paracoccus denitrificans shows the activity as high as ≈1400 mol NO (mol enzyme) −1 min −1 [50] (see also [54,55]).
Taken together, the novel findings reported here, particularly the high resistance to NO inhibition of the mycobacterial bcc-aa 3 supercomplex and its unexpected NO-metabolizing activity, suggest a role for this enzymatic complex in the defence against NO, a major effector in the immune response to M. tuberculosis infection [56]. The resistance of bcc-aa 3 to NO inhibition may help the pathogen survive acute NO stress before transcriptional and late phase responses take place [57]. All in all, this study reveals unexpected properties of the mycobacterial bcc-aa 3 supercomplex, further pointing to this protein as a valuable drug target.

Supercomplex Purification and Concentration Determination
The cytochrome bcc-aa 3 supercomplex was purified from a strain of M. smegmatis expressing the hybrid supercomplex, as described by Kim et al. [30] with modifications. Cells were collected by centrifugation. Membranes were prepared by sonication of cells in 50 mM K-Pi pH 7.4, 0.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), followed by differential centrifugation, resuspended to 35 g/L protein in the same buffer, and frozen. For extraction, membranes were treated with DDM at a concentration of 10 g/L protein, 5 g/L detergent in 50 mM KPi pH 7.4, 100 mM NaCl, 0.1 mM PMSF. Insoluble material was separated from the extract by centrifugation. In this case the initial extract contained little soluble supercomplex but did have a large amount of soluble protein containing flavin and cytochrome b 557 . Therefore, the pellet was resuspended in buffer without detergent, protein content was measured, and the material was re-extracted as before but with 1 g/(g protein) of added DDM. This extract was diluted with 1 4 volume of water, applied to DEAE-Sepharose CL6B column, and eluted with a gradient from 100 to 500 mM NaCl in 5-mM KPi, 0.5 mM EDTA, and 0.1 g/L DDM. Fractions containing bcc-caa 3 were identified by UV-visible spectra and haem-stained gels [58,59]. The pooled fractions were concentrated by ultrafiltration and layered on glycerol density step-gradients with 60, 40, and 20% w/w glycerol in 20 mM K-MPOS, 100 mM NaCl, and 0.5 mM EDTA, pH 7.2. The gradients were centrifuged at 30,000 rpm in the Beckman SW-32.Ti rotor for 72 h. Fractions containing bcc-aa 3 were pooled and chromatographed on Sepharose CL-6B size-exclusion medium equilibrated with 20 mM K MOPS, 100 mM NaCl, 0.5 mM EDTA, and 0.1 g/L DDM (pH 7.2). Fractions containing pure supercomplex, as assessed by SDS-PAGE (Supplementary Figure S2), were pooled and concentrated. Concentration of the supercomplex was estimated using the extinction coefficient for the oxidized form ∆ε 413-371 nm = 485 mM −1 cm −1 .

Catalytic Assays
Oxygraphic measurements were performed at 25 • C with a high-resolution respirometer (Oxygraph-2k, Oroboros Instruments) equipped with two 1.5-mL gas-tight thermostated chambers. The assays were carried out at 25 • C in 20 mM K/MOPS buffer (pH 7.3) containing 100 mM NaCl, 0.5 mM EDTA and 0.01% dodecyl-β-d-maltoside. Changes in the O 2 and NO levels in solution were recorded in real time simultaneously. The NO concentration was recorded with the aid of a NO-selective electrode (World Precision Instruments, Sarasota FL, USA). The electrode was calibrated by sequential additions of NO from a stock solution, prepared by equilibrating degassed water at room temperature with pure NO (Air Liquide, Paris, France) at 1 atm. The O 2 reductase activity of purified mycobacterial cytochrome bcc-aa 3 supercomplex was measured with an excess of the reductants DTT (5 mM) and MD (0.26 mM). Mycobacterial bcc-aa 3 preparations are indeed active with menadiol as substrate, which has a higher solubility in water solutions compared to menaquinol [60]. DTT was used to keep MD in the reduced state during the oxygraphic assays (Supplementary Figure S3). In the presence of DDT and MD, 520 units/mL catalase and 60 units/mL SOD were added to the solution to reduce the non-enzymatic background oxygen consumption.

Data Analysis
The catalytic NO-consuming activity was obtained by subtracting from the rate of NO consumption measured after addition of bcc-aa 3 from that of the non-enzymatic NO consumption. The latter was obtained by analyzing either the same trace prior to the enzyme addition (anaerobic reducing conditions with excess DTT and MD) or a trace independently collected under identical experimental conditions in the absence of bcc-aa 3 (aerobic conditions with excess DTT and MD). Origin software (OriginLab Corporation) was used for data analysis and figure preparation.

Supplementary Materials:
The following are available online at http://www.mdpi.com/1422-0067/21/22/8521/s1. Figure S1. Control NO trace acquired under anaerobic conditions in the absence of the supercomplex but in the presence of DTT/MD, Figure S2. SDS-PAGE analysis of the purified chimeric bcc-aa 3