Nitrosylation Mechanisms of Mycobacterium tuberculosis and Campylobacter jejuni Truncated Hemoglobins N, O, and P

Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.


Introduction
Based on phylogeny, the globin superfamily contains three lineages: flavohemoglobins and single domain globins (lineage 1), protoglobins (Pgb) and globin coupled sensors (lineage 2), and truncated hemoglobins (trHbs; lineage 3). Members of the globin superfamily belong to two structural classes: one showing the classical 3-on-3 a-helical sandwich (lineages 1 and 2) and one having the 2-on-2 a-helical sandwich (lineage 3) (Fig. 1). Although no definitive conclusion can be drawn about the ancestral state of the globin fold, the occurrence of the 2-on-2 fold, but not of an isolated 3-on-3 fold, in all three kingdoms of life suggests that the 2-on-2 is the ancestral fold. In an evolutionary perspective, the predominant function of globins is (pseudo-)enzymatic, with O 2 transport and storage being specialized functions associated with the evolution of metazoans [1][2][3][4][5][6].
TrHbs are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. They are distantly related to the 3-on-3 globins, showing less than 20% overall identity with the latter. Phylogenetic analysis of protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). TrHbs belonging to groups N and O separate into two and four subgroups, respectively; trHbs belonging to group P display a level of conservation higher than those of groups N and O (Fig. 1). The overall sequence identity between trHbs from different groups is #20%, but may be higher than 80% within a given group. Some bacteria display multiple trHbs belonging to different groups, suggesting a scenario for the evolution of the different groups where the group O gene is the ancestor, and group N and P genes are the results of duplications and transfer events [1][2][3][4][5][6].
TrHbs fold as a 2-on-2 a-helical sandwich characterized by a very short or absent A-helix, a brief CE inter-helical region, and most of the F-helix occurring as a loop, with only the B, E, G, and H a-helices surrounding the heme group. Specific residue deletions and substitutions distributed throughout the trHb sequence allow the achievement of the simplified fold, keeping at the same time a high affinity for the heme, a suitable ligand access to the heme-Fe atom, and the proper heme-Fe oxidation state. However, specific features, such as ligand entry/exit mechanisms holding to promote diffusion of ligands to/from the heme, the heme distal pocket structure and polarity, as well as ligand stabilization mechanisms distinguish members of the three trHb groups (Fig. 2) [5].
Most trHbs are involved in the O 2 /NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host [5,[11][12][13]. Noteworthy, Mt-trHbN binds reversibly isoniazid, a first-line anti-tuberculosis medication in prevention and treatment of tuberculosis, highlighting a direct role of the pro-drug to impair fundamental functions of mycobacteria, e.g. scavenging of reactive nitrogen and oxygen species, and metabolism [14].
Studies performed with Mycobacterium bovis Calmette-Guerin (BCG) demonstrated that the inactivation of the glbN gene impairs the ability of stationary phase cells to protect aerobic respiration from NO inhibition, suggesting that Mt-trHbN may play a vital role in protecting M. tuberculosis from NO toxicity in vivo [12,15]. This functional assessment is supported by the observation that Mt-trHbN catalyzes the rapid oxidation of NO into nitrate [13,16]. Heterologous expression of Mt-trHbN has also been shown to protect Escherichia coli against nitrosative stress [17]. On the other hand, Mt-trHbO does not detoxify NO efficiently [18], but displays peroxidase activity, suggesting an oxidation/reduction function [19]. Lastly, Cj-trHbP has been proposed to play a prominent role in C. jejuni respiration rather than in protection against reactive nitrogen and oxygen species. A strain of C. jejuni lacking trHbP turned out to be disadvantaged with respect to wild-type cells when grown under high aeration, achieving lower growth yields and consuming O 2 at approximately half the rate displayed by wild-type cells. Although Cj-trHbP mutated cells are equally sensitive as the wild-type to NO and oxidative stress, the actual functional role of Cj-trHbP remains elusive [7,8,11].
Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP (trHbN(II), Mt-trHbO(II), and Cj-trHbP(II), respectively) nitrosylation, (ii) nitrite-mediated nitrosylation of Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II), and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP (trHbN(III), Mt-trHbO(III), and Cj-trHbP(III), respectively) is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO, the conversion of nitrite to NO being facilitated primarily by ferrous Mt-trHbN. This reflects the great ligand accessibility to the heme center of Mt-trHbN, and Cj-trHbP by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.
In view of the linear relationship between the absorbance change and the protein concentration change, values of the pseudo-first-order rate constant k were obtained according to Eqn.

Stopped-flow apparatus
Kinetic experiments have been carried out spectrophotometrically with the BioLogic SFM 2000 (Claix, France) rapid-mixing stopped-flow apparatus at single wavelength between 360 nm and 460 nm; the dead-time of the stopped-flow apparatus was ,1 ms and the observation chamber was 1 cm.

Data analysis
Kinetic data obtained at different wavelengths have been normalized each other on the basis of the total absorbance change at the specific wavelength. The results are given as mean values of at least four experiments plus or minus the corresponding standard deviation. All data were analyzed using the MATLAB program (The Math Works Inc., Natick, MA, USA).
Under all experimental conditions, the time course of NO binding to Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II) corresponds to a mono-molecular process for more than 80% of its course (Fig. 3, and Figs S1 and S2 in File S1, panels B and C). Values of the pseudo-first-order rate constant for Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II) nitrosylation (i.e., k; Eqn. (1)) are wavelength-independent at fixed NO concentration (data not shown).
Values of k increase linearly with the NO concentration (Fig. 3, and Figs S1 and S2 in File S1, panels D and E). The analysis of data reported in Figure 3, and Figures S1 and S2 in File S1 (panels D and E), according to Eqn. (2), allowed the determination of values of the second-order rate constant for Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II) nitrosylation (i.e., k on ; corresponding to the slope of the linear plots), which are essentially pHindependent (Fig. 3, and Figs S1 and S2 in File S1, panels D and E). The y intercept of the linear plots appears very close to zero (Fig. 3, and Figs S1 and S2 in File S1, panels D and E), indicating that values of k off for Mt-trHbN(II)-NO, Mt-trHbO(II)-NO, and Cj-trHbP(II)-NO denitrosylation are lower by at least two-orders of magnitude than values of k obtained at the lowest NO concentration (i.e., k off ,1610 21 s 21 ).
Values of k on for NO binding to Mt-trHbN(II) and Cj-trHbP(II) vary between 1.1610 7 M 21 s 21 and 2.1610 7 M 21 s 21 at pH 7.0 and pH 9.0 (Fig. 3, and Figs S1 and S2 in File S1, panels D and E, and Table 2). Values of k on for NO binding to Mt-trHbO(II) here determined at pH 7.0 and 9.0 ( = 1.9610 5 M 21 s 21 and 2.3610 5 M 21 s 21 , respectively; Fig. S1 in File S1, panels D and E, and Table 2) are in agreement with data referring of the slow nitrosylation course, reflecting 80% of the whole process, previously reported at pH 7.5 ( = 1.8610 5 M 21 s 21 ) [18]. Values of k on for NO binding to Mt-trHbO(II) and Cj-trHbP(II) increase changing the pH from 7.0 to 9.0, whereas values of k on for Mt-trHbN(II) nitrosylation decrease. However, the small magnitude of the pH-dependent change, essentially within the error limit, might suggest that these variations are not statistically relevant.
Values of k on for nitrosylation of ferrous heme-proteins span over eight orders of magnitude (Table 2) ( [36,[41][42][43][47][48][49][50][51][52][53] and present study), mainly reflecting the ligand accessibility to the heme distal pocket and the coordination of the heme-Fe(II) atom. Of note, H-bond interactions locking the heme distal residues TyrCD1 and TrpG8 of Mt-trHbO limits ligand access to the heme distal pocket (Fig. 2) [18]. In contrast, the low values of k on for ferrous horse heart cytochrome c and rabbit hemopexin-heme-Fe (HPX-heme-Fe) nitrosylation reflect the slow rate of hexa-to penta-coordination transition of the heme-Fe(II) atom, which precedes ligand binding ( [36,52] and present study). On the other hand, the fast hexa-to penta-coordination conversion of the heme-Fe(II) atom of Arabidopsis thaliana hemoglobin (Hb) class 1 and Glycine max Lb does not affect NO binding [31,34]. Accordingly, the decrease of the k on value for the nitrosylation of ferrous human serum heme-Fe-albumin (HSA-heme-Fe) upon binding of warfarin and ibuprofen to the fatty acid binding site 2 (FA2) has been attributed to the allosteric drug-dependent hexa-coordination of the heme-Fe(II) atom [54][55][56]. Values of h increase linearly with the nitrite concentration (Fig. 4, and Figs S3 and S4 in File S1, panel C). The analysis of data reported in Figure 4, and Figure S3 and S4 in File S1 (panel C) according to Eqn. (4) allowed the determination of values of the second-order rate constant for the nitrite-mediated conversion of Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II) to Mt-trHbN(II)-NO, Mt-trHbO(II)-NO, and Cj-trHbP(II)-NO, respectively, (i.e., h on ; corresponding to the slope of the linear plots). The y intercept of the linear plots corresponds to zero, indicating that the nitritemediated conversion of Mt-trHbN(II), Mt-trHbO(II), and Cj-trHbP(II) to Mt-trHbN(II)-NO, Mt-trHbO(II)-NO, and Cj-trHbP(II)-NO, respectively, can be considered as an irreversible process.
As reported for most heme-proteins [24][25][26][27][28][29][30][31][32]34,35], the nitritemediated conversion of Mt-trHbN(III), Mt-trHbO(II), and Cj-trHbP(II) to Mt-trHbN(II)-NO, Mt-trHbO(II)-NO, and Cj-trHbP(II)-NO, respectively, requires one proton for the NO and OH 2 formation (Scheme B). Indeed, on increasing the proton concentration by one pH unit, the rate of the nitrite-mediated conversion of trHbN(II) to trHbP(II)-NO (i.e., Log h on ) increases by one-order of magnitude (Fig. 4, and Figs S3 and S4 in File S1, panel D). However, the increase of h on for the nitrite-mediated conversion of human cytoglobin on pH decrease has been interpreted accounting for the reversible pH-dependent penta-tohexa-coordination transition of the heme-Fe(II) atom [33]. Values of h on for nitrite-mediated conversion of ferrous hemeproteins to their ferrous nitrosylated derivatives range between 7.0610 22 M 21 s 21 and 6.8610 1 M 21 s 21 (  (Table 3). Of note, the low reactivity of Mt-trHbO reflects the unfavorable ligand accessibility to the heme pocket due to the locked conformation(s) of the heme distal residues TyrCD1 and TrpG8 (Fig. 2) [18]. The very different values of the interconversion rate for the hexa-to penta-coordination of the heme-Fe(II) atom modulates kinetics of nitrite binding to Synechocystis Hb, Arabidopsis thaliana nonsymbiotic Hbs classes 1 and 2, and rice nonsymbiotic Hb class 1, human Cygb, and horse heart cytochrome c [18,31,33,34]. Accordingly, changes of the h on values for the nitrite-mediated conversion of ferrous human neuroglobin (Ngb) to its ferrous nitrosylated derivative (from 1.2610 22 M 21 s 21 to 1.2610 21 M 21 s 21 ) reflect the reversible redox-linked hexa-topenta-coordination transition of the heme-Fe(II) atom. In fact, under oxidative conditions, the formation of the Cys46-Cys55 bridge stabilizes the high-reactive penta-coordinated heme-Fe(II) atom, thus facilitating the reaction. In contrast, under reductive conditions, the cleavage of the Cys46-Cys55 bridge leads to the formation of the low-reactive hexa-coordinated heme-Fe(II) atom [32]. Similarly, the inhibition of nitrite-dependent conversion of ferrous HSA-heme-Fe to the nitrosylated derivative reflects warfarin binding to the FA2 site with the concomitant penta-to-hexa coordination interconversion of the heme-Fe(II) atom [35], Lastly, the nitrite-mediated conversion of ferrous human Hb to the nitrosylated derivative is modulated allosterically, inositol hexakisphosphate impairing the reactivity of ferrous human Hb stabilizing the low-reactivity T-state [25,26].
Over the whole NO concentration range explored, the time course for Mt-trHbN(III), Mt-trHbO(III), and Cj-trHbP(III) reductive nitrosylation corresponds to a biphasic process (Fig. 5, and Figs S5 and S6 in File S1, panel B); values of l and b are wavelength-independent at fixed NO concentration (data not shown).
The first step of kinetics for Mt-trHbN(III), Mt-trHbO(III), and Cj-trHbP(III) reductive nitrosylation (indicated by l in Scheme C) is a bimolecular process as observed under pseudo-first order conditions (Fig. 5, and Figs S5 and S6 in File S1, panel C) at all the pH values investigated. Plots of l versus [NO] are linear (Eqn. (6)), the slope corresponding to l on . Values of the second-order rate constant l on over the pH range explored (i.e., from pH 8.4 to 9.4; Tables S1-S3 in File S1) vary between 1.   -trHbP(III)). According to the trHb(III):NO 1:1 stoichiometry of reaction Ca in Scheme C, the Hill coefficient n is 1.0060.02. As expected for a simple system [23], values of L correspond to those of l off /l on , under all experimental conditions (Table 4 and Tables S1-S3 in File S1).
The second step of kinetics for Mt-trHbN(III), Mt-trHbO(III), and Cj-trHbP(III) reductive nitrosylation (indicated by Cb-Cd in Scheme C) follows a [NO]-independent monomolecular behavior (Fig. 5, and Figs S5 and S6 in File S1, panel E) at all the pH values investigated. According to Scheme C, the value of b increases linearly on increasing [OH 2 ] (i.e., from pH 8.4 to 9.4; Fig. 5, and Figs S5 and S6 in File S1, panel F, and Tables S1-S3 in File S1).  (Table 4).

Conclusion and Perspectives
The occurrence of different types of trHbs (i.e., trHbN, trHbO and trHbP) in bacteria, plants and some unicellular eukaryotes ( Fig. 1) opens the question of their role, also in view of their frequently simultaneous presence in the same organism; this envisages the possibility that different types of trHbs reflect different physiological roles in these organisms [1][2][3][4][5][6]11].
The high reactivity of Mt-trHbN and Cj-trHbP reflects both the penta-coordination of the heme-Fe-atom and the ligand accessibility to the heme pocket (Fig. 2). Indeed, the ligand access pathway through protein matrix tunnels in penta-coordinated Mt-trHbN (Fig. 2) [5,83] and the E7-path in penta-coordinated Cj-trHbP (Fig. 2) [5,22,66] is characterized by the lowest energy barrier, with values close to those displayed by penta-coordinated sperm whale Mb. Of note, ligand entry to and exit from the heme distal pocket of sperm whale Mb is modulated by gating movement of the HisE7 residue [75]. In contrast, the low reactivity of penta-coordinated Mt-trHbO [21] reflects H-bond interactions that lock the heme distal residues TyrCD1 and TrpG8 into a conformation(s) that limits ligand access to the heme distal pocket (Fig. 2) [18,70]. Of note, the E7-path appears to sustain ligand diffusion to the heme distal cavity of Mt-trHbO; indeed, Mt-trHbO hosts two protein matrix cavities instead of the protein matrix tunnel occurring in Mt-trHbN (Fig. 2) [84].
M. tuberculosis is massively exposed to NO during its intramacrophagic life, and Mt-trHbN has been shown to play a prominent role in protection from nitrosative stress [16]. Therefore, Mt-trHbN is most likely to function as NO dioxygenase that converts toxic NO into harmless nitrate in the presence of oxygen, and relieves toxicity due to NO and nitrosative stress. This is also consistent with the NO-inducible response of the gene encoding for trHbN in M. tuberculosis, both in vitro and inside infected macrophages [85]. Of note, also the promoter of the trHbO gene is induced during macrophage infection, though it poorly responds to NO induction [85]. Based on biochemical data, the evidence of trHbO in protection from NO is weak, while it is more likely that this protein is involved in oxygen sensing and aerobic respiration [86]. Similar considerations could hold true for the enteric pathogen C. jejuni, which colonizes the intestinal tract of birds and can cause enteritis in humans. Like other pathogens, C. jejuni is exposed to NO and other nitrosating species during host infection [87], the expression of both bacterial Cj-trHbP and 3-on-3 globin being upregulated by NO [11]. The 3-on-3 globin plays the major role in resistance to nitrosative stress and aerobically converts NO to nitrate [88], whereas the contribution of trHbP is less prominent. In contrast, both globins are devoid of NO-protective activity under oxygen-limited conditions that normally exist in vivo [89]. Therefore, the role of Cj-trHbP is clearly distinct form that of the 3-on-3 globin, being related to O 2 metabolism [7,8], likely performing a peroxidase or P450-type of oxygen chemistry [9].
As a whole, the comparison of the biochemical properties of microbial globins, including trHbs, will allow to shed light on their functional diversification and on the molecular bases of microbe ecology.

Supporting Information
File S1 Supporting tables and figures. (DOCX)