Nitrite-Reductase and Peroxynitrite Isomerization Activities of Methanosarcina acetivorans Protoglobin

Within the globin superfamily, protoglobins (Pgb) belong phylogenetically to the same cluster of two-domain globin-coupled sensors and single-domain sensor globins. Multiple functional roles have been postulated for Methanosarcina acetivorans Pgb (Ma-Pgb), since the detoxification of reactive nitrogen and oxygen species might co-exist with enzymatic activity(ies) to facilitate the conversion of CO to methane. Here, the nitrite-reductase and peroxynitrite isomerization activities of the CysE20Ser mutant of Ma-Pgb (Ma-Pgb*) are reported and analyzed in parallel with those of related heme-proteins. Kinetics of nitrite-reductase activity of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) is biphasic and values of the second-order rate constant for the reduction of NO2 – to NO and the concomitant formation of nitrosylated Ma-Pgb*-Fe(II) (Ma-Pgb*-Fe(II)-NO) are k app1 = 9.6±0.2 M–1 s–1 and k app2 = 1.2±0.1 M–1 s–1 (at pH 7.4 and 20°C). The k app1 and k app2 values increase by about one order of magnitude for each pH unit decrease, between pH 8.3 and 6.2, indicating that the reaction requires one proton. On the other hand, kinetics of peroxynitrite isomerization catalyzed by ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) is monophasic and values of the second order rate constant for peroxynitrite isomerization by Ma-Pgb*-Fe(III) and of the first order rate constant for the spontaneous conversion of peroxynitrite to nitrate are h app = 3.8×104 M–1 s–1 and h 0 = 2.8×10–1 s–1 (at pH 7.4 and 20°C). The pH-dependence of h on and h 0 values reflects the acid-base equilibrium of peroxynitrite (pK a = 6.7 and 6.9, respectively; at 20°C), indicating that HOONO is the species that reacts preferentially with the heme-Fe(III) atom. These results highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.


Introduction
Phylogenetic analysis revealed that members of the globin superfamily evolved from an ancestral monomeric flavo-hemoglobin and were arranged in three globin lineages and two structural classes. The first lineage includes flavo-hemoglobins and related single domain globins, the second lineage embraces truncated hemoglobins, and the third lineage encompasses two-domain globin-coupled sensors, single-domain sensor globins and related single-domain protoglobins (Pgb) [1,2]. All members of the first and third lineage belong to the same structural class, showing a long amino acid sequence (.145 residues) and the classical 3-on-3 a-helical fold found in myoglobin, with the heme group surrounded by the A, B, and E a-helices on one side and the F, G, and H a-helices on the other [3,4]. Conversely, all members of the second lineage, consisting of three subgroups, belong to the same structural class and show a short amino acid sequence (,135 residues), adopting the 2-on-2 a-helical sandwich fold, 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 [5].
Methanosarcina acetivorans protoglobin (Ma-Pgb), generally taken as the molecular model of Pgbs, shows a homodimeric quaternary structure mostly based on the inter-molecular four-helix bundle built by the G and H a-helices of each protomer. The threedimensional structure of the Ma-Pgb* monomer (a site-directed mutant of Ma-Pgb displaying the CysE20Ser mutation to prevent the formation of intermolecular disulphide bonds) shows that the 195 amino acid chain can be considered an expanded version of the classical 3-on-3 a-helical fold globin fold, being characterized by the presence of a 20-residue N-terminal extension and a pre-A a-helix Named Z-helix). Moreover, the heme of Ma-Pgb* is markedly distorted and fully buried in the protein matrix, due to extended CE and FG loops and the 20-residue N-terminal extension (Fig. 1, panel A). Therefore, the access of ligands to the heme distal pocket is granted by two protein matrix tunnels, which are located at the B/G (tunnel 1) and B/E (tunnel 2) a-helix interfaces (Fig. 1, panel B) [11][12][13][14][15][16].
Multiple functional roles have been postulated for Ma-Pgb, since the detoxification of reactive nitrogen and oxygen species, which appears pivotal in the physiology of the strictly anaerobe Methanosarcina acetivorans, might co-exist with enzymatic activities facilitating the conversion of acetate, methanol, CO 2 , and CO to methane [17]. As expected for a strict anaerobe, the generation of reduced Ma-Pgb* requires an O 2 -free environment as the heme-Fe(II) atom autoxidizes rapidly (t 1/2 = 3.6 min) [7,8]. On the other hand, ferric Ma-Pgb* (Ma-Pgb*-Fe(III)) undergoes reductive nitrosylation through the transient formation of the ferric nitrosylated species, which is converted to the ferrous form that in turn binds NO very rapidly [16].
Here, kinetics of the NO 2 --mediated conversion of ferrous Ma-Pgb* (Ma-Pgb*-Fe(II)) to the ferrous nitrosylated derivative (Ma-Pgb*-Fe(II)-NO) and of the ferric Ma-Pgb*-Fe(III)-mediated peroxynitrite isomerization are reported and analyzed in parallel with those of related heme-proteins to highlight the potential role of Pgbs in the biosynthesis and scavenging of reactive nitrogen and oxygen species.

Materials
Ma-Pgb*-Fe(III) was expressed in Escherichia coli cells Bl21(DE3)-pLysS, collected, and purified as previously reported [11,15]. The analysis of the structure of Ma-Pgb*-Fe(II) and Ma-Pgb*-Fe(III) derivatives indicates that the SerE20 residue is not involved in any specific interaction critical for structural stability, but is partly exposed to the solvent (see Fig. 1). Moreover, SerE20 falls at the Cterminal end of the E-helix, in a location 18 Å away from the heme-ligand binding site, from which this residue is isolated by the protein matrix (see Fig. 1). Thus, the CysE20Ser mutation is justifiable to ease crystallization [14,17,18]. Furthermore, values the second-order rate constants for CO binding to ferrous wild type Ma-Pgb and Ma-Pgb*, obtained by rapid-mixing stoppedflow and laser photolysis techniques, are closely similar [11,15]. The different values of the first-order rate constants for CO dissociation from ferrous carbonylated Ma-Pgb and Ma-Pgb*, obtained by different methods based on the conversion of the CObound protein to the NO-bound form (i.e., by mixing the carbonylated protein with dithionite/nitrite or the NO donor MAHMA NONOate solution) [11,15], may reflect different steps or mechanisms of CO escaping from the heme pocket to the solvent.
The Ma-Pgb*-Fe(III) concentration was determined spectrophotometrically using the extinction coefficient at 399 nm (i.e., e = 1.38610 5 M 21 cm 21 ), pH 7.0 (5.0610 22 M 1,3-bis(tris(hydroxymethyl)methylamino)propane (bis-tris-propane) buffer) and 20uC (see [18] and present study). Ma-Pgb*-Fe(II) was obtained, under anaerobic conditions, by adding Na 2 S 2 O 4 (final concentra- The secondary structure elements are labeled A-H'. The 20 N-terminal residues and the extended CE and FG loops that seal the heme pocket and prevent the access of small ligands to the heme distal cavity are in orange. The pre-A Z-helix is in green. The heme (red) is displayed edge on. The proximal HisF8 residue is shown on the left hand side of the heme. The picture includes the mutated SerE20 residue, located at the C-terminus of the E-helix. The HisF8 and SerE20 side chains and residues building up the heme distal pocket are drawn as skeletal models (C atoms yellow, N atoms blue, and O atoms red) and labeled. (Panel B) Mono views of ''tunnel 1'' (top) and ''tunnel 2'' (bottom) access sites in Ma-Pgb*. Helices flanking the tunnel entries are labelled. The heme group (seen through the tunnel apertures) is shown in red. The protein is correctly oriented in both images, to bring each tunnel in the direction of sight. The images are rotated by 90u. The pictures have been drawn by UCSF -Chimera [55]. For details, see ref. [11]. doi:10.1371/journal.pone.0095391.g001 tion, 1.0610 -3 M to 5.0610 -3 M). The Ma-Pgb*-Fe(II) concentration was determined spectrophotometrically using the pHindependent (5.0610 22 M bis-tris-propane buffer) extinction coefficient at 432 nm (i.e., e = 1.25610 5 M 21 cm 21 ), 20uC [15].
CO was purchased from Linde AG (Höllriegelskreuth, Germany) or Rivoira (Milan, Italy). The CO stock solution was prepared by keeping anaerobically distilled water in a closed vessel under CO at P = 760.0 mm Hg (T = 20uC). The solubility of CO in water is 1.03610 23 M, at P = 760.0 mm Hg and 20uC [33].
All chemicals where of analytical grade and were used without further purification unless stated.

Methods
The Although the sodium dithionite concentration lower than 1.0610 -2 M has been reported not to reduce significantly to NO [34], the appropriate control was performed as follows. Five mL of the NO 2 solution (final concentration, 1.0610 -2 M) were reacted with 5.0 mL of the sodium dithionite solution (final concentration, 2.0610 -3 M), at pH 7.4 and 20uC for 10 min, under anaerobic conditions. Then, the concentration of NO 2 was determined spectrophotometrically at 543 nm by using the Griess reagent [35] and by reductive chemiluminescence [34]. -mediated conversion of Ma-Pgb*-Fe(II) to Ma-Pgb*-Fe(II)-NO in the absence and presence of CO was analyzed in the framework of the minimum reaction mechanism depicted by Scheme 1 [16,34,[36][37][38][39][40][41][42][43][44][45]:

Scheme 1
It is important to outline that the two processes described by k app1 and k app2 correspond to the same reaction for the two molecular species observed (see below). The process depicted by fast corresponds to the very fast nitrosylation of Ma-Pgb*-Fe(II) in the presence of sodium dithionite [16].
Values of the pseudo-first-order rate constant (i.e., k obs1 and k obs2 ) and of the amplitude (i.e., [Ma-Pgb*-Fe(II)] i1 and [Ma-Pgb*-Fe(II)] i2 ) of the fast and slow phases, respectively, for the NO 2 -mediated conversion of Ma-Pgb*-Fe(II) to Ma-Pgb*-Fe(II)-NO were determined, in the absence and presence of CO (final concentration, 1.0610 24 M), from data analysis, according to Eqs 1a and 1b, depending on the wavelength (i.e., on the increase or the decrease of the spectral change; see Fig. S1) [36,42,43,45,46]: Values of the second order rate constant for the NO 2 --mediated conversion of Ma-Pgb*-Fe(II) to Ma-Pgb*-Fe(II)-NO (i.e., k app1 and k app2 ) were obtained from the dependence of k 1 and k 2 on the NO 2 concentration (i.e., [NO 2 -]), according to Eqs 2 and 3, respectively [36,42,43,45,46]: According to literature [36,42,43,45,46], the values of k app1 and k app2 refer to the interaction of NO 2 with Ma-Pgb-Fe*(II) under the general assumption that this process represents the ratelimiting step of the overall reaction reported in Scheme 1.

Data Analysis
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 the NO 2 -mediated conversion of Ma-Pgb*-Fe(II) to Ma-Pgb*-Fe(II)-NO corresponds to a biphasic process, the amplitude of the fast and of the slow phase corresponding to 5765% and 4365%, respectively, of the whole process, at all NO 2 concentrations (Fig. 3). Values of the pseudo-first-order rate constant for the NO 2 --mediated conversion of Ma-Pgb*-Fe(II) to Ma-Pgb*-Fe(II)-NO (i.e., k 1 and k 2 ; see Eq. 1) are wavelength-independent at fixed NO 2 concentration (data not shown). Of note (see [34] and present study), values of k 1 and k 2 are unaffected by the dithionite concentration between 1.0610 -3 M and 5.0610 -3 M (data not shown).
Values of k app1 and k app2 increase linearly with the NO 2 concentration (Fig. 4). The analysis of data reported in Fig. 4 according  Fig. S2. Therefore, the NO 2 --mediated nitrosylation of Ma-Pgb*-Fe(II) reflects the reaction of NO 2 with the heme-Fe(II) atom, as reported for plant and cyanobacterial hemoglobins [42].
To identify tentatively the species that preferentially react(s) with Ma-Pgb*-Fe(III), the effect of pH on kinetics of peroxynitrite isomerization in the absence and presence of Ma-Pgb*-Fe(III) (i.e., on h 0 and h on ) was examined. As shown in Fig. 8, values of h 0 and h on increase on lowering pH from 8.3 to 6.2; the analysis of data, according to Eq. 6, allowed the estimation of pK a values for the pH dependence of h 0 and h on values for peroxynitrite isomerization in the absence and presence of Ma-Pgb*-Fe(III). The pH dependence   of h 0 and h on for peroxynitrite isomerization in the absence and presence of Ma-Pgb*-Fe(III) is similar, the pK a values being 6.9 and 6.7, respectively (Fig. 8). The pK a values for the pH dependence of h 0 and h on here determined are in excellent agreement with pK a values reported in the literature [28,48]. According to literature [22,24,28], the close similarity of the pH dependence of h 0 for peroxynitrite isomerization in the absence of Ma-Pgb*-Fe(III) (pK a = 6.9) (Fig. 8, panel A) and of h app for peroxynitrite isomerization by Ma-Pgb*-Fe(III) (pK a = 6.7) (Fig. 8,  panel B) suggests that HOONO is the species that reacts preferentially with the heme-Fe(III) atom.

Discussion
The observation, reported in this work, that Ma-Pgb* displays in the ferrous form (i.e., Ma-Pgb*-Fe(II)) a nitrite-reductase activity and in the ferric form (i.e., MaPgb*-Fe(III)) catalyzes peroxynitrite isomerization opens a new scenario on the physiological role played by this Pgb in the framework of Methanosarcina acetivorans metabolism. The efficiency of these two processes catalyzed by Ma-Pgb* indeed suggests that this heme-protein might play a role in the metabolism of reactive nitrogen and oxygen species facilitating, under reducing conditions, NO synthesis from NO 2 and, under oxidative conditions, peroxynitrite conversion to NO 3 -. In Table 1, the ligand-linked reactions of the reduced and oxidized forms of MaPgb* are summarized.
Unlike all known globins, Ma-Pgb*-specific loops and the Nterminal extension completely bury the heme within the protein matrix (Fig. 1, panel A). Therefore, the access of ligands to the heme distal pocket is granted by the apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces (Fig. 1, panel B) [11]. The presence of the two tunnels within the protein matrix may be partly responsible for the biphasic ligand binding behavior of Ma-Pgb*-Fe(II) towards NO 2 -(present study) and CO [15,18] (see Table 1). In contrast, azide binding to Ma-Pgb*-Fe(III) [18], cyanide dissociation from the Ma-Pgb*-Fe(III)cyanide complex [17], Ma-Pgb*-Fe(III) nitrosylation [16], and Ma-Pgb*-Fe(III)-mediated peroxynitrite isomerization (present study) correspond to a monophasic process (see Table 1). Taken together, these data suggest the occurrence of the interplay between the oxidation state of the heme-Fe-atom and the modulation of ligand binding kinetics, which might be related to some conformational and/or dynamic differences between Ma-Pgb*-Fe(II) and Ma-Pgb*-Fe(III) and/or an oxidation state-linked different role of the apolar tunnels in modulating the ligand pathway. In this respect, TyrB10 and IleG11 residues, located in the heme distal site and lining the protein matrix tunnels 1 and 2, respectively, display a crucial role on the heme distal site structural organization and on the modulation of ligand binding to the heme-Fe-atom. In particular, the ligand accessibility to the heme distal site through tunnel 1 is modulated by the ligand-dependent reorganization of the TrpB9 and PheE11 side-chains, triggered by the TyrB10 and IleG11 residues. In this scenario, the PheE11 residue acts as the ligand sensor and controls the ligand accessibility to the heme distal pocket by modifying the conformation of the TrpB9 side chain [10,14,17]. Therefore, subtle different geometries of these residues in Ma-Pgb*-Fe(II) and Ma-Pgb*-Fe(III) could be reflected in a different regulation of ligand binding, as observed for kinetics of Ma-Pgb*-Fe(III) reactivity towards azide, cyanide, NO, and peroxynitrite (which is monophasic), and for kinetics of Ma-Pgb*-Fe(II) reactivity towards CO and NO 2 2 (which is biphasic) (see Table 1). In this respect, the only exception is represented by the monophasic very fast reaction of Ma-Pgb*-Fe(II) with NO (see Table 1) [16], which may either (i) reflect the loss of the first very fast step in the deadtime of the rapid-mixing stopped-flow apparatus, or else (ii) the existence of a ligand-linked conformational change(s) slower than NO binding and faster than CO binding, this resulting in a monophasic process for Ma-Pgb*-Fe(II) nitrosylation and of a biphasic process for Ma-Pgb*-Fe(II) carbonylation.
The k app values for the NO 2 --mediated conversion of the ferrous deoxygenated HisE7Ala and HisE7Leu mutants of sperm whale myoglobin to their ferrous nitrosylated derivatives are significantly lower than that of the wild type protein, reflecting the different  Table 2) [43].
The low k app values for the NO 2 --mediated conversion of ferrous human cytoglobin to the ferrous nitrosylated derivative could reflect the hexa-coordination of the heme-Fe(II) atom (see Table 2) [44]. However, the NO 2 --mediated conversion of ferrous human neuroglobin to the ferrous nitrosylated derivative reflects the reversible redox-linked hexa-to-penta-coordination transition of the heme-Fe(II) atom. Therefore, under oxidative conditions, the formation of the CysCD4-CysD5 bridge stabilizes the highreactive penta-coordinated heme-Fe(II) atom facilitating the reaction. In contrast, under reductive conditions, the cleavage of the CysCD4-CysD5 bridge leads to the formation of the lowreactive hexa-coordinated heme-Fe(II) atom. Accordingly, the CysCD4Ala and CysD5Ala mutations, impairing the formation of the CysCD4-CysD5 bridge and stabilizing the hexa-coordinated heme-Fe(II) atom, slow down the NO 2 --mediated conversion of ferrous human neuroglobin to its ferrous nitrosylated derivative. Also the HisE7Leu and HisE7Gln mutations, leading to a stable penta-coordinated heme-Fe(II) atom, facilitate the nitrite-reductase activity of ferrous human neuroglobin [43].
Although the heme-Fe atom of Synechocystis hemoglobin, Arabidopsis thaliana nonsymbiotic hemoglobins classes 1 and 2, and rice nonsymbiotic hemoglobin class 1 has been reported to be basically hexa-coordinated, k app values for the NO 2 --mediated conversion of the ferrous deoxygenated derivative to the ferrous nitrosylated species range between 4.9 M -1 s -1 and 6.86 10 1 M -1 s -1 (see Table 2). This finding has been interpreted assuming that a substantial fraction of the ferrous derivative of these proteins may display a penta-coordinated heme geometry [42,45]. In the case of Ma-Pgb*-Fe(II) nitrite reductase activity, the observed rate constants are k app1 = 9.660.2 M -1 s -1 and k app2 = 1.260.1 M -1 s -1 ; therefore, their values appear to fall in the average range observed for other heme-proteins, being somewhat slower that for Synechocystis hemoglobin, Arabidopsis thaliana nonsymbiotic hemoglobin class 1 and rice non symbiotic Hb class 1, but significantly faster than human cytoglobin and human neuroglobin (see Table 2). Such behavior suggests that the access of NO 2 to the heme, and heme reactivity, indeed meet some energetic barrier (due to the limited access through the apolar tunnels), but the heme itself should be mostly penta-coordinated.
Lastly, the NO 2 --mediated conversion of ferrous human hemoglobin to its ferrous nitrosylated derivative is impaired allosterically by inositol hexakisphosphate binding to the central cavity of the tetramer, stabilizing the low reactive T-state (see Table 2) [37,38]. Also the NO 2 --mediated conversion of ferrous human serum heme-albumin to its ferrous nitrosylated derivative is inhibited allosterically by warfarin binding to the fatty acid binding site 2. In fact, warfarin binding to ferrous human serum heme-albumin induces the hexa-coordination of the heme-Fe atom which becomes unreactive (see Table 2) [46]. Peroxynitrite isomerization is facilitated by Ma-Pgb*-Fe(III), whereas the Ma-Pgb*-Fe(III)-azide derivative is non-reactive, clearly demonstrating that the efficiency of the isomerization process reflects the heme-Fe(III) reactivity; moreover, HOONO appears to be the species that preferentially reacts with Ma-Pgb*-Fe(III). Peroxynitrite isomerization by Ma-Pgb*-Fe(III), Mycobacterium tuberculosis truncated-hemoglobin N, Pseudoalteromonas haloplanktis TAC125 truncated-hemoglobin O, horse heart myoglobin, sperm whale myoglobin, human hemoglobin, human serum heme-albumin, cardiolipin-bound horse heart cytochrome c as well as cardiolipin-free and -bound carboxymethylated horse heart cytochrome c (see Table 3) represents a common feature of pentacoordinated heme-proteins (see [22,24,28,29,31,32] and present study). In contrast, hexa-coordinated ferric horse heart cytochrome c and ferric human neuroglobin do not catalyze peroxynitrite isomerization [29,50]. Notably, cardiolipin acts as an allosteric effector of horse heart cytochrome c inducing the cleavage of the sixth coordination bond of the heme-Fe atom (i.e., Met80-Fe), thus stabilizing the penta-coordinated derivative [29].
The value of h on for peroxynitrite isomerization by MaPgb*-Fe(III) (see Table 3) falls in the same range as observed for other heme-proteins from different sources (such as Mycobacterium tuberculosis truncated-hemoglobin N and mammalian myoglobins and hemoglobins; see Table 3), clearly indicating that the binding process is closely similar, and confirming that MaPgb*-Fe(III) is essentially penta-coordinated [10,11]. In this respect, the analysis of kinetics for peroxynitrite isomerization by ferric sperm whale myoglobin mutants (see Table 3) suggests that the heme-Fe(III) reactivity towards peroxynitrite is regulated either by steric factors modulating the ligand accessibility to the metal center (in the absence of a hydrogen-bonding residue, i.e. His E7) or by the Lewis acidity of the heme-Fe(III) atom [24]. Thus, the two most  [51], and the values of h on for the heme-based conversion of peroxynitrite to NO 3 -(see Table 3) [24]. It is worth noting that also azide binding to Ma-Pgb*-Fe(III) displays a behavior closely similar to what has been observed for other heme-proteins [18], indeed suggesting that in the ferric form steric factors posed by the two apolar tunnels do not dramatically alter the energetics of the ligand binding pathway. Sperm whale myoglobin f 6.0

Conclusions
Ma-Pgb shows a selectivity ratio for O 2 /CO binding that favors O 2 ligation [11] and displays anti-cooperativity in CO binding [15]. This very unusual behavior could be related to the fact that Methanosarcina acetivorans takes advantage of acetate, methanol, CO 2 and CO as carbon sources for methanogenesis; methane production occurs simultaneously with the formation of a proton gradient that is essential for energy harvesting [9,52,53]. Therefore, the capability to convert CO to methane suggests that CO is the actual ligand of Ma-Pgb in vivo, this being in agreement with the hypothesis of the very ancient origin for this metabolic pathway(s) [9,54].
However, an additional Ma-Pgb role, which has been already postulated for other bacterial heme-proteins, is that of detoxifier to preserve the environment free of oxygen and reactive nitrogen and oxygen species. This hypothesis, which is obviously not in contrast with the metabolic role of Ma-Pgb, is supported by the present data. We show that Ma-Pgb* is able to play such scavenging role both in the reduced form, whereby under reducing environmental conditions Ma-Pgb*-Fe(II) may behave as a nitrite-reductase, and in the oxidized form, Ma-Pgb*-Fe(III) being able to catalyze the isomerization of peroxynitrite to nitrate in an oxidizing atmosphere. Such multiple roles may indeed reflect the adaptation of this ancient protein to different environmental conditions met during evolution.   [31]. d pH 7.0 and 20uC. From [22]. e pH 7.5 and 20uC. From [24]. f pH 7.5 and 20uC. From [22]. g pH 7.2 and 22uC. From [28]. h pH 7.0 and 20uC. Cardiolipin was 1.6610 -4 M. From [29]. doi:10.1371/journal.pone.0095391.t003