Comparison of the TvPaR and TvNiR structures
The translated nucleotide sequence of TvPaR (GenBank accession number HQ665012.1) contains 553 residues, including 28 residues of the signal peptide. The amino acid sequence alignment of TvPaR and TvNiR (GenBank accession number AJ880678.2) shows 81% identity.
The structures of the free form of TvPaR and the TvPaR complex with sulfite as the substrate (TvPaR–SO3) were refined to Rf 0.138 (1.9-Å resolution) and 0.134 (2.0-Å resolution), respectively. There are two TvPaR subunits per asymmetric unit (A and B). The subunits of TvPaR form a hexamer with point group symmetry 32 (Fig. S1), similar to the TvNiR hexamer . In each subunit, 519 residues are visible in the electron density (residues 5–523).
The structures of the TvPaR and TvNiR subunits are very similar; 95 amino acid substitutions in the structure of the TvPaR subunit as compared with TvNiR are accompanied by no changes in the secondary structure. The subunits of TvPaR and TvNiR [Protein Data Bank (PDB) ID: 2OT4] can be superimposed by use of the Cα atoms of 519 residues with an rmsd of 0.49 Å. The sulfite binding in the active site of TvPaR is similar to that in TvNiR (PDB ID: 3FO3)  (Fig. 1). All features that distinguish TvNiR from NrfA were found in the TvPaR structures as well.
Figure 1. Binding of the sulfite ion in the active site of TvPaR. The 2Fo − Fc electron density map for Tyr303 and Cys305 (1σ) and the OMIT electron density map for the sulfite and cobalt ions (5σ) are shown. The OMIT map corresponds to the 2Fo − Fc map in these regions contoured at 1σ. Hydrogen bonds and coordination bonds are indicated by dashed lines. The protoporphyrin of heme 4 and the calcium ion are shown in gray.
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Substantial differences between TvPaR and TvNiR are observed in the vicinity of heme 6 (Fig. 2). The replacement of Pro400 and Val225 in TvPaR by Gln400 and Leu225 in TvNiR results in rotation of the imidazole ring of His119 coordinated to heme 6 at the distal side about the CB–CG bond by ~ 30° (Fig. 2). Also, the Pro400Gln substitution leads to a change in the side chain conformation of Phe77, which is, in turn, associated with the replacement of Pro63 in TvPaR by Leu63 in TvNiR. Because of the above-mentioned structural changes, the redox properties of heme 6 in TvPaR may differ from those in TvNiR.
Figure 2. Superposition of the structures of TvPaR (green) and TvNiR (pink), by use of the atoms of hemes 6. Ser399 and Gln400 in TvNiR have two conformations.
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The only substantial difference concerning the substrate and product channels is, apparently, the replacement of Leu88 in TvNiR by Glu88 in TvPaR. This replacement leads to an increase in the negative charge at the exit from the product channel into the cavity in the hexamer, which may facilitate the directed transport of the reaction product (ammonium ion) from the active site.
There are two cobalt ions per subunit in the structures of TvPaR and TvPaR–SO3. The crystallization solution served as a source of cobalt ions. One cobalt ion (Fig. 1) is bound in the active site pocket of the enzyme and interacts with propionate D of heme 4 and the side chain of His113 located at the beginning of the product channel. In TvPaR–SO3, this ion is characterized by an octahedral environment and an occupancy of 0.5. The second cobalt ion is bound at the exit from the product channel, and is coordinated by the side chains of Glu88 and His85. The binding of both of these ions may interfere with ammonium ion transport from the active site, which accounts for the observed inhibition of the nitrite reductase activity by cobalt ions (Fig. S2).
In the structures of TvPaR and TvPaR–SO3, a sulfate ion is located in the vicinity of heme 1 of subunit A (Fig. 3). This ion is hydrogen-bonded to the ND1 atom of His18, ligating heme 1. In the structures of TvNiR complexes with sulfite (PDB IDs: 3LGQ and 3FO3) [19, 22], sulfite and sulfate ions are located in the same position.
Figure 3. Binding of the sulfate ion near heme 1 in the structure of TvPaR. The 2Fo − Fc electron density map for the sulfate ion is shown (1σ). The coordination bond and hydrogen bonds are indicated by dashed lines. The side chain of Thr19 has two conformations.
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Catalytic properties of TvPaR and TvNiR
TvPaR and TvNiR exhibit high nitrite reductase activity, being the most active among cytochrome c nitrite reductases (Table 1). As in the case of NrfAs, ammonium is the final reaction product .
Table 1. Kinetic parameters of the nitrite and sulfite reduction catalyzed by TvPaR, TvNiR, and NrfAs
|Enzyme||Nitrite reduction||Sulfite reduction|
|kcat (s−1)||Km (mm)||kcat/Km (m−1·s−1)||kcat (s−1)||Km (mm)||kcat/Km (m−1·s−1)|
|TvPaR||4160 ± 320||0.10 ± 0.02||4.2 × 107||0.06 ± 0.02||0.08 ± 0.04a||7.5 × 102a|
|TvNiR||3100 ± 300||0.18 ± 0.05||1.7 × 107||0.04 ± 0.01 || || |
|NrfA from D. desulfuricans [24-27]||415||1.140||3.6 × 105||0.63||0.75||9 × 102|
|NrfA from E. coli [24, 28]||769||0.022 ± 0.007||3.5 × 107|| ||0.070 ± 0.015||4.3 × 102|
|NrfA from Sulfospirillum deleyianum ||962|| || ||0.34|| || |
|NrfA from Wolinella succinogenes ||380|| || ||0.4|| || |
|NrfA from Desulfovibrio vulgaris ||639|| || ||0.3|| || |
In spite of the high structural similarity of TvPaR and TvNiR, there are some differences in their catalytic properties. For TvNiR, substantial inhibition by the substrate was observed at a nitrite concentration > 1 mm. The inhibition constant (Ks′) is 2.5 ± 0.5 mm. For TvPaR, no inhibition by the substrate was found up to a nitrite concentration of 3 mm (Fig. S3).
The catalytic constant (kcat) for nitrite reduction by TvPaR is 1000 units higher than that for by nitrite reduction TvNiR (Table 1). This difference was reproduced for different samples of both enzymes, and reflects the more efficient catalysis of the nitrite reductase reaction by TvPaR. This may be associated with the structural differences between the enzymes in the environment of heme 6 (Fig. 2), which is located at a distance of 9.6 Å (Fe–Fe) from the catalytic heme 4 and mediates the electron transfer to the catalytic heme. The redox properties of heme 6 can play a key role in the catalysis of the nitrite reductase reaction by influencing the rate of electron transfer to nitrite, as well as the rate of intramolecular electron transport. A higher negative charge at the exit of the product channel resulting from the replacement of Leu88 in TvNiR by Glu88 in TvPaR may contribute to the high nitrite reductase activity of TvPaR. The importance of the charge of the channel for efficient catalysis is confirmed by the inhibitory effect of cobalt(II) ions, which are bound at both ends of the product channel, thus decreasing the negative potentials in these regions.
The sulfite reductase activity of TvPaR, measured at a saturating concentration of sulfite (1 mm, 10 times larger than the Ki for sulfite; see below), is identical to that of TvNiR  (Table 1). This activity is similar to the sulfite reductase activity of NrfA from Escherichia coli, which, like Thioalkalivibrio species, belongs to the γ-proteobacteria, and is lower than the activities of NrfAs from δ-proteobacteria and ε-proteobacteria (Table 1). The nitrite activity/sulfite activity ratio for TvPaR and TvNiR (6–7 × 104) is much higher than that for NrfAs (0.6–5.5 × 103), which may be indicative of the finer tuning of the active sites of TvPaR and TvNiR to the catalysis of nitrite reduction.
For TvNiR [19, 20, 22], it was shown that sulfite and nitrite are bound to the catalytic heme, and hence sulfite should act as a competitive inhibitor in measurements of the nitrite reductase activity in the presence of sulfite. The concentration curves for TvPaR (v versus substrate concentration) measured at different sulfite concentrations, and represented as linear Lineweaver–Burk plots, intersect in the left upper quadrant (Fig. S4). This indicates the mixed type of inhibition, which is close to the competitive one but implies that there is another sulfite-binding site, where sulfite can be bound to the enzyme—substrate complex TvNiR—NO2-. The competitive inhibition constant is 0.08 ± 0.04 mм. Therefore, NO2− and HSO3− have almost equal affinities for the active site of TvPaR, and sulfite is an efficient inhibitor of the nitrite reductase reaction. This fact may be important in regulating the nitrite reductase activity of octaheme nitrite reductases in cells.
The Michaelis constant for nitrite reduction determined earlier for TvNiR (16.7 ± 4.0 mm)  is overestimated, owing to the competitive inhibition by sulfite that is formed in the course of oxidation of dithionite, which was used as an electron source. The fact that we obtained sulfite complexes of TvNiR  or TvPaR after soaking of crystals of these enzymes in the free form with dithionite strongly suggests that sulfite, rather than sulfate or another decomposition product of dithionite, is responsible for the observed inhibition of the enzyme.
The dissociation constant (Ki′) for the second sulfite-binding site is approximately 1.3 ± 0.3 mm. The additional sulfate- and sulfite-binding site found in the TvPaR and TvNiR structures in the vicinity of heme 1 may correspond to this second sulfite-binding site (Fig. 3). Heme 1 is one of the most solvent-exposed hemes , and can serve as one of the primary electron acceptors from such a donor as reduced methyl viologen (MV). The binding of negatively charged ions can lead to a change in the potential of this heme, thus influencing the rate of the nitrite reductase reaction.
Effect of pH and ionic strength on the nitrite reductase activity of TvPaR
Like TvNiR, TvPaR is a periplasmic protein from haloalkaliphilic bacteria, whose optimal growth conditions are pH 10–10.2 and a salt concentration of ~ 0.5 м (NaHCO3/Na2CO3) . The examination of the influence of the pH value on the rate of the nitrite reductase reaction (Vm) catalyzed by TvPaR showed that the pH optimum is within a neutral and weakly alkaline pH range (7.0–7.5) (Fig. 4A). In alkaline media (pH 9.5–10.5), the activity is high enough and equal to 15–20% of the initial value.
Figure 4. (A) The pH dependence of the nitrite reductase activity of TvPaR. The reaction was performed in 50 mm potassium phosphate/Tris (pH 6.5–9.5) and sodium borate (pH 9.5–10.5) buffer systems; nitrite, 1 mm; TvPaR, 0.036 μg·mL−1. The activity at pH 7.0 was taken as 100%. (B) The influence of NaCl concentration on the nitrite reductase activity of TvPaR. The conditions were as follows: 50 mm potassium phosphate buffer, pH 7.0; TvPaR, 0.036 μg·mL−1; nitrite, 1 mm. The activity at 0 m NaCl was taken as 100%.
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An increase in the NaCl concentration in solution to 1–2 m led to a decrease in the nitrite reductase activity of TvPaR (Fig. 4B). At 0.5 m NaCl, the activity was ~ 60% of the initial activity. This effect can be attributed to the fact that electrostatic interactions essential for the transport of charged substrate and product molecules play a smaller role in salt solutions. Therefore, the specific activating effect of the high-salinity and alkaline pH medium on the activity of TvPaR was not observed in the model nitrite reduction reaction with MV.
A decrease in the rate of the nitrite reductase reaction at alkaline pH values can be attributed to a combination of several factors: the stoichiometry of the reaction (the six-electron reduction of nitrite is accompanied by the binding of eight/seven protons, whose concentration decreases with increasing pH), the less efficient transport of an uncharged ammonia molecule (pKa 9.3) through the product channel, and the deprotonation of the active site residues essential for the catalysis.
The active sites of TvPaR and TvNiR contain two groups whose deprotonation occurs in the pH range where the enzymes exhibit catalytic activity (7.0–10.0), and this can influence the efficiency of the catalysis. One of these groups is the imidazole group of His361. It was suggested that the catalytic histidine is the most probable proton donor at the first stage of nitrite reduction by NrfAs . Another group is the hydroxyl of Tyr303, which is hydrogen-bonded to the O1 atom of nitrite in the active site of TvNiR , and can also be involved in proton transfer to the substrate. Owing to the formation of a covalent bond between Tyr303 and Cys305, the pKa of the Tyr303 hydroxyl group can decrease by 0.5–1 [30-32]. Actually, the inflection point in the right branch of the pH dependence is equal to 8.5–8.6 (Fig. 4A), and is in the range of the possible pKa of Tyr303. This is indirect evidence in favor of the participation of Tyr303 in catalysis as a proton donor. The role of Tyr303 as the proton donor is also supported by the fact that the TvNiR and TvPaR structures have a channel through which protons from the protein surface could be transferred to the hydroxyl group of Tyr303 . In the NrfA structures, this channel is absent. Therefore, the involvement of Tyr303 linked to Cys305 in the catalytic actions of TvPaR and TvNiR might enable the extension of the pH range in which the enzymes exhibit high catalytic activity, and this may be of essential importance for enzymes from haloalkaliphilic bacteria.
Sequence analysis of proteins homologous to TvPaR and TvNiR
A search for sequences homologous to TvNiR and TvPaR in the UniProt Knowledgebase (Swiss-Prot) revealed genes of 16 proteins with an identity of 40% and higher (Fig. S5). One of these proteins (G4DHD8) was discovered in the genome of another bacterium of the genus Thioalkalivibrio – Thioalkalivibrio thiocyanoxidans. According to blast, this protein has 100% identity with TvPaR. Representatives of the lineage T. thiocyanoxidans were isolated together with T. paradoxus from a medium containing thiocyanate as the only source of energy, nitrogen, and sulfur. The presence of octaheme nitrite reductase in yet another organism of the genus Thioalkalivibrio suggests the importance of this class of proteins for cell viability.
Among homologs from the genomes of bacteria belonging to other genera and classes of proteobacteria, the highest homology was found with octaheme cytochromes c from the haloalkaliphilic δ-proteobacterium Desulfurivibrio alkaliphilus (D6Z5C1; 53% identity) and the Desulfobulbaceae bacterium MLMS1 (Q1NQZ7; 54% identity) isolated from soda lake sediments, like the γ-proteobacteria T. nitratireducens and T. paradoxus. Homologous proteins were also found in the genomes of obligately anaerobic δ-proteobacteria of the genus Geobacter (Q74G90, D7AEA4, A5G8N0, C6E3S2, E8WT73, B5EIZ4, B9M3N3, and B3E660; 48–46% identity), Pelobacter propionicus (A1ATC6; 49% identity), aerobic β-proteobacteria of the genus Burkholderia (F3QGY9, E7H1H6, and D9Y3D5; 40–41% identity), and the anaerobic bacterium Calditerrivibrio nitroreducens belonging to the phylum Deferribacteres (E4TEZ1; 43% identity).
All translated amino acid sequences contain (Fig. S5) seven CXXCH heme-binding motifs and one CXXCK motif unique to cytochrome c nitrite reductases, the latter motif in all sequences being the fourth from the N-terminus of the molecule. The sequence alignment of the structures superimposed by use of the heme-binding motifs showed that all sequences contain: (a) the conserved catalytic residues corresponding to the active site residues His361, Arg131 and Tyr303 of TvNiR and TvPaR (hereinafter, the numbering of the sequence for TvNiR and TvPaR is used), as well as Phe109 and Lys358, which are involved in the active site pocket of TvNiR and TvPaR; (b) the conserved residues Glu302, Gln360 and the above-mentioned Lys358, which are responsible for the binding of the Ca2+ that is present in all pentaheme and octaheme nitrite reductases; and (c) Cys305, which is characteristic only of octaheme nitrite reductases. All distal bis-histidine heme-ligating histidines, with only one exception, are also conserved. The exception is the protein D6Z5C1, in which one of these histidines is replaced by methionine.
All of these data suggest that the above-mentioned proteins belong to a new family of octaheme nitrite reductases, whose representatives are found in different classes of proteobacteria. The physiological function of these homologous proteins in cells is unknown. The question is whether this function is associated with respiratory nitrite reduction under anaerobic growth conditions. The presence of this protein in cells of T. paradoxus, which cannot grow under anaerobic conditions with nitrate as the electron acceptor and is even unable to utilize nitrate or nitrite as a nitrogen source for growth , rules out rather than supports this possibility. Another possible function of octaheme nitrite reductases in cells is based on their ability to reduce compounds toxic to cells, such as nitrite, nitric oxide, hydroxylamine, and hydrogen peroxide , and is associated with their involvement in cell detoxification and oxidative and nitrosative stress defense. The key role of NrfAs in these processes is well documented .