Structure–function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures

Abstract

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe–4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k_cat/K_m). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k_cat) and Michaelis constant (K_m) of Nii4-WT were lower than those of Nii3-WT. However, the k_cat/K_m of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k_cat/K_m was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.

Introduction

Nitrogen is an essential element required for the synthesis of physiological substrates, including nucleic acids and proteins. Higher plants acquire nitrogen, primarily as NO₃^–, from the soil. The NO₃^– is preserved in the vacuoles¹ before being sequentially reduced to NH₄⁺ by two enzymes: assimilatory nitrate reductase and assimilatory nitrite reductase (aNiR).2–5 The produced NH₄⁺ is used by glutamine synthetase and glutamate synthetase in amino acid synthesis. These processes are known as nitrogen assimilation. An alternative inorganic nitrogen metabolic pathway, nitrogen dissimilation, also exists. One of the major dissimilatory processes, starting from nitrate or nitrite, is denitrification³ in which a dissimilatory nitrite reductase (dNiR) plays a role. The dNiR enzyme has been classified into three types: Cu-NiR (molecular mass of 37–41 kDa), which has a Cu atom in the active site³ and for which a number of high-resolution structures are available6–8; cd1-NiR (118–134 kDa), a homodimer in which each subunit contains a heme c and a heme d₁³; and the penta-heme cytochrome c NiRs (55–65 kDa) that reduce nitrite to ammonia or ammonium.^{3, 9} Cu-NiR and cd1-NiR both reduce nitrite to NO. On the other hand, aNiR, which has a siroheme and a [4Fe–4S] cluster as its prosthetic groups, reduces nitrite to ammonia or ammonium and sulfite to hydrogen sulfide.^{2, 10}

aNiRs are expressed in several species including algae, cyanobacteria, and higher plants. In some plants, distinct types of aNiR are expressed in leaves and roots.¹¹ However, the reason for this tissue-specific expression pattern is poorly understood. In aNiR, the binding and reduction of the substrates occur at the distal position of the siroheme. Ferredoxin (Fd) serves as the electron donor for electron transfer. Fd is reduced by photosystem I² in the leaf and by ferredoxin-NADP(+) reductase, which receives electrons from NADPH, in the root.¹² The electrons are then provided to the siroheme via the [4Fe–4S] cluster.² Kuznetsova et al.^{13, 14} showed that nitrite reduction to ammonia/ammonium is a five-step process. In recent years, the crystal structure of aNiR from spinach (SpNiR) was solved at 2.8 Å resolution, and a docking-model for Fd binding to aNiR was proposed.¹⁵ This model combined with site-directed mutagenesis studies was used to predict the amino acid residues in aNiR that could be involved in the binding of Fd.^{16, 17} Because aNiR can effectively reduce low-molecular weight environmental pollutants such as NO₂^–, it would be very valuable to know its mechanism. However, this is still poorly understood mainly because of the absence of high-resolution structures of aNiR.

In this study, we determined the crystal structures of tobacco aNiRs from leaf (Nii3) and root (Nii4).¹⁸ The Nii3 structure was determined to a higher resolution than the previously solved aNiR, assimilatory sulfite reductase (aSiR) and dissimilatory sulfite reductase (dSiR) structures,¹⁵, 19–25 and therefore precise geometries for the [4Fe–4S] cluster and siroheme could be determined. In addition, based on the detailed structures and on kinetic parameters, comparisons among wild-type Nii3 (Nii3-WT), Nii4-WT, and their variants (Nii3-M175G, Nii3-M175E, Nii3-M175K, Nii3-Q448K, and Nii4-K449Q) showed that the hydrophobicity of the Met175 side-chain was important for the efficient activity of aNiR and revealed that Gln448 of Nii3 and Lys449 of Nii4 play a role in enzyme efficiency (k_cat/K_m). Finally, a possible explanation for the tissue-specific expression of aNiR in tobacco is presented.

Results

Comparison of the primary structures of four aNiRs in tobacco

To verify the conservation of the primary structures of the four homologs of tobacco aNiR that are expressed in leaf (Nii1 and Nii3) and root (Nii2 and Nii4),¹⁸ their amino acid sequences were aligned using ClustalX²⁶ (Fig. 1). The identity and similarity of the amino acid sequences for the leaf aNiRs (Nii1 and Nii3) were both 99%, and for the root aNiRs (Nii2 and Nii4), the identity and similarity were 96% and 98%, respectively. The identity and similarity between Nii3 and Nii4 were 86% and 93%, respectively. Thus, the sequences of the aNiRs from the same tissue are highly conserved, while for the aNiRs isolated from different tissues, the sequences are less conserved. Overall, about 84 residues were variable when Nii3 and Nii4 were compared. Nii3 and Nii4 in which the transit peptides located at the N-termini were removed (18 residues)²⁷ were used in this study. Nii3 and Nii4 are 569 and 566 residues in length and have estimated molecular weights of 63.7 and 63.5 kDa, respectively.

Figure 1.

Multiple sequence alignment of Nii1, Nii2, Nii3 and Nii4 from tobacco. The amino acid sequence data were obtained from PubMed. Residues 175 and 448 are indicated by the broken and straight arrows, respectively. The three active site residues (Arg109, Arg179, and Lys224) are indicated by the black arrowheads. The three Cys residues (Cys440, Cys446, and Cys481) that bind the iron atoms of the [4Fe–4S] cluster are indicated by asterisks (*). Cys485, which binds both the iron atom of the [4Fe–4S] cluster and the iron atom of the siroheme, is indicated by *’. The sequence numbers for Nii3 and Nii4 in this figure are 25 and 22 higher, respectively, than in crystal structures. The figure was prepared with the BOXSHADE program.

To make it easier to compare structures, in this study, the numbering used for the Nii3 and Nii4 sequences is the same as the SpNiR sequence. Thus, the sequence numbers shown for Nii3 and Nii4 in Figure 1 are higher, by 25 and 22, respectively, than those in the Nii3 and Nii4 crystal structures.

Structure analysis

The crystal structures of Nii3-WT and Nii4-WT were determined at 1.25 and 2.3 Å resolution, respectively. The crystals of Nii3-WT and all Nii3 variants belonged to the space group P42₁2 (lattice parameters: a = b = 134.05 Å, c = 77.80 Å) and contained only one molecule per asymmetric unit, while Nii4-WT belonged to P2₁2₁2₁ (a = 99.34 Å, b = 112.06 Å, c = 92.50 Å) and contained two molecules per asymmetric unit. After crystallographic refinement, the R/R_free factors of Nii3-WT and Nii4-WT converged to 0.160/0.168 and 0.178/0.241, respectively. Neither the anisotropic B-factor nor the (Translation, Libration and Screw-rotation displacements of a pseudo-rigid body) TLS refinements were applied and no special weights between the geometry and the X-ray term were used except for Nii4-WT, where the geometry terms were stressed slightly. The Nii3 variants were analyzed by the same procedure, and the qualities of the reflection data and the refinement statistics are listed in Table I. Ramachandran plots indicated that the geometries of all the amino acid residues were within the allowed regions. The electron densities of all the protein molecules, cofactors, and solvent were well defined in the difference Fourier maps.

Table I.

Crystallographic Parameters and Refinement Statistics

	Nii3-WT	Nii4-WT	Nii3-Q448K	Nii3-M175E	Nii3-M175G	Nii3-M175K
Space group	P4212	P212121	P4212	P4212	P4212	P4212
A (Å)	134.05	99.34	133.46	133.55	133.27	133.15
B (Å)	134.05	112.06	133.46	133.55	133.27	133.15
C (Å)	77.80	92.50	77.66	77.81	77.73	77.59
X-ray Source	SPring-8	SPring-8	PF-AR	PF-AR	PF-AR	PF-AR
	BL44XU	BL44XU	NW12A	NW12A	NW12A	NW12A
Wavelength (Å)	0.9	0.9	1.0	1.0	1.0	1.0
Resolution (Å)	50-1.25	50-2.3	50-2.0	50-1.7	50-1.7	50-1.9
No. of reflections	1233291	175194	640930	905676	404012	696303
No. of unique refls	193657	45971	47915	76243	74629	55053
Completeness (%)	99.9 (99.9)	99.4 (99.8)	99.8 (99.1)	98.4 (96.8)	96.4 (96.5)	99.1 (96.3)
I/σ(I)	15.2 (1.3)	7.9 (1.4)	13.4 (2.0)	11.9 (1.7)	6.8 (1.5)	12.6 (2.1)
Rmergea	0.066 (0.492)	0.106 (0.426)	0.088 (0.416)	0.078 (0.431)	0.061 (0.370)	0.089 (0.367)
B from Wilson plot (Å)2	10.00	28.16	20.30	14.28	15.98	16.85
Rb	0.160	0.178	0.154	0.157	0.177	0.163
Rfreec	0.168	0.241	0.186	0.176	0.204	0.197
RMSD of geometry
Bond length (Å)	0.007	0.014	0.015	0.011	0.011	0.014
Bond angle (°)	1.218	1.553	1.419	1.293	1.325	1.429
Chiral volume (Å3)	0.083	0.104	0.098	0.087	0.092	0.099
Average B factors (Å)2
Main chain atoms	9.94	25.32	23.06	12.68	15.01	18.29
Side chain atoms	11.98	29.32	25.48	15.05	17.24	20.70
Cofactors	6.65	19.88	17.18	8.98	10.93	12.02
Solvent	28.48	29.31	40.31	32.77	34.57	37.69
PDB ID	3B0G	3B0H	3B0N	3B0J	3B0L	3B0M

^aR_merge = Σ_hΣ_i|I_i(h) – <I(h)>|/Σ_h I(h), where I_i(h) is the ith measurement of reflection h, and <I(h)> is the mean value of the symmetry related reflection intensities. Values in brackets are for the shell of the highest resolution.

^bR = Σ||F_o|–|F_c ||/ Σ|F_o |, where F_o and F_c are the observed and calculated structure factors used in the refinement, respectively.

^cR_free is the R-factor calculated with 5% of the reflections chosen at random and omitted from the refinement.

The B-factors of Nii4 from the Wilson plot were higher than those of Nii3 (Table I), probably because the Nii4 plate crystal was too thin (∼20 μm); the Nii3 plate crystal was ∼100 μm. Thus, because of the X-ray decay during data collection, the difference between R and R_free was larger for Nii4 than it was for Nii3. Because Nii4 contains two molecules per asymmetric unit, we also used the non-crystallographic symmetry (NCS) refinement; however, this did not improve the electron densities and B-factors. At the final refinement, all NCS restrictions were removed.

For the crystallographic refinement of the siroheme, the CCP4 dictionary files (named SRM and SF4) were employed. However, because the SRM dictionary made siroheme completely planar, the planar restrictions for the single-bonded carbon and for the Fe-coordination were removed from the SRM dictionary at the crystallographic refinement step. On the other hand, the final Fe–S_γ bond lengths to cysteine are related not only to the dictionary but also to the weight of geometry versus the X-ray data. To validate the effect of this weight, refinements for five different weights were carried out (data not shown). The different weights resulted in estimated standard deviations that were smaller than those listed in Table II.

Table II.

Geometries of the [4Fe-4S] cluster of Nii3-WT and SiR-HP (PDB ID: 1AOP)

A. Bond length (Å)			B. Bond angles (°)
Fe-S bond length	Nii3-WT	SiR-HP20 (1AOP)	S-Fe-S angle	Nii3-WT	SiR-HP20 (1AOP)
Fe1-S2	2.33	2.41	S2-Fe1-S3	104.7	102.1
Fe1-S3	2.28	2.30	S2-Fe1-S4	106.8	106.1
Fe1-S4	2.32	2.34	S3-Fe1-S4	101.7	102.3
Fe2-S1	2.30	2.25	S1-Fe2-S3	110.7	109.5
Fe2-S3	2.32	2.35	S1-Fe2-S4	104.6	103.9
Fe2-S4	2.26	2.29	S3-Fe2-S4	102.5	102.2
Fe3-S1	2.26	2.33	S1-Fe3-S2	103.3	102.2
Fe3-S2	2.31	2.33	S1-Fe3-S4	103.3	102.1
Fe3-S4	2.32	2.36	S2-Fe3-S4	107.6	107.8
Fe4-S1	2.32	2.34	S1-Fe4-S2	103.7	104.5
Fe4-S2	2.26	2.25	S1-Fe4-S3	108.6	107.6
Fe4-S3	2.36	2.41	S2-Fe4-S3	104.4	103.8
Average ± S.D.	2.30 ± 0.03	2.33 ± 0.05	Average ± S.D.	105.2 ± 2.6	104.5 ± 2.5
Fe-Sγ			Fe-S-Fe angle
Fe1-Sγ (C481(C479))	2.28	2.23	Fe2-S1-Fe3	72.9	73.8
Fe2-Sγ (C440(C434))	2.30	2.23	Fe2-S1-Fe4	69.5	71.0
Fe3-Sγ (C446(C440))	2.29	2.20	Fe3-S1-Fe4	72.7	72.7
Fe4-Sγ (C485(C483))	2.29	2.16	Fe1-S2-Fe3	71.8	71.9
Average ± S.D.	2.29 ± 0.01	2.21 ± 0.03	Fe1-S2-Fe4	73.0	74.6
Fe-Fe bond length			Fe1-S3-Fe2	73.9	74.4
Fe1-Fe2	2.76	2.81	Fe1-S3-Fe4	72.2	73.6
Fe1-Fe3	2.72	2.79	Fe2-S3-Fe4	68.6	69.5
Fe1-Fe4	2.73	2.82	Fe1-S4-Fe2	74.1	74.7
Fe2-Fe3	2.72	2.80	Fe1-S4-Fe3	71.8	72.7
Fe2-Fe4	2.63	2.71	Fe2-S4-Fe3	72.9	74.1
Fe3-Fe4	2.73	2.77	Average ± S.D.	72.2 ± 1.6	73.1 ± 1.6
Average ± S.D.	2.72 ± 0.04	2.78 ± 0.04

Crystal structures of Nii3-WT and Nii4-WT

The overall structure of Nii3-WT is shown in Figure 2(A). The [4Fe–4S] cluster is located in Domain 3 (blue) and the distal position of the siroheme spans Domain 1 (green) and Domain 2 (orange) [Fig. 2(A)]. The three domain structure of Nii3-WT is similar to the SpNiR structure.¹⁵ The positions of the two mutated residues (Met175 and Gln448) at the distal and proximal sides of the siroheme are shown in Figure 2(B).

Figure 2.

Crystal structure of Nii3-WT. (A) The overall structure of Nii3-WT. Based on the similar SpNiR structure,¹⁵ the protein was divided into Domain 1 (Residues 18–166 and 353–431, green), Domain 2 (Residues 167–352, orange), and Domain 3 (Residues 432–555, blue), surrounding the cofactors (magenta). (B) A detail of the Nii3-WT showing the positions of the two mutated residues (Met175 and Gln448) in Domain 2 and Domain 3. Met175 and Gln448 are shown (stick model) in relation to the siroheme (magenta).

Nii3 and Nii4 contain a His-tag region at their N-terminus. In both structures, this region is distal from the active site (>25 Å) and is invisible in the electron-density maps. It has been shown that, in such a case, the His-tag does not interfere with the native protein structure.²⁸

The two molecules in the asymmetric unit of Nii4-WT were located side-by-side in identical orientations without any rotation. From the gel-filtration analysis, the molecular weight of the Nii4-WT was estimated to be 61.8 kDa. This indicated that the Nii4-WT is monomer in solution. An examination of dimeric formation in the Nii4-WT crystal using the PISA web server²⁹ predicted a complexation significance score (CSS) value for the interface of 0.00, indicating that in the crystal the dimer interface was not physiologically relevant. The residues at the dimer interface are shown in Supporting Information Figure S1, and the results of the PISA analysis are summarized in Supporting Information Table SI. The CSS values for all other interface regions were less than 0.1. These results support the suggestion that Nii4-WT is a monomer in solution.

Structural comparison between the leaf (Nii3) and root (Nii4) aNiRs

The structural differences in Nii3-WT and Nii4-WT were analyzed by superimposing their structures on the SpNiR structure as shown in Figure 3(A). The structures were similar overall as indicated by the root mean square deviation (RMSD) values for the C_α atoms, all of which were <0.50 Å (the RMSD value between SpNiR and Nii3-WT, SpNiR and Nii4-WT, Nii3-WT and Nii4-WT was 0.44, 0.37, and 0.35 Å, respectively). The only region in which there was a noticeable difference between the proteins was the loop region marked by a circle in Figure 3(A). Swamy et al.¹⁵ predicted that the loop region (Residues 498–512) in SpNiR may function by “locking in Fd.” The average B-factor values of this loop region (Residues 502–508) in Nii3-WT, Nii4-WT and the variants (Nii3-M175G, Nii3-M175E, Nii3-M175K, and Nii3-Q448K) were 1.3 times higher than the corresponding B-factor values for the overall structures. This indicated that the loop region is more flexible than other regions and, therefore, may recognize other proteins such as Fd. For all of these structures, the electron-density map for the main chain in this loop region was well defined.

Figure 3.

Comparison of the crystal structures Nii3-WT and Nii4-WT. (A) The superimposed backbone structures of Nii3-WT (cyan), Nii4-WT (green), and SpNiR (orange). The circled loop region differs significantly in the three structures. (B) The active site of Nii3-WT (C) The active site of Nii4-WT. Potential hydrogen bonds (broken lines) less than 3.4 Å are shown. The 2F_o−F_c maps (blue) of water molecules are contoured at the 2.0σ level for Nii3-WT and at 1.0σ for Nii4-WT.

In the active site structures of both Nii3-WT and Nii4-WT [Fig. 3(B,C) respectively], globular electron densities were observed at the distal position of the siroheme-Fe, indicating that the siroheme-Fe was in a hexa-coordinated state in both active sties. Based on the density height and the shape of the electron-density map, the ligand appears to be a water molecule. This was consistent with the results from spectroscopy that aNiR is in a high spin hexa-coordinated state.^{13, 14} The bond lengths between the water molecule and the siroheme-Fe in Nii3-WT and Nii4-WT were 2.18 and 2.35 Å, respectively. It has been reported that the siroheme-Fe of SpNiR is in a penta-coordinated state in the crystal structure¹⁵; however, this may be because the structure of SpNiR was solved only at a moderate resolution (2.8 Å). Moreover, Swamy et al.¹⁵ indicated that the sixth-coordinated molecule would appear if the resolution limit of the data was improved.

Geometries of the siroheme and the [4Fe–4S] cluster of Nii3-WT

Because a high-resolution structure of Nii3-WT was obtained, accurate geometries for the coordination of the prosthetic groups ([4Fe–4S] cluster and siroheme) are available. The structures and nomenclature for the atoms of these prosthetic groups are shown in Figure 4.

Figure 4.

Structures of the cofactors and neighboring residues in Nii3-WT. The atom names match those that are used to define the geometries in Tables II and III. (A) The [4Fe-4S] cluster. The S_γ atom of Cys485 (shown by the arrow) connects the iron atom of the siroheme to the iron atom of the [4Fe–4S] cluster. (B) The siroheme. Possible hydrogen bond interactions between the carboxylate group of siroheme and the surrounding residues are shown by broken lines.

We first compared the geometries of the [4Fe–4S] cluster for Nii3-WT to that of sulfite reductase hemoprotein subunit from Escherichia coli (SiR-HP)¹⁹ (1AOP) because the SiR-HP structure has been determined at high resolution (1.6 Å). SiR-HP reduces sulfite to sulfide and has the same cofactors as Nii3-WT.¹⁹ The geometries of the [4Fe–4S] clusters in Nii3-WT are shown in Table II. A comparison of the bond lengths of the [4Fe–4S] cluster in the two structures revealed that the four iron atoms of the [4Fe–4S] cluster formed weaker interactions with the sulfur atoms (S_γ) of the four Cys residues in Nii3-WT than in SiR-HP. Indeed, the average Fe–S_γ bond length in Nii3-WT was about 0.08 Å longer than in SiR-HP (Table II). The difference in the bond lengths may explain the differences in the efficiency of electron transfer to siroheme among the aNiRs and SiRs. Even after imposing the different weights on the geometry during the structure refinement, the geometries were not changed. No noticeable differences were found when the S—Fe—S and the Fe—S—Fe bond angles in Nii3-WT and SiR-HP were compared.

We next compared the siroheme geometries in Nii3-WT and SiR-HP. The geometries for Nii3-WT are shown in Table III. The CHA–SFe–CHC and CHB–SFe–CHD bond angles were 165.7° and 194.3°, respectively, indicating that the siroheme ring was distorted. In SpNiR structure, the ring had very planar shape.¹⁵ This discrepancy may be due to the difference in the resolution of the electron-density maps between Nii3 and SpNiR, and Swamy et al. also described this possibility in their article.¹⁵ In SiR-HP, the angles of CHA–SFe–CHC and CHB–SFe–CHD were 181.0° and 228.7°, respectively. This difference may be due to the difference in the hydrogen bond interactions between the siroheme and the protein molecule among aNiR, dSiR, and aSiR enzymes.¹⁹ The angles were estimated from the side to the distal position of the siroheme.

Table III.

Geometries of the siroheme of Nii3-WT

A. The geometry of the siroheme ring.
Bond length (Å)		Bond angle (°)
SFe1-NAa	2.04	CHB-SFe-CHD	198.3
SFe-NB	2.01	CHA-SFe-CHC	165.8
SFe-NC	2.05
SFe-ND	2.03
SFe-Sγ(C485)	2.38

B. The coordination between the carboxylate group of siroheme and the surrounding residues.
Hydrogen bonds length (Å)
O1A-NH1 (R149)	2.92	O1C-NZ (K224)	2.73
O2A-NH2 (R149)	2.82	O2C-NH1 (R309)	2.84
O3A-NH2 (R98)	2.89	O3C-NH2 (R143)	2.91
O4A-NH1 (R98)	3.24	O4C-NE2 (Q487)	2.87
O2B-ND2 (N145)	3.01	O1D-NH2 (R309)	2.91
O3B-N (T441)	3.23	O4D-NH1 (R223)	3.03
O4B-N (T142)	2.89	O4D-NH2 (R223)	3.08

^aThe “SFe” represents the iron atom of siroheme.

The coordination between the carboxylate groups of the siroheme and the residues that form the hydrogen bonds was also measured (Table III). In Nii3-WT, 10 residues (Arg98, Thr142, Arg143, Asn145, Arg149, Arg223, Lys224, Arg309, Gln487, and Thr491) could form hydrogen bonds via their nitrogen atoms with the carboxylate groups of the siroheme (Table III). Except for Thr491, all of these residues interacted with the carboxylate groups of the siroheme in the SpNiR structure.¹⁵ Siroheme has eight carboxylate groups and 16 oxygen atoms 13 of which formed hydrogen bonds with the surrounding residues. The three oxygen atoms that did not form hydrogen bonds were O1B, O2D, and O3D.

Structural comparison among the three Met175 variants (Nii3-M175G, Nii3-M175E, and Nii3-M175K)

Met175 is located on the positively charged tunnel that is thought to be the substrate entrance pathway,¹⁵ and may be one of the shielding residues for the substrate binding site.²² To investigate the role of Met175 as the main affinity-regulating residue in the aNiRs, we determined the crystal structures of three Nii3-M175 variants (Nii3-M175G, Nii3-M175E, and Nii3-M175K).

In the Nii3-Met175 variants, the conformation of the Gln47 and Lys91 side-chains changed significantly [Fig. 5(A)]. Compared with the conformation in Nii3-WT, in Nii3-M175K, the Gln47 side-chain rotates by about 120° (χ₂), and in Nii3-M175E and Nii3-M175G, there is a rotation in χ₁ of 120° [Fig. 5(A)]. The amino group in the Lys91 side-chain moved toward the side-chain of Residue 175 in all three Nii3-M175 variants [Fig. 5(A)]. Compared with its position in Nii3-WT, the Lys91 amino group moved by ∼0.2 Å in Nii3-M175K, 0.8 Å in Nii3-M175E, and 1.5 Å in Nii3-M175G.

Figure 5.

Comparison of the active site structures of the Nii3-Met175 variants. (A) The superimposed active site structures of Nii3-WT (green), Nii3-M175G (cyan), Nii3-M175E (magenta), and Nii3-M175K (yellow) showing the Gln47 and Lys91 conformational changes. (B) The hydrogen bond network for Nii3-M175G. (C) The hydrogen bond network for Nii3-M175E. (D) The hydrogen bond network for Nii3-M175K. All hydrogen bond distances (dotted lines) are less than 3.4 Å.

The replacement of the Met175 to another residue brought about changes in the hydrogen bond interactions around the replaced residue [Fig. 5(B–D)]. In the active site of Nii3-M175G, because of the absence of the hydrophobic Met side-chain, two water molecules formed hydrogen bonds with Gln47 and Lys91 [Fig. 5(B)]. In the Nii3-M175E active site, the Glu175 side-chain could form new hydrogen bonds with Lys91 and Arg179 [Fig. 5(C)] and in Nii3-M175K, a hydrogen bond with Arg305 could form via a water molecule [Fig. 5(D)].

Structural comparison among the Nii3-Q448K, Nii3-WT, and Nii4-WT proteins

The results of a BLASTP³⁰ search revealed that in aNiRs, Residue 448 (Gln448 in Nii3 and Lys449 in Nii4) is highly conserved and that only Nii4 has a Lys at this position. Thus, it is likely that Residue 448 is a functionally important residue in aNiRs.

Comparison of the overall structures of Nii3-Q448K and Nii3-WT [Supporting Information Fig. S3(A)] revealed that the RMSD value calculated between the C_α atoms was about 0.12 Å, indicating that the mutation had only a negligible effect on the overall fold of the protein. In addition, the mutation did not affect the hydrogen bond interaction at the distal position of the siroheme [Supporting Information Fig. S3(B)].

The structure of the side-chains around Residue 448 differed in Nii3-WT, Nii3-Q448K, and Nii4-WT (Fig. 6). In Nii3-WT, the Gln448 residue could assume two conformations: in one, a hydrogen bond with the Asn483 side-chain could form; and in the other, no hydrogen bond could form [Fig. 6(A)]. In both Nii3-Q448K and Nii4-WT, Residue 448 (Lys448 in Nii3-Q448K and Lys449 in Nii4-WT) adopted a single conformation and no hydrogen bonds with other residues could be formed. When the Nii3-WT, Nii3-Q448K, and Nii4-WT structures were superimposed [Supporting Information Fig. S3(C)], we found that the residues located near Residue 448 had almost the same conformation. This indicated that the replacement of Gln by Lys did not cause structural changes in neighboring residues.

Figure 6.

Structures of the side-chains around Residue 448 in Nii3-WT, Nii3-Q448K, and Nii4-WT. (A) The side-chains of Residues 448 and 483 in Nii3-WT are in green. The Q448 side-chain can adopt multiple conformations. (B) The side-chains of Residues 448 and 483 in Nii3-Q448K are in magenta. (C) The side-chains of Residues 449 (equivalent to 448 in Nii3) and 484 (equivalent to 483 in Nii3) in Nii4-WT are in orange.

Kinetic studies of Nii3-WT, Nii4-WT, and the variants

The kinetic parameters for Nii3-WT, Nii4-WT, and the five variants (Nii3-Q448K, Nii4-K449Q, Nii3-M175G, Nii3-M175E, and Nii3-M175K) were analyzed, and initial velocity curves of their enzymatic activity are shown in Figure 7. The kinetic parameters for the reduction of nitrite to ammonia are listed in Table IV.

Figure 7.

Initial velocity curves for the reduction of nitrite by Nii3-WT, Nii4-WT, and the five variants. (A) Initial velocity plots for Nii3-Q448K, Nii4-WT, and Nii4-K449Q using a nitrite concentration range of 60–750 μM. (B) Initial velocity plots for Nii3-WT, Nii3-M175G, Nii3-M175E, and Nii3-M175K using a nitrite concentration range of 0.2–2.5 mM. The different nitrite concentration ranges that were used are based on differences in the measured K_m values of the enzymes. Each measurement was carried out >3 times. The error bars were estimated by Student's t test (reliability = 95%). The kinetic parameters that were obtained are listed in Table IV.

Table IV.

Kinetic parameters of Nii3-WT, Nii4-WT and the five mutants^a

	kcat (/sec)	Km (mM)	kcat/Km (/sec*mM)
Nii3-WT	200 ± 7	587 ± 58	341 ± 36
Nii4-WT	73 ± 4	156 ± 24	467 ± 76
Nii3-Q448K	107 ± 7	226 ± 37	473 ± 83
Nii4-K449Q	92 ± 4	277 ± 26	332 ± 34
Nii3-M175G	227 ± 8	1087 ± 81	208 ± 17
Nii3-M175E	96 ± 8	767 ± 159	125 ± 28
Nii3-M175K	102 ± 4	760 ± 81	134 ± 15

^aAll of the data in these columns are represented as the mean ± standard error (standard error of the mean, S.E.M). The K_m and k_cat values were estimated from iterating experiments and calculations of more than three times using a non-linear least-square method. The standard errors were calculated by the Student's t test and all values in each column were significantly different at P < 0.05. The initial velocities are plotted in Fig. 4.

A comparison of the k_cat and K_m values of Nii3-WT and Nii4-WT revealed that they were three times higher for Nii3-WT (k_cat = 200, K_m = 587 μM) than for Nii4-WT (k_cat = 73, K_m = 156 μM); the enzyme efficiency of Nii4-WT (k_cat/K_m = 467) was ∼1.4 times higher than the enzyme efficiency of Nii3-WT (k_cat/K_m = 341).

When we compared the kinetic parameters of Nii3-WT and Nii3-Q448K, we found that the k_cat and K_m values of Nii3-Q448K (k_cat = 107, K_m = 226 μM) were lower than those of Nii3-WT; the enzyme efficiency (k_cat/K_m) of Nii3-Q448K was about 1.4 times higher than for Nii3-WT. Compared with the k_cat and K_m values for Nii4-WT, those of Nii4-K449Q (k_cat = 92, K_m = 277 μM) were higher; the enzyme efficiency (k_cat/K_m) of Nii3-Q448K was about 1.4 times lower than for Nii4-WT. These results indicate that Residue 448 is involved in regulating the k_cat/K_m values. The k_cat/K_m values for Nii3-Q448K and Nii4-WT that have a Lys as Residue 448 were ∼1.4 times higher than the corresponding values for Nii3s and Nii4-K449Q that had a Gln as Residue 448, indicating that Residue 448 is involved in regulating the enzyme efficiency.

When the parameters for the three Met175 variants of Nii3 (Nii3-M175G, Nii3-M175E, and Nii3-M175K) were compared with those of Nii3-WT, we found that while the k_cat value of the Nii3-M175G (k_cat = 227) was almost the same as the Nii3-WT value, the K_m value (K_m = 1087 μM) was about twice as high. The k_cat values for Nii3-M175E (k_cat = 96) and Nii3-M175K (k_cat = 102) were about half the k_cat for Nii3-WT and the K_m values (K_m = 767 μM for Nii3-M175E, K_m = 760 μM for Nii3-M175K) were about 1.3 times higher than for Nii3-WT. Therefore, the k_cat/K_m values for these variants (k_cat/K_m = 208, 125, and 134 for Nii3-M175G, Nii3-M175E, and Nii3-M175K, respectively) were lower than the k_cat/K_m value determined for Nii3-WT.

Here, we used methyl viologen (MV) and sodium dithionite as the reductants. Dithionite produces a sulfite ion (oxidation of the dithionite ion), which may inhibit Nii3 and Nii4. However, the enzyme kinetic measurements indicate that, in this case, the sulfite ion did not inhibit the enzymes because the enzymatic curves for Nii3 and Nii4 could be fitted using the Michaelis-Menten equation (Fig. 7). Moreover, Bellissimo et al. successfully determined the kinetic parameters of SpNiR using MV and dithionite as the reductant,³¹ supporting our finding that dithionite is an appropriate agent to measure the kinetic parameters.

Discussion

By combining the information obtained from the crystal structures and kinetic data of Nii3-WT, Nii4-WT and their variants, we have assigned possible functions to the hydrophobic Met175 side-chain and to Residue 448.

Function of the Met175 residue in Nii3-WT

Because of the observed high K_m value of Nii3-M175G (Table IV), we initially suggested that the hydrophobicity provided by Met175 in Nii3-WT was important for the enzymatic efficiency of the aNiRs. In the Nii3-M175G crystal, two additional water molecules were present in the space created by replacing the hydrophobic side-chain of Met175 with Gly [Fig. 5(B)], suggesting that Met175 might protect the active site from the water solvent.

The replacement of Met175 with Lys in Nii3-M175K did not improve the k_cat and K_m values compared with the values for Nii3-WT (Table IV); the kinetic parameters for Nii3-M175E were almost the same as those for Nii3-M175K (Table IV). These observations were unexpected because the location of a positively (Lys) and negatively (Glu) charged residue at Position 175 should improve and worsen the kinetic parameters, respectively. This is derived from the concept that the Met175 is located at the surface of the positively charged pathway, and the replacement of Met175 to other charged residues would affect NO₂⁻ recognition.

We then examined whether the observed decrease in the kinetic parameters for Nii3-M175E and Nii3-M175K may have arisen from hydrogen bond interactions that disrupt proton or electron translation at the active site. Indeed, in the active sites of both Nii3-M175E and Nii3-M175K, the Glu and Lys side-chains could form new hydrogen bonds with water molecules and neighboring residues [Fig. 5(C,D)].

Gln448 is important in controlling the enzyme efficiency (k_cat/K_m) of aNiR

By comparing the kinetic parameters of Nii3-WT, Nii4-WT, Nii3-Q448K, and Nii4-K449Q, we found that the enzymatic efficiency of Nii3 and Nii4 was about 1.4 times higher when Residue 448 was replaced Gln with Lys (Table IV); thus, the enzyme efficiency of the aNiRs seems to improve when Lys replaces the native residue at Position 448. For the soluble forms of the variants that we could obtain, the only improvement in enzyme efficiency that we could accomplish was when Residue 448 was replaced.

It is unclear why, in Nii3-WT, the replacement of Residue 448 with Lys improved enzyme efficiency (k_cat/K_m). Here, we propose two possible explanations based on the location of Gln448 in Nii3-WT. One possible explanation might be that in the Nii3-Q448K and Nii4-K449Q variants, the redox potential of the active sites is altered. In an earlier study, Marshall et al. found that the redox potential of some metalloproteins changed when an amino acid close to the active site was replaced.³² In Nii3-WT and Nii4-WT, Residue 448 is located close to the [4Fe–4S] cluster (Fig. 6). The second possible explanation is that the k_cat/K_m values are affected by changes to Residue 448 because this residue is directly located at the substrate entrance pathway to the active site. This explanation is supported by the electrostatic potential maps of Nii3-WT and Nii4-WT, which show that Gln448 in Nii3-WT and Lys449 in Nii4-WT are located at the positively charged cleft that has been predicted to be the substrate entrance channel.¹⁵

In the root aNiRs (Nii2 and Nii4) in tobacco, Residue 448 is Gln in Nii2 and Lys in Nii4; therefore, our findings suggest that Nii2 may have a lower k_cat/K_m value than Nii4.

Nii3 (leaf) has low affinity and Nii4 (root) has high affinity for NO₂^–

Our study of the structure and enzyme kinetics of Nii3 and Nii4 has shown that the k_cat and K_m values of Nii3-WT are about three times higher than the corresponding values for Nii4-WT. Similar examples have been found in other species that express multiple types of nitrate transporters, some of which have higher V_max and K_m values than others.^{33, 34} Based on the classification of these nitrate transporters, we propose that Nii3-WT can be classified as a low-affinity aNiR for NO₂^– and Nii4-WT as a high-affinity aNiR for NO₂^–.

To understand why tobacco might express low-affinity and high-affinity aNiRs, we examined the advantages that expressing different aNiRs might have for the plant. In nitrite-poor conditions, tobacco could reduce the substrate more efficiently using the high-affinity Nii4, which displayed a k_cat/K_m value that was about 1.4 times higher than the value for the low-affinity Nii3. In relatively nitrite-rich conditions, on the other hand, the tobacco could reduce NO₂^– more rapidly using the low-affinity Nii3, which had a k_cat value that was about three times higher than the value for the high-affinity Nii4. Therefore, a plant that can express both low-affinity and high-affinity aNiRs can readily adjust to changes in nitrite concentrations by regulating the expression of the aNiRs. Plants that can express only one kind of aNiR would have to respond to changes in nitrite concentrations by changing the expression rate of the one aNiR. Therefore, it is possible that tobacco may select and use the different aNiRs under different nitrite concentration conditions. In a similar study reported by Unkles et al., they mentioned that the expression of two types of nitrate transporters would be advantageous to cope with different environments such as rapid changes in NO₃^– concentrations.³⁴

Here, we have suggested why tobacco might express distinct aNiRs in leaf and root. This postulate is yet to be validated; however, it represents an interesting theme for future investigations.

Materials and Methods

Overproduction and purification of proteins

The pET16b-Nii3 and pET16b-Nii4 plasmids containing a His-tag (MGHHHHHHHHHHSSGHIEGRHM) at the N-terminus were each transformed into the BL21(DE3)pLysS E. coli strain. We used the cDNAs of nii3 and nii4 that had been obtained previously.¹⁸ The transformed cells were cultured at 21°C in 5L of LB medium with 50 μg/mL ampicillin. Protein expression was induced by adding 0.1 mM isopropyl-1-thio-β-D-glucopyranoside. After 30 h of incubation, the cells were harvested, suspended in buffer N (50 mM sodium phosphate at pH 7.0, 300 mM NaCl, and 0.02% sodium azide) and sonicated. The insoluble fraction was excluded by centrifugation. The lysate was loaded onto a Ni²⁺-Sepharose column (GE Healthcare) and the proteins were eluted using a stepwise-gradient and buffer N containing imidazole. To improve purity, the obtained sample was applied to a hydroxyapatite column (BioRad). For Nii3, Nii4 and their variants, the same purification procedure was employed. The purity of Nii3-WT and Nii4-WT was examined using SDS-PAGE [Supporting Information Fig. S4(A)], and the optical spectra for Nii3-WT and Nii4-WT are shown in Figure S4(B,C), respectively.

Site-directed mutagenesis

Site-directed mutagenesis of Nii3 and Nii4 was performed using the KOD-plus mutagenesis kit (TOYOBO). For the expression and purification of the variants, we used the same method as the one employed for production of Nii3-WT. The variants were sequenced across the region of the desired mutation to confirm the amino acid replacement.

Molecular weight estimations of Nii3-WT and Nii4-WT by gel-filtration chromatography

The molecular weights of Nii3-WT and Nii4-WT in solution were estimated by gel-filtration chromatography using a Superdex-75pg column (GE Healthcare). The elution buffer was 25 mM Tris–HCl (pH 8.0) and 150 mM NaCl. The flow rate was 0.50 mL/min, and protein detection was measured using the absorbance at 280 nm.

To create the calibration curve, bovine serum albumin (66.8 kDa), hen egg white ovalbumin (45.0 kDa), and hen egg white lysozyme (14.3 kDa) were used [Supporting Information Fig. S2(A)]. The elution volume was measured from the UV spectrum for the proteins and the elution patterns for Nii3-WT and Nii4-WT were recorded [Supporting Information Fig. S2(B,C)]. The estimated molecular weights of Nii3-WT and Nii4-WT were 60.9 and 61.8 kDa, respectively.

Enzyme activity assay

The enzyme activity assay of NiR was performed as described previously.³⁵ For Nii3-WT, Nii4-WT and the variants, the non-physiological electron donor MV, which is reduced by sodium dithionite,³⁵ was used for the assays. Assay buffer [150 μL; 50 mM potassium phosphate (pH 7.0), 2 mM MV, 0.12–6.0 mM NaNO₂] and 90 μL of an enzyme solution were mixed and incubated at 30°C for 30 min. The reaction was initiated by adding 60 μL of a sodium dithionite solution (1% w/v sodium dithionite). The amount of nitrite in the solution was determined by spectrophotometry at 540 nm after the addition of 1% sulfanilamide and 0.02% N-(1-naphtyl) ethylenediamine. To determine the enzyme concentration of Nii3, Nii4, and the variants, we measured the maxima peak of siroheme at around 390 nm (the Soret band) using a nano-drop UV–Vis spectrophotometer (Thermo Fisher Scientific). All concentrations of the enzymes were calculated using a molar absorption coefficient at 390 nm of 40,000 M^–1 cm^–1.¹³ The initial velocities were measured with a UV–Vis spectrophotometer (JASCO V-560). The k_cat and K_m values were determined using the Michaelis-Menten equation, and applying a non-linear least square method.

Crystallization and data collection

Before crystallization, the samples were dialyzed against a buffer of 25 mM Tris–HCl (pH 8.0) and 150 mM NaCl. Nii3-WT was crystallized using the sitting drop vapor diffusion method. Briefly, the protein sample was concentrated to 15–20 mg/mL using Centricut (Kurabou, Tokyo). Then 3 μL of the sample was mixed with 3 μL of a reservoir solution [30% polyethylene glycol 4000, 0.1M Tris–HCl at pH 8.5, 0.2M MgCl₂, and 3% methyl pentane diol (MPD)]. After a week at 4°C, brown crystals in the shape of thin plate clusters appeared. Single crystals of sufficient size for X-ray data collection were obtained by streak seeding in newly made drops equilibrated for 6 h against the reservoir solution described above. After about 2 weeks, crystals of 400 × 400 × 100 μm grew. For cryo-protection, the crystals were transferred to the reservoir solution, which had been supplemented with 20% MPD. Crystallization of the Nii3-Q448K variant was performed by hetero seeding using Nii3-WT crystals as the seeds. The reservoir conditions were the same as for Nii3-WT.

Nii4-WT was crystallized by the sitting drop vapor diffusion method. The Nii4-WT sample was concentrated to 10 mg/mL, and 4 μL of the sample was mixed with 2 μL of a reservoir solution [25% PEG4000, 0.1M Tris–HCl (pH 8.5), 0.3M MgCl₂, 12 mM EDTA, and 3% PEG400]. After 2 months at 4°C, clusters of thin plate crystals of 200 × 50 × 20 μm appeared. For cryo-protection, the Nii4-WT crystals were transferred to the reservoir solution supplemented with 20% PEG400.

Characterization of the crystals was performed using an imaging plate diffractometer, RaxisVII (Rigaku). The Nii3-WT and Nii4-WT diffraction data were collected at the beamline BL44XU in SPring-8 (Hyogo, Japan) using the imaging plate detector DIP6040 (Bruker-AXS). The diffraction data for the Nii3-Q448K variant crystals were collected at the beamline NW12A and BL17A in the Photon Factory Advanced Ring (PF-AR, Tsukuba, Japan) using the CCD detector Quantum 210r (ADSC). Crystals were fixed in the nitrogen cryostream at 110K. The X-ray wavelength was fixed to 0.9 Å at BL44XU and to 1.0 Å at NW12A and BL17A. Diffraction data were processed and scaled with the HKL2000 and Scalepack programs,³⁶ respectively. The data collection statistics are summarized in Tables I and Supporting Information.

Structure determination and crystallographic refinement

Initial phases were determined by the MolRep program³⁷ implemented in the CCP4 suite³⁸ and using the of SpNiR coordinates¹⁵ (PDB access code: 2AKJ). Crystallographic refinement was performed using the program REFMAC5.³⁹ Difference Fourier maps, with 2F_o − F_c and F_o − F_c coefficients, and omit maps were calculated and fitted to the electron densities using the three-dimensional display interface in XtalView.⁴⁰ Figures were prepared using the PyMOL program.⁴¹ The initial phases of the Nii3 variants and Nii4-WT were determined by molecular replacement using the coordinates of Nii3-WT (PDB access code: 3B0G). The refinement calculations were performed using the method described for Nii3-WT. The refinement statistics are summarized in Table I.

Protein Data Bank Accession Codes

All coordinates and structure factors have been deposited to the protein data bank with accession codes: 3B0G (Nii3-WT), 3B0H (Nii4-WT), 3B0N (Nii3-Q448K variant), 3B0L (Nii3-M175G variant), 3B0J (Nii3-M175E variant), and 3B0K (Nii3-M175K variant).

Glossary

Abbreviations:

aNiR

assimilatory nitrite reductase

aSiR

assimilatory sulfite reductase

CCS

complexation significance score

dNiR

dissimilatory nitrite reductase

dSiR

dissimilatory sulfite reductase

Fd

ferredoxin

MPD

methyl pentane diol

MV

methyl viologen

NCS

non-crystallographic symmetry

PEG

polyethylene glycol

RMSD

root mean square deviation

SiR-HP

sulfite reductase hemoprotein subunit from Escherichia coli

SpNiR

nitrite reductase from spinach.

Supplementary material

Additional Supporting Information may be found in the online version of this article.