Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin-like mediator

Abstract

We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key β-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.

Keywords: NirK; Sinorhizobium meliloti 2011; X-ray crystal structure; copper; nitrite reductase; pseudoazurin.

FIGURE 1

SmNirK overall structure. (a) Top and side views of the trimeric structure. The threefold symmetry axis is indicated by a black triangle (Top view) and thick lines (Side view). (b) Domain distribution within each SmNirK subunit. Domain I (blue) harbors the two copper centers and is connected by the linker loop (red) to domain II (green). The Cys–Met loop and the tower loop located in Domain I and Domain II, respectively, play a key role in the enzyme–ED interaction and electron transfer processes. (c) Coordination around each copper center and pathways linking T1Cu and T2Cu. The amino acids in blue belong to the same monomer whereas those in green to a neighbor monomer. Other residues involved in catalysis are depicted with subindexes CAT (catalytic proton channel) and PPC (primary proton channel). Catalytically relevant occluded water molecules are in red

FIGURE 2

SmNirK proton channels. (a) Primary and secondary proton channel (PPC and SPC, respectively) accessed from the bulk solution. SPC coincides with the substrate channel (SC). SPC/SC (red arrows in bottom view and red circle in side view) and PPC (magenta circles in bottom view) are indicated. Subunits are shown in different colors. (b) SC/SPC mouth architecture in resting as‐isolated structures of rhizobial NirKs. The bulky Phe348 residue in SmNirK defines a narrow mouth compared to that observed in Br ^2DNirK (PDB ID: 6ZAS), which has a less bulky Ala residue in that position. Waters within the channel are indicated as spheres. (c) Comparison of proton channels in SRX structure of SmNirK (blue or green depending on the subunit) with XFEL‐FRIC structure of Br ^2DNirK (cyan). SPC walls architecture in SmNirK: Ile293 (Ile_CAT), Ile336, Ala338, Val340, Leu344, Phe348, Ala353, Ala354, and His355 from subunit A (transparent yellow surface); Asn132, Asp134 (Asp_CAT), Leu142, Gly145, Ala146, Val149, His171, Ala173, Pro175, Gly176, Val178, and Pro179 from subunit B (transparent orange surface). Dashed lines indicate the SPC path. Amino acids related to T2Cu active site are indicated, as well as all the residues involved in the PPC and SPC (SmNirK numbering). Variations on the SPC/SC mouth entry among different NirKs are indicated: Phe348 and Val149 (SmNirK numbering). SmNirK (green) and Br ^2DNirK (cyan) SPC waters are indicated. Occluded water molecules that connect His_CAT with Glu–Thr residues are indicated in red

FIGURE 3

Kinetic assays. (a) Electron donation progress curves of pseudoazurins to SmNirK. Green, SmPaz2 (ε₅₉₀ = 1.51 mM⁻¹ cm⁻¹); blue, SmPaz1 (ε₅₉₇ = 3.17 mM⁻¹ cm⁻¹). Absorbance values were converted to μmol of protein. (b) Currents recorded at different nitrite concentrations by chronoamperometry. The solid line was obtained by least squares analysis assuming a Michaelis–Menten model

FIGURE 4

The intermolecular electron transfer process. T1Cu surface area analysis. “Open‐book unfolding” of enzyme–electron donor complex indicating: (a) the conservation of residues on surfaces covering T1Cu centers in both two‐domain NirKs (left) and pseudoazurins (right) and, (b) surface electrostatic analysis depicted by coulombic surface coloring in UCSF Chimera: SmNirK (left); SmPaz1 (homology model constructed by Swiss‐Model) and SmPaz2 (PDB ID: 3TU6) (pseudoazurins, right). The potential contours are shown on a scale from −10.0 k B T (red) to +10.0 k B T (blue). The open‐book unfolding was made after structural alignment based on the X‐ray crystal structure of the AxNirK–HdPaz complex (PDB ID: 5B1J). Amino acids that are essential for the AfNirK–AfPaz interaction, as shown by mutagenesis studies, are indicated. The amino acids that are not preserved compared to the AfNirK–AfPaz complex are indicated in red

FIGURE 5

The intermolecular electron transfer process: in‐silico models for T1Cu _SmPaz2 → T1Cu _SmNirK ET. Top panel shows a close‐up view of interacting surfaces and the putative ET pathways are indicated in ball‐stick. The jumps are indicated by dashed lines. Amino acids taking part in the ET process are indicated in black. Bottom panel shows a top‐view of the complex. The homotrimeric structure of SmNirK is depicted in yellow. The tower loop and the Cys–Met loop (both indicated in red) take part in the interaction with SmPaz2 (green). (a) Molecular arrangement obtained after structural alignment of SmNirK and SmPaz2 structures on the AfNirK–AfPaz complex structure (PDB ID: 2P80). (b) SmNirK–SmPaz2 complex arrangement obtained using the AxNirK–HdPaz complex (PDB ID: 5B1J) as template. (c) Model obtained after docking performed by ClusPro