Atomic resolution structures of resting-state, substrate- and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism

Abstract

Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using "crystal harvesting" and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 A, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 A and 1.15 A, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 A resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.

Fig. 1.

The T2Cu site of the resting state enzyme at 0.9 Å resolution is shown with 2F_obs – F_calc electron density contoured at 1.2σ. The site is well ordered, with isotropic B factors of 5.3 Ų for the Cu²⁺ atom and an average of 5.4 Ų for the NE2 atoms of the three ligating histidine ligands (100, 135, 306B), coordinated at 1.99 Å. A water molecule, W1, is the fourth Cu ligand, at 2.2 Å with a B factor of 5.9 Ų. The Ile257B residue from the second monomer provides a steric constraint (W1 to Ile257B is ≈3.4 Å) on the substrate binding site (8). A well ordered network of water molecules, W3–W8, leads from the substrate-binding site to the protein surface along the probable route of nitrite entry. W2, W4, and W5 have partial or dual occupancy, depending on the orientation of the Asp-98 side chain, either toward the substrate-binding pocket (proximal orientation) or into the substrate-entry channel (gatekeeper orientation). In the resting enzyme, the gatekeeper orientation has lower occupancy, as suggested by the weaker electron density for this position of the Asp-98 side chain. The Leu-106 residue also exhibits a dual conformation, corresponding to the different positions of Asp-98, W4 and W5. Water molecule W0 belongs to the proton-transfer water network.

Fig. 2.

The T2Cu site of the nitrite-soaked enzyme at 1.10 Å resolution, shown with 2F_obs – F_calc electron density contoured at 1.2σ. Either a water or nitrite molecule occupies the substrate-binding site, with the nitrite OD2/OD1 atoms at 1.98/2.19 Å and the N atom at 2.15 Å or a water molecule, W1 at 2.0 Å from T2Cu. The B factors of the Cu atom and nitrite ligand are 10.1 Ų and 16.0 Ų, respectively, compared with the B factors for ligating N(His), which average 10.0 Ų. The Cu occupancy of the protein was 90%. The nitrite is oriented so that the O1 and O2 atoms form H-bonds with water W2 at 2.4 Å and with water W3, at 2.6 Å. W2 is 2.4 Å from the conserved proton channel water molecule, W0. The substrate-entry water network of the resting-state enzyme is conserved in this structure, while the gatekeeper conformation of the Asp-98 is more pronounced in the electron density map.

Fig. 3.

The T2Cu site of the enzyme with endogenously bound NO at 1.12 Å resolution, shown with 2F_obs – F_calc electron density contoured at 1.2σ. The Cu atom is coordinated by either water or NO. The B factors of the Cu, N_NO, and O_NO atoms are 10.2, 10.9, and 12.6 Ų, respectively. The close correspondence of these B values indicates the correctness of the atomic assignments of the diatomic molecule. In molecules with water bound to T2Cu in place of NO, W1 is at 2.2 Å from the metal, with a B factor of 10.2 Ų. The proton channel water, W0, is indirectly linked to the NO via H-bonding through Asp-98: the W0—OD1(Asp-98) distance is 3.0–3.17 Å. The direct separation between the NO and W0 is 3.8 Å. The N atom of NO also has an H-bond to W3, at 2.6 Å. The His-135 is coordinated at 2.04 Å with the other histidine ligands at 2.0 Å; the average N(His) B factor is 9.8 Ų. The Asp-98 side chain is oriented toward the proton delivery channel, where it is H-bonded to the N atom of NO. The gatekeeper orientation is absent in this structure. Correspondingly, there is only a single conformation of Leu-106 and a single chain of water molecules in the substrate-entry channel. W4 refers to two distinct water molecules separated by 2.3 Å and not a single water with dual occupancy.

Fig. 4.

Comparison of T2Cu site in resting, nitrite-bound, NO bound, or mixed (NO and NO₂) forms. (A) The catalytic T2 Cu site of AcNiR with endogenously bound nitrite and NO in the same crystal, with the NO removed. The nitrite ligand has been modeled in the 2F_obs – F_calc electron density map, while the presence of the NO ligand is evident in the resulting F_obs – F_calc difference electron density map, contoured at 3.2σ. (B) Comparison of the T2Cu site of the enzyme in the resting state (red), nitrite soaked (green), endogenously bound NO (yellow), and endogenously bound nitrite and NO trapped in the same crystal (blue). A perspective view is shown from the surface of the protein into the substrate-entry channel. For clarity, all water molecules except the bound water in the resting state have been omitted. The flexibility of the Leu-106 residue and the nearly 90° flip of the Asp-98 side chain, between the gatekeeper orientation (pointing out of the page) and the proximal orientation, are clearly illustrated.

Scheme 1.