Copper‐Containing Nitrite Reductase Employing Proton‐Coupled Spin‐Exchanged Electron‐Transfer and Multiproton Synchronized Transfer to Reduce Nitrite

Abstract

The possible catalytic mechanism of the reduction of nitrite by copper-containing nitrite reductases (CuNiRs) is examined by using the M06 function according to two copper models, which include type-one copper (T1Cu) and type-two copper (T2Cu) sites. Examinations confirm that the protonation of two residues, His255 and Asp98, near the T2Cu site, can modulate the redox states of T1Cu and T2Cu, but cannot directly cause electron transfer from T1Cu to T2Cu. The electron hole remains at the T2Cu site when only one residue, His255 or Asp98, is protonated. However, the hole resides at the T1Cu site when both His255 and Asp98 are protonated. Then, the first protonation of nitrite takes place through indirect proton transfer from protonated His255 through the bridging H O and Asp98 with three protons moving together, which cannot cause the cleavage of the HO-NO bond. Subsequently, the substrate is required to obtain another proton from reprotonated His255 through the bridging H O. The reprotonation of nitrite induces the generation of nitric oxide (NO) and H O at the T2Cu site through a special double-proton-coupled spin-exchanged electron-transfer mechanism with indirect proton transfer from His255 to the substrate, a beta-electron of T2Cu shift to the NO cation, and the remaining alpha-electron changing spin direction at the same time. These results may provide useful information to better understand detailed proton-/electron-transfer reactions for the catalytic processes of CuNiR.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase

“…MSOX shows a side-on nitrosyl product bound to T2Cu as is observed in NO-soaked CuNIR crystals (15,18). However, several computational studies have suggested that direct release of NO without forming nitrosyl copper species is energetically favorable and hydroxyl T2Cu remains as a stable product (29)(30)(31)(32). The calculated activation energy shows good agreement with an activation energy value determined experimentally.…”

A three-domain copper-nitrite reductase with a unique sensing loop

“…The TsNirK crystal structure also shows additional residues postulated to be relevant for catalysis in two-domain NirKs, with Gln239 and Thr240 [ Fig. 2(d)] catalytically equivalent to Glu279 and Thr280 in AfNir (Boulanger et al, 2000;Fukuda et al, 2016) and in AcNir (Qin et al, 2017). There is a hydrogen bond between His80 and the side chain of Gln239.…”

Remote Water‐Mediated Proton Transfer Triggers Inter‐Cu Electron Transfer: Nitrite Reduction Activation in Copper‐Containing Nitrite Reductase

“…These results indicate that ET from T1Cu to T2Cu occurs after the T2Cu site receives the first proton and before obtains the second proton from the surroundings, which implies that the PT from Asp98 to the nitrite substrate are not the direct driving force for the inter-Cu ET. [26] Therefore, we infer that the proton conveying along the proton channel occurs from the surface of protein, which is far away from Asp98 or His255, essentially triggers the inter-Cu ET. The long-range PT pathway includes several PT steps, however, which step actually induces ET from T1Cu to T2Cu is unknown.…”

“…Thus, the computational model containing T1Cu, T2Cu, all residues and H 2 O molecules as mentioned above, named EÀ W E3 À D, was constructed to explore the possible PT steps along the high-pH channel (Figure 1). The structure of EÀ W E3 À D not only contains all the residues of our previous models, [26] but also includes the relative residues constructing the high-PH channel.…”

“…[25] Our previous DFT (density functional theory) study revealed that the protonation states of neighboring Asp98 and His255 in the T2Cu active site can modulate the distribution of electron hole. [26] If only one residue (Asp98 or His255) is protonated, the electron hole entirely distributes in the T2Cu site. However, if both Asp98 and His255 are protonated, the hole predominantly resides in the T1Cu site.…”