Page 59 - ILL Annual Report 2019

SCIENTIFIC HIGHLIGHTS
56-57
Figure 2
The AcNiR crystal used for neutron data collection.
While the benefits of neutrons are clear, they bring their own specific challenges. These include deuteration and the need to grow large crystals (0.1 to 1 mm3). For the production of perduterated AcNiR, we benefitted from the excellent assistance and facilities of the Deuteration Laboratory platform. Large crystals of perduterated AcNiR were then grown in Liverpool, using hanging-drop vapour diffusion methods in combination with seeding/feeding (figure 2). Neutron diffraction data were collected at room temperature using LADI-III to a resolution of 1.8 Å (figure 3) from a 0.36 mm3 crystal.
The resulting structure revealed that in the resting state of AcNiR, at pH values close to the optimum for activity, Aspcat is observed in a charged state (i.e. deprotonated) as expected, while Hiscat is unexpectedly seen in a neutral state (i.e. singly protonated). In addition, the catalytic T2Cu-ligated water (D1 in figure 4) is observed as a neutral D2O molecule—as opposed to D3O+ or OD–, which have previously been proposed as possible alternatives—while the bridging water (D2 in figure 4)
is also observed as a neutral D2O molecule, positioned with one deuteron directed towards Aspcat O+δ1 and one towards Hiscat Nε2. These details have allowed us to revisit the catalytic mechanism. We suggest that upon the binding of nitrite, a proton is transferred from the bridging water (D2) to the Oδ2 atom of Aspcat, prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from Aspcat to the bound nitrite, enabling the reaction to proceed.
Figure 3
A neutron diffraction pattern (exposure time of 18h) from the AcNiR crystal.
Figure 4
Nuclear scattering density at the T2Cu site and its surroundings. The protonation states of key residues, including Aspcat and Hiscat, are shown in grey; the positions and orientations of bound water molecules, including the T2Cu-ligated water (D1) and the bridging water (D2), are shown in turquoise.
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