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

The copper-containing nitrite reductase (CuNiR) catalyzes the biological conversion of nitrite to nitric oxide, in which the key long-range electron/proton transfers are involved in the catalysis. However, the details of the electron/proton transfer mechanism are still unknown. In particular, the driving force of the electron transfer from the type-1 copper (T1Cu) site to the type-2 copper (T2Cu) site is ambiguous. Here, we explored the two possible proton transfer channels by using two-layered ONIOM calculations, including the high-pH proton channel and the primary proton channel. Our calculation results reveal that the driving force for electron transfer from T1Cu to T2Cu comes from a remote water-mediated triple-proton transfer, which can be termed as a water-mediated triple-proton-coupled electron transfer mechanism. In the high-pH proton channel, the water-mediated triple-proton transfer occurs from Glu113 to an intermediate water molecule, whereas in the primary channel the transfer is performed from Lys128 to His260. Subsequently, the two channels employ another two or three distinct proton transfer steps to deliver the proton to the nitrite substrate at the T2Cu site. These findings explain the detailed proton/electron transfer mechanisms of copper-containing nitrite reductase and may extend our understanding of the diverse proton-coupled ET mechanisms in complicated proteins.