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

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been intensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies, and hence the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation has arisen from that even atomic resolution X-ray crystallography fails to determine positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs. We determined the 1.50 Å resolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ~ 106 kDa) in its resting state at low pH. Our neutron structure reveals protonation states of catalytic residues (deprotonated aspartate and protonated histidine), providing insights into the catalytic mechanism. We found that a hydroxide ion, not a water molecule, can exist as a ligand to the catalytic Cu atom in the resting state even at low pH. This OH-bound Cu site is unexpected given previous X-ray structures, but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.CBI学会2020年大