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内容記述 |
Metalloproteins play fundamental roles in organisms. Knowing strategies of metalloproteins, by which they exquisitely tune their activities, will not only lead to the understanding of biochemical phenomena but also contribute to various applications. Obtaining precise enzyme structures that include all the H atoms, account for about half the atoms in biomacromolecules, allows a deeper understanding of their structure-function relationships.Copper-containing nitrite reductases (CuNIRs) catalyze transformation of nitrite to nitric oxide, which has impacts on geochemical, agricultural, and medical health fields. Despite intense research efforts, the dynamics of H atoms during the enzymatic reaction of CuNIRs are unknown and hence the catalytic mechanism remains unclear. We performed neutron crystallography to shoot a single H-atom resolution picture of a CuNIR in complex with nitrite. We found that nitrite binds on the catalytic Cu center as NO2- and not as protonated HNO2. Our X-ray data and quantum chemical calculation show that NO2- is in an electron-localized state that can facilitate N-O bond cleavage after receiving an electron. The catalytic residues, AspCAT and HisCAT, are deprotonated and protonated, respectively, suggesting that HisCAT is the point of departure of the proton transfer sequence. Subatomic resolution X-ray structures of the AspCAT-to-Asn mutants, which mimic the protonated state of AspCAT, were also determined to investigate the involvement of protonated AspCAT in the reaction. Our crystallographic data and quantum chemical calculations reveal in detail the first step of the CuNIR reaction.Pseudoazurin (Paz), a blue copper protein, mediates electron transfer in the bacterial anaerobic respiratory chain. Its redox potential is finely tuned by hydrogen (H) bond networks; however, difficulty in visualizing H atom positions in the protein hinders the de-tailed understanding of the protein's structure-function relationship. We here used neutron and sub-ångström resolution X-ray crystallography to directly observe H atoms in Paz. The 0.86-Å-resolution X-ray structure shows that the peptide bond between Pro80 and the His81 Cu ligand deviates from the ideal planar structure. The 1.9-Å-resolution neutron structure confirms a long-overlooked H bond formed by the amide of His81 and the S atom of another Cu ligand Cys78. Quantum mechanics/molecular mechanics calculations show that this H bond increases the redox potential of the Cu site and explains experimental results well. Our study demonstrates the potential of neutron and sub-ångström resolution X-ray crystallography to understand the chemistry of metalloproteins at atomic and quantum levels. |
