Active-site protein dynamics and solvent accessibility in native Achromobacter cycloclastes copper nitrite reductase

• Main Authors: Kakali Sen, Sam Horrell, Demet Kekilli, Chin W. Yong, Thomas W. Keal, Hakan Atakisi, David W. Moreau, Robert E. Thorne, Michael A. Hough, Richard W. Strange
• Format: Article
• Language: English
• Published: International Union of Crystallography 2017-07-01
• Series: IUCrJ
• Subjects: serial crystallography; high temperature; catalysis; molecular dynamics; density functional theory; denitrification; copper nitrite reductase; radiolysis; synchrotron radiation
• Online Access: http://scripts.iucr.org/cgi-bin/paper?S2052252517007527

Microbial nitrite reductases are denitrifying enzymes that are a major component of the global nitrogen cycle. Multiple structures measured from one crystal (MSOX data) of copper nitrite reductase at 240 K, together with molecular-dynamics simulations, have revealed protein dynamics at the type 2 copper site that are significant for its catalytic properties and for the entry and exit of solvent or ligands to and from the active site. Molecular-dynamics simulations were performed using different protonation states of the key catalytic residues (AspCAT and HisCAT) involved in the nitrite-reduction mechanism of this enzyme. Taken together, the crystal structures and simulations show that the AspCAT protonation state strongly influences the active-site solvent accessibility, while the dynamics of the active-site `capping residue' (IleCAT), a determinant of ligand binding, are influenced both by temperature and by the protonation state of AspCAT. A previously unobserved conformation of IleCAT is seen in the elevated temperature series compared with 100 K structures. DFT calculations also show that the loss of a bound water ligand at the active site during the MSOX series is consistent with reduction of the type 2 Cu atom.