Abstact

Metalloproteins represent a major fraction of the protein kingdom and often exploit the redox chemistry of transition metals to drive key biological events involving proton and electron transfer. Copper is one of the most widely used transition metals whose redox properties are utilised in both electron transfer and catalysis of chemical substrates. Copper nitrite reductases (CuNiRs) utilise two types of copper centres and have become a model system for studying complex biological events that underpin the reaction mechanisms of redox enzymes, including proton-coupled electron transfer and substrate gating. We utilised the higher X-ray energy (13 keV) available at the SACLA X-ray Free Electron Laser (XFEL) and SHELXL refinement to obtain accurate atomic resolution structures of CuNiRs at ~1 A from three organisms - in the oxidised (low and high pH), reduced and substrate-bound states. A consistent picture now emerges with the observation of a pentacoordinated oxidised catalytic type-2 Cu (T2Cu(2+)) centre in all cases. A tetracoordinated reduced T2Cu(+) site with a single solvent ligand has also been captured, giving structural support to the random-sequential scheme with ordered pathway being dominant.