Insight into Catalysis of Nitrous Oxide Reductase from High-resolution Structures of Resting and Inhibitor-bound Enzyme from Achromobacter cycloclastes Konstantinos Paraskevopoulos 1,2 , Svetlana V. Antonyuk 2 , R. Gary Sawers 3 , Robert R. Eady 2 and S. Samar Hasnain 1,2 ⁎ 1 School of Biomolecular Sciences, Liverpool John Moores University , Liverpool L3 5AF, UK 2 Molecular Biophysics Group, CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD, UK 3 Max-Planck-Institute for Terrestrial Microbiology, D-35043 Marburg, Germany The difficult chemistry of nitrous oxide (N 2 O) reduction to gaseous nitrogen (N 2 ) in biology is catalysed by the novel μ 4 -sulphide-bridged tetranuclear Cu z cluster of the N 2 O reductases (N 2 OR). Two spectroscopically distinct forms of this cluster have been identified as Cu Z and Cu Z *. We have obtained a 1.86 Å resolution crystal structure of the pink-purple species of N 2 OR from Achromobacter cycloclastes (AcN 2 OR) isolated under aerobic conditions. This structure reveals a previously unobserved ligation with two oxygen atoms from H 2 O/OH – coordinated to Cu1 and Cu4 of the catalytic centre. We ascribe this structure to be that of the Cu Z form of the cluster, since the previously reported structures of two blue species of N 2 ORs, also isolated aerobically, have characterised the redox inactive Cu Z * form, revealing a single water molecule at Cu4. Exposure of the as-isolated AcN 2 OR to sodium iodide led to reduction of the electron-donating Cu A site and the formation of a blue species. Structure determination of this adduct at 1.7 Å resolution showed that iodide was bound at the Cu Z site bridging the Cu1 and Cu4 ions. This structure represents the first observation of an inhibitor bound to the Cu1-Cu4 edge of the catalytic cluster, providing clear evidence for this being the catalytic edge in N 2 ORs. These structures, together with the published structural and spectroscopic data, give fresh insight into the mode of substrate binding, reduction and catalysis. © 2006 Elsevier Ltd. All rights reserved. *Corresponding author Keywords: denitrification; catalysis; nitrous oxide binding; copper chem- istry; electron gating Introduction The biochemical conversion of “inert gaseous substrates” is of substantial importance to life on the planet. A number of metalloenzymes have evolved to activate the otherwise inert substrates N 2 and N 2 O, enabling their biological utilisation. The active sites of these enzymes often involve complex metal–ligand clusters. How these clusters perform the difficult chemistry of such conversions has attracted the attention of a wide range of disciplines. Nitrogenase is a paradigm example, where structural studies have enabled the rationa- lisation of a tremendous body of spectroscopic data providing insightful understanding of how nitrogen fixation is achieved at ambient temperature and pressure by a unique metallo-cluster. Likewise, the crystal structures of N 2 OR from Pseudomonas nautica and Paracoccus denitrificans at 2.4 Å and 1.6 Å resolutions, have revealed a novel μ 4 -sulphide- bridged tetranuclear Cu Z cluster. 1 However, under- standing how this cluster, unique in biology, performs the difficult chemistry of N 2 O reduction remains a challenge. Recent spectroscopic and computational Abbreviations used: AcN 2 OR, Achromobacter cycloclastes nitrous oxide reductase; PnN 2 OR, Pseudomonas nautica nitrous oxide reductase; PdN 2 OR, Paracoccus denitrificans nitrous oxide reductase; AxN 2 OR, Alcaligenes xylosoxidans nitrous oxide reductase; N 2 OR, nitrous oxide reductase; DFT, density functional theory; EPR, electron paramagnetic resonance. E-mail address of the corresponding author: s.hasnain@dl.ac.uk doi:10.1016/j.jmb.2006.06.064 J. Mol. Biol. (2006) 362, 55–65 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.