title: Structural insights into the function of a thermostable copper-containing nitrite reductase.
abstract: Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO(-)2) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate-bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η(1)-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen-bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. On the basis of these observations, the reaction mechanism of GtNIR through the η(1)-O coordination manner is proposed.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan; Department of Applied Chemistry, School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Faculty of Life and Environmental Sciences, Department of Environmental Information, Kyoto Prefectural University, Shimogamo-Hanki-cho, Sakyou, Kyoto 606-8522, Japan; Molecular-Recognition Structure Analysis Team, Molecular Profiling Research Center for Drug Discovery (molprof) National Institute of Advanced Industrial Science and Technology (AIST) 2-3-26 Aomi, Koto-ku, Tokyo 135-0064, Japan; Microbial Genome Research Group, Japan Agency of Marine-Earth Science and Technology, 2-15 Natsushima, Yokosuka, Kanagawa 237-0061, Japan; and RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yohta
authors.initials: Y
authors.lastname: Fukuda
authors.forename: Ka Man
authors.initials: KM
authors.lastname: Tse
authors.forename: Masami
authors.initials: M
authors.lastname: Lintuluoto
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Fukunishi
authors.forename: Eiichi
authors.initials: E
authors.lastname: Mizohata
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Hideto
authors.initials: H
authors.lastname: Takami
authors.forename: Masaki
authors.initials: M
authors.lastname: Nojiri
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Inoue
mesh_terms: D000333:Aeromonas / Q000737:chemistry / Q000201:enzymology*; D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D013816:Thermodynamics
chemical_list: D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing
keywords: Copper; Enzyme; Metallo; Metals; Methods; Oxidation-Reduction; Thermostable enzyme; X-ray Crystallography