title: New insights into the catalytic active-site structure of multicopper oxidases.
abstract: Structural models determined by X-ray crystallography play a central role in understanding the catalytic mechanism of enzymes. However, X-ray radiation generates hydrated electrons that can cause significant damage to the active sites of metalloenzymes. In the present study, crystal structures of the multicopper oxidases (MCOs) CueO from Escherichia coli and laccase from a metagenome were determined. Diffraction data were obtained from a single crystal under low to high X-ray dose conditions. At low levels of X-ray exposure, unambiguous electron density for an O atom was observed inside the trinuclear copper centre (TNC) in both MCOs. The gradual reduction of copper by hydrated electrons monitored by measurement of the Cu K-edge X-ray absorption spectra led to the disappearance of the electron density for the O atom. In addition, the size of the copper triangle was enlarged by a two-step shift in the location of the type III coppers owing to reduction. Further, binding of O2 to the TNC after its full reduction was observed in the case of the laccase. Based on these novel structural findings, the diverse resting structures of the MCOs and their four-electron O2-reduction process are discussed.
authors.affiliation: Faculty of Education, Kagawa University, 1-1 Saiwai-cho, Takamatsu, Kagawa 760-8522, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Komori
authors.affiliation: Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Sugiyama
authors.affiliation: Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Kunishige
authors.initials: K
authors.lastname: Kataoka
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Sapporo, Hokkaido 062-8517, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Miyazaki
authors.affiliation: RIKEN SPring-8 Center, 1-1-1 Koto, Mikazuki-cho, Sayo-gun, Hyogo 679-5198, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Higuchi
authors.affiliation: Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakurai
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000528:radiation effects; D055162:Biocatalysis; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000528:radiation effects; D042845:Laccase / Q000737:chemistry* / Q000528:radiation effects; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000528:radiation effects; D010100:Oxygen / Q000737:chemistry / Q000528:radiation effects; D011485:Protein Binding / Q000528:radiation effects; D013379:Substrate Specificity; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D003300:Copper; D010088:Oxidoreductases; C499679:cueO protein, E coli; D042845:Laccase; D010100:Oxygen
keywords: X-ray absorption spectroscopy; bioinorganic chemistry; enzyme catalysis; metalloproteins