title: X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states.
abstract: During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2 to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2 reduction was found in a double conformation: Glu451a (∼7 Å from the TNC) and Glu451b (∼4.5 Å from the TNC). A positive peak of electron density above 3.5σ in an Fo - Fc map for Glu451a O(ℇ2) indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.
authors.affiliation: Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
authors.forename: Hugo
authors.initials: H
authors.lastname: Serrano-Posada
authors.affiliation: Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
authors.forename: Sara
authors.initials: S
authors.lastname: Centeno-Leija
authors.affiliation: Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
authors.forename: Sonia Patricia
authors.initials: SP
authors.lastname: Rojas-Trejo
authors.affiliation: Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
authors.forename: Claudia
authors.initials: C
authors.lastname: Rodríguez-Almazán
authors.affiliation: NSLS, Brookhaven National Laboratory, 75 Brookhaven Avenue, Building 725D, Upton, NY 11973-5000, USA.
authors.forename: Vivian
authors.initials: V
authors.lastname: Stojanoff
authors.affiliation: Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
authors.forename: Enrique
authors.initials: E
authors.lastname: Rudiño-Piñera
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D020134:Catalytic Domain; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011522:Protons; D011994:Recombinant Proteins; D003300:Copper; D010088:Oxidoreductases; D010100:Oxygen
keywords: X-ray-induced reduction; copper depletion; dioxygen reduction; laccase; multicopper oxidase; proton-relay mechanism; radiation damage