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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 anFo-Fc map for Glu451a Oε2 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 structuresmore » (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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1354625
Report Number(s):
BNL-113142-2016-JA
Journal ID: ISSN 1399-0047
DOE Contract Number:  
SC00112704
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section D: Biological Crystallography (Online)
Additional Journal Information:
Journal Volume: 71; Journal Issue: 12; Journal ID: ISSN 1399-0047
Publisher:
International Union of Crystallography
Country of Publication:
United States
Language:
English
Subject:
copper depletion; dioxygen reduction; laccase; multicopper oxidase; proton-relay mechanism; radiation damage; X-ray-induced reduction

Citation Formats

Serrano-Posada, Hugo, Centeno-Leija, Sara, Rojas-Trejo, Sonia Patricia, Rodríguez-Almazán, Claudia, Stojanoff, Vivian, and Rudiño-Piñera, Enrique. X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2 -reduction states. United States: N. p., 2015. Web. doi:10.1107/S1399004715018714.
Serrano-Posada, Hugo, Centeno-Leija, Sara, Rojas-Trejo, Sonia Patricia, Rodríguez-Almazán, Claudia, Stojanoff, Vivian, & Rudiño-Piñera, Enrique. X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2 -reduction states. United States. https://doi.org/10.1107/S1399004715018714
Serrano-Posada, Hugo, Centeno-Leija, Sara, Rojas-Trejo, Sonia Patricia, Rodríguez-Almazán, Claudia, Stojanoff, Vivian, and Rudiño-Piñera, Enrique. 2015. "X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2 -reduction states". United States. https://doi.org/10.1107/S1399004715018714.
@article{osti_1354625,
title = {X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2 -reduction states},
author = {Serrano-Posada, Hugo and Centeno-Leija, Sara and Rojas-Trejo, Sonia Patricia and Rodríguez-Almazán, Claudia and Stojanoff, Vivian and Rudiño-Piñera, Enrique},
abstractNote = {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 anFo-Fc map for Glu451a Oε2 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.},
doi = {10.1107/S1399004715018714},
url = {https://www.osti.gov/biblio/1354625}, journal = {Acta Crystallographica. Section D: Biological Crystallography (Online)},
issn = {1399-0047},
number = 12,
volume = 71,
place = {United States},
year = {Thu Nov 26 00:00:00 EST 2015},
month = {Thu Nov 26 00:00:00 EST 2015}
}