Direct electron transfer from graphite and functionalized gold electrodes to T1 and T2/T3 copper centers of bilirubin oxidase |
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Authors: | Pablo Ramí rez,Rafael Andreu,Adam Heller,Sergey Shleev |
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Affiliation: | a Departamento de Química Física, Universidad de Sevilla, 41012 Sevilla, Spain b Centre de Recherche Paul Pascal (CRPP), Université Bordeaux I, UPR 8641, Avenue Albert Schweitzer, 33600 Pessac, France c Biomedical Laboratory Science, Malmö University, 20506 Malmö, Sweden d Department of Chemical Engineering and The Texas Material Institute, The University of Texas, 78712 Austin, USA e Department of Analytical Chemistry, Lund University, 221 00 Lund, Sweden f Laboratory of Chemical Enzymology, Institute of Biochemistry, 119071 Moscow, Russia |
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Abstract: | Direct electron transfer (DET) from bare spectrographic graphite (SPGE) or 3-mercaptopropionic acid-modified gold (MPA-gold) electrodes to Trachyderma tsunodae bilirubin oxidase (BOD) was studied under anaerobic and aerobic conditions by cyclic voltammetry and chronoamperometry. On cyclic voltammograms nonturnover Faradaic signals with midpoint potentials of about 700 mV and 400 mV were clearly observed corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively. The immobilized BOD was differently oriented on the two electrodes and its catalysis of O2-electroreduction was also massively different. On SPGE, where most of the enzyme was oriented with the T1 copper site proximal to the carbon with a quite slow ET process, well-pronounced DET-bioelectroreduction of O2 was observed, starting already at > 700 mV vs. NHE. In contrast, on MPA-gold most of the enzyme was oriented with its T2/T3 copper cluster proximal to the metal. Indeed, there was little DET-based catalysis of O2-electroreduction, even though the ET between the MPA-gold and the T2/T3 copper cluster of BOD was similar to that observed for the T1 site at SPGE. When BOD actively catalyzes the O2-electroreduction, the redox potential of its T1 site is 690 mV vs. NHE and that of one of its T2/T3 copper centers is 390 mV vs. NHE. The redox potential of the T2/T3 copper cluster of a resting form of BOD is suggested to be about 360 mV vs. NHE. These values, combined with the observed biocatalytic behavior, strongly suggest an uphill intra-molecular electron transfer from the T1 site to the T2/T3 cluster during the catalytic turnover of the enzyme. |
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Keywords: | BOD, bilirubin oxidase NI, native intermediate PI, peroxy intermediate FR, fully reduced BOD RF, resting form DET, direct electron transfer ET, electron transfer IET, intra-molecular electron transfer CV, cyclic voltammogram Em, midpoint redox potential ET1, ET2, ET3, and ET2/T3, redox potentials of the T1, T2, T3 sites and the T2/T3 cluster, respectively ΔEp, peak separation between anodic and cathodic peaks k0, standard electron transfer rate constant kDET, heterogeneous DET rate constant Γ, surface concentration jcat, biocatalytic current density mmlsi1" onclick=" submitCitation('/science?_ob=MathURL& _method=retrieve& _eid=1-s2.0-S0005272808006373& _mathId=si1.gif& _pii=S0005272808006373& _issn=00052728& _acct=C000053510& _version=1& _userid=1524097& md5=608db25aaeaf439da555a7f4579c3c5e')" style=" cursor:pointer " alt=" Click to view the MathML source" title=" Click to view the MathML source" > 20" border=" 0" style=" vertical-align:bottom" width=" 32" alt=" View the MathML source" title=" View the MathML source" src=" http://ars.els-cdn.com/content/image/1-s2.0-S0005272808006373-si1.gif" >, maximum biocatalytic current density Δjcat(+ F&minus ), differences in biocatalytic current densities in the absence and presence of F&minus EDC, N-(3-dimethylaminopropyl)-N&prime -ethylcarbodiimide hydrochloride NHS, N-hydroxysuccinimide AMTP, 4-aminothiophenol MHOL, 6-mercapto-1-hexanol MPA, 3-mercaptopropionic acid DT, 1-decanethiol SPGE, spectrographic graphite electrode MPA-gold, 3-mercaptopropionic acid-modified gold electrode |
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