Electrochemical characterization of purified Rhus vernicifera laccase: Voltammetric evidence for a sequential four-electron transfer

Daniel L. Johnson, Janene L. Thompson, Sandra M. Brinkmann, Kathryn A. Schuller, Lisandra L. Martin

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Rhus vernicifera (Rv) laccase was purified to electrophoretic homogeneity by hydrophobic interaction chromatography. A comprehensive study of the direct electrochemistry of Rv laccase covalently immobilized at a gold electrode using alkanethiol monolayers was undertaken. The observed midpoint potential was 410 mV versus the normal hydrogen electrode (NHE), consistent with reduction potentials obtained by potentiometric titration for the T1 copper site. Evidence is presented for a concerted 4-electron reversible process at slow scan rates (v) on the basis of peak current ratios (ipa/i pc). Catalytic currents were observed in the presence of the biological substrate oxygen, indicating that laccase activity is retained throughout the immobilization process. Electrochemical characteristics of the immobilized laccase were essentially invariant across the pH range 5.5-8.5 and the temperature range 5-35 °C. The purified enzyme displayed a pH optimum of 9.0, when assayed spectrophotometrically with syringaldazine as a substrate. Inhibition of the laccase activity with azide or fluoride showed an I 50(NaN3) of 2.5 mM and an I50(NaF) of 18.5 mM. Electrochemistry in the presence of azide reduces the anodic current by ca. one-half, consistent with the 4-electron process decreasing to a 2-electron process. However, fluoride has no effect on anaerobic electrochemistry. These electrochemical results suggest that the pH dependence of laccase activity is related to the effects of pH on the structure or binding of the substrate.

LanguageEnglish
Pages10229-10237
Number of pages9
JournalBiochemistry
Volume42
Issue number34
DOIs
Publication statusPublished - 2 Sep 2003
Externally publishedYes

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Rhus
Laccase
Electrons
Electrochemistry
Azides
Fluorides
Electrodes
Substrates
Sodium Azide
Chromatography
Titration
Hydrophobic and Hydrophilic Interactions
Immobilization
Gold
Copper
Hydrogen
Monolayers
Oxygen
Temperature
Enzymes

Cite this

Johnson, Daniel L. ; Thompson, Janene L. ; Brinkmann, Sandra M. ; Schuller, Kathryn A. ; Martin, Lisandra L. / Electrochemical characterization of purified Rhus vernicifera laccase : Voltammetric evidence for a sequential four-electron transfer. In: Biochemistry. 2003 ; Vol. 42, No. 34. pp. 10229-10237.
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Electrochemical characterization of purified Rhus vernicifera laccase : Voltammetric evidence for a sequential four-electron transfer. / Johnson, Daniel L.; Thompson, Janene L.; Brinkmann, Sandra M.; Schuller, Kathryn A.; Martin, Lisandra L.

In: Biochemistry, Vol. 42, No. 34, 02.09.2003, p. 10229-10237.

Research output: Contribution to journalArticleResearchpeer-review

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