Electrochemical characterization of purified Rhus vernicifera laccase: voltammetric evidence for a sequential four-electron transfer.
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 (i(pa)/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 degrees 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)(NaN(3)) of 2.5 mM and an I(50)(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.
Johnson
Daniel L
DL
School of Biological Sciences and Chemistry, Faculty of Science and Engineering, Flinders University, GPO Box 2100, Adelaide 5001, South Australia, Australia.
Thompson
Janene L
JL
Brinkmann
Sandra M
SM
Schuller
Kathryn A
KA
Martin
Lisandra L
LL
D002384:Catalysis; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D042845:Laccase; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D029045:Rhus / Q000201:enzymology*; D019810:Sodium Azide / Q000494:pharmacology; D012969:Sodium Fluoride / Q000494:pharmacology; D013696:Temperature
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D006046:Gold; D012969:Sodium Fluoride; D019810:Sodium Azide; D010088:Oxidoreductases; D042845:Laccase; D010100:Oxygen