Effect of cysteine mutations on direct electron transfer of horseradish peroxidase on gold.
Surface exposed cysteines were genetically engineered in the structure of recombinant horseradish peroxidase (rHRP). Recombinant forms of HRP with either a His-tag or a Strep-tag at the C-terminus were produced, which additionally had cysteines at positions 57, 189 or 309 (C-terminus) of the polypeptide chain. An E. coli expression system was exploited. The effect of these mutations on the direct electron transfer (ET) between Au and the enzyme was studied in the reaction of the bioelectrocatalytic reduction of H(2)O(2), at -50 mV versus Ag/AgCl, on rHRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Adsorptive immobilisation of rHRPs on pre-oxidised Au from the protein solution at pH 6.0 provided a high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct (mediatorless) ET between Au and the active site of the rHRPs. Comparative analysis of the direct ET rate constants, estimated from the amperometric data on direct and mediated ET in the presence of catechol at pH 7.4 and 6.0, gave evidence that the introduction of the His-tag or cysteine in the C-terminal area of the enzyme resulted in an increased efficiency of direct ET due to a favourable coupled electron and proton transfer pathway. Due to the high efficiency of direct ET, the sensitivity was independent on the addition of the mediator or change of pH indicating that the response to H(2)O(2) is determined solely by the mass transfer of the analyte to the active site of HRP. The sensitivities obtained for the Au electrodes modified with rHRPs (2.0+/-0.1 A M(-1) cm(-2)) and the low detection limit for H(2)O(2) (10 nM) paves the way to develop the P-chip (peroxidase chip)--a biosensors system of a microscopic size for a mediatorless detection of H(2)O(2) based on direct ET between Au and the recombinant forms of HRP.
Ferapontova
Elena
E
Department of Analytical Chemistry, University of Lund, P.O. Box 124, SE 221 00 Lund, Sweden. elena.ferapontova@analykem.lu.se
Schmengler
Kai
K
Börchers
Torsten
T
Ruzgas
Tautgirdas
T
Gorton
Lo
L
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004579:Electron Transport; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry; D004926:Escherichia coli / Q000145:classification / Q000201:enzymology / Q000235:genetics; D006046:Gold; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D006046:Gold; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D003545:Cysteine