Reconstitution of peroxidase onto hemin-terminated alkanethiol self-assembled monolayers on gold

• Horseradish peroxidase was reconstituted onto hemin-terminated alkanethiol SAMs on gold.
• Treatment with apo-enzyme did not affect non-catalytic electrochemistry of hemin SAMs.
• Changes in the hemin reaction kinetics and potentials alone did not evidence HRP reconstitution.
• Reconstitution efficiency was correlated with the bioelectrocatalytic activity of reconstituted HRP.
• Bioelectrocatalytic reduction of H2O2 at rHRP-electrodes started from 0.4 V, 0.5 V higher than at hemin-electrodes.

Reconstitution of enzymes onto surface tethered cofactors may be considered as a powerful tool both for design of biorecognition interfaces with a molecular-level control over spatial distribution and orientation of biosensing elements and for fundamental studies of their structure–function relationships. Here, reconstitution of horseradish peroxidase (HRP) on the heme cofactor covalently attached to the mixed self-assembled monolayers (SAM) on gold electrodes was studied. The efficiency of the holoenzyme formation was assessed by analysis of the hemin electrode reaction and bioelectrocatalytic activity of HRP. The formal potential, E0′, of the electrode-tethered hemin approached −340 mV vs. Ag/AgCl, while the heterogeneous electron transfer (ET) rate constant, ks, for its redox transformation ranged between 245 and 340 s−1, depending on the SAM composition. After enzyme reconstitution, both the E0′ and ks values for the hemin ET reaction showed a very little variation, while the onset of electrocatalytic reduction of H2O2 shifted by ca. 500 mV more positive by this approaching the potentials of compound I of native HRP. Both the onset potential and bioelectrocatalytic currents were lower than observed with native HRP adsorbed on gold (Ferapontova and Gorton, 2001), being consistent with a lower surface population of the holoenzyme and/or less efficient ET pathway.

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