Investigating the Reversible Inhibition Model of Laccase by Hydrogen Peroxide for Bioelectrocatalytic Applications

Abstract

The reversible inhibition of laccase by H 2 O 2 as a bioelectrocatalyst was studied in mediated-and direct electron transfer-based configurations to understand the differences in mechanism. The reversible inhibition of laccase follows a noncompetitive inhibition model when 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is used as an electron mediator, whereas laccase is inhibited by an uncompetitive inhibition model when anthracene-moieties are used to intelligently "dock" laccase to electrodes (consisting of multi-walled carbon nanotubes, MWCNTs) which afford direct electron transfer (DET). This further confirms the efficient orientation of laccase onto suitably-designed docking moieties for bioelectrocatalysis applications. 1-5 Along with some other MCOs (such as bilirubin oxidase, BOx), laccase contains 3 different copper centers. A type-1 copper center (T1 Cu) is proximally located within the enzyme structure and is responsible for the single-electron oxidation of phenolic-type substrates, and a further type-2 copper center (T2 Cu) and 2 further type-3 copper centers (T3 Cu) are combined in a tri-nuclear cluster (TNC), which is responsible for the 4-electron reduction of O 2 to H 2 O. Following the oxidation of substrates at the T1 Cu site, electrons are quickly and individually transferred to the TNC via a His-Cys-His tripeptide chain.6,7 Although laccase (from Trametes versicolor) is reported to optimally reduce O 2 at acidic pH (which may limit its incorporation within implantable EFCs) further complications can be found within a physiological setting, where laccase can be inhibited by Cl − (approximately 150 mM physiological concentration) via a competitive inhibition model whereby Cl − can compete against certain electron mediators at the T1 Cu site of the enzyme. 8,9 Laccase can be intelligently orientated (or "docked") onto electrode architectures whereby moieties with structural similarities to the natural substrates of laccase (oxidized at the T1 site) are incorporated on specifically-designed electrode architectures, which subsequently allow direct electron transfer (DET).2,4,10-14 Efficient mediated electron transfer (MET) has also been demonstrated using mediators such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or osmium-based complexes 1,[15][16][17] and comparison between orientationally-driven DET and MET of laccase (with ABTS) has been reported, contrasting catalytic current densities that can be achieved with both electron transfer methods.18 Such electrode architectures can be capable of demonstrating increased resistance to Cl − inhibition, since the moieties used for docking can efficiently out-compete Cl − at the T1 site and therefore permit electron transfer. 9,13,19 Recent studies, however, determined that certain membrane-less EFC configurations (favorable to lower overall device cost and size) can present further problems for laccase-based biocathodes, whereby oxidoreductases utilized at EFC bioanodes (such as glucose oxidase * Electrochemical Soc...

Synthesis and Structure-Activity Relationship Studies of Hydrazide-Hydrazones as Inhibitors of Laccase from Trametes versicolor

“…Factors affecting laccase activity were identified as the following: heavy metals, chelating agents, halides, ionic strength, organic solvents, and surfactants. Inorganic species such as hydrogen peroxide [67], bases [68], and organic inhibitors such as dipeptides [69], β-mercaptoethanol [70] as well as hardly available natural compounds such high-molecular-weight humic acids [71], ptilomycalin A [72], and medicarpin [73,74] complete the above list. To the best of our knowledge, only the semi-synthetic hydrazide-hydrazone ether derivative of coumarin and gentisaldehyde (2,5-dihydroxybenzaldehyde) [41] is known to inhibit laccase, with a mixed type of inhibition (K iu = 35 µM and K ic = 490 µM).…”

Comparison of Direct and Mediated Electron Transfer for Bilirubin Oxidase from Myrothecium Verrucaria. Effects of Inhibitors and Temperature on the Oxygen Reduction Reaction

“…These same authors reported the inhibition of laccase with H 2 O 2 under both conditions: DET and MET with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as redox mediator. They indicated that laccase under DET follows an uncompetitive inhibition by H 2 O 2 binding to the T2/T3 cluster [54]. Calvo showed the inhibition of laccase by H 2 O 2 using an multilayer osmium derivative poly (allylamine) redox mediator and found that exogenous H 2 O 2 inhibits laccase due to T3 Cu +1 [31].…”

High Performance Platinum Group Metal-Free Cathode Catalysts for Microbial Fuel Cell (MFC)

“…5,6 The theoretical value for ORR at pH 7.5 is 0.606 V (vs Ag/AgCl). Except for selective enzymes (laccase and bilirubine oxidase) performing ORR, in which the overpotentials are limited to 50-100 mV, [7][8][9] higher overpotentials are measured using abiotic and inorganic catalysts. 5,6 Unfortunately, enzymes are not stable in polluted environments as previously demonstrated.…”