Unraveling the mechanism of catalytic reduction of O2 by microperoxidase-11 adsorbed within a transparent 3D-nanoporous ITO film.

Nanoporous films of indium tin oxide (ITO), with thicknesses ranging from 250 nm to 2 µm, were prepared by Glancing Angle Deposition (GLAD) and used as highly sensitive transparent 3D-electrodes for quantitatively interrogating, by time-resolved spectroelectrochemistry, the reactivity of microperoxidase-11 (MP-11) adsorbed within such films. The capacitive current densities of these 3D-electrodes as well as the amount of adsorbed MP-11 were shown to be linearly correlated to the GLAD ITO film thickness, indicating a homogeneous distribution of MP-11 across the film as well as homogeneous film porosity. Under saturating adsorption conditions, MP-11 film concentration as high as 60 mM was reached. This is equivalent to a stack of 110 monolayers of MP-11 per micrometer film thickness. This high MP-11 film loading combined with the excellent ITO film conductivity has allowed the simultaneous characterization of the heterogeneous one-electron transfer dynamics of the MP-11 Fe(III)/Fe(II) redox couple by cyclic voltammetry and cyclic voltabsorptometry, up to a scan rate of few volts per second with a satisfactory single-scan signal-to-noise ratio. The potency of the method to unravel complex redox coupled chemical reactions was also demonstrated with the catalytic reduction of oxygen by MP-11. In the presence of O(2), cross-correlation of electrochemical and spectroscopic data has allowed us to determine the key kinetics and thermodynamics parameters of the redox catalysis that otherwise could not be easily extracted using conventional protein film voltammetry. On the basis of numerical simulations of cyclic voltammograms and voltabsorptograms and within the framework of different plausible catalytic reaction schemes including appropriate approximations, it was shown possible to discriminate between different possible catalytic pathways and to identify the relevant catalytic cycle. In addition, from the best fits of simulations to the experimental voltammograms and voltabsorptograms, the partition coefficient of O(2) for the ITO film as well as the values of two kinetic rate constants could be extracted. It was finally concluded that the catalytic reduction of O(2) by MP-11 adsorbed within nanoporous ITO films occurs via a 2-electron mechanism with the formation of an intermediate Fe(III)-OOH adduct characterized by a decay rate of 11 s(-1). The spectroelectroanalytical strategy presented here opens new opportunities for characterizing complex redox-coupled chemical reactions not only with redox proteins, but also with redox biomimetic systems and catalysts. It might also be of great interest for the development and optimization of new spectroelectrochemical sensors and biosensors, or eventually new photoelectrocatalytic systems or biofuel cells.

Cyclic voltammetric responses of horseradish peroxidase multilayers on electrodes.

The catalytic responses obtained with step-by-step neutravidin-biotin deposition of successive monolayers of HRP are analyzed by means of cyclic voltammetry. The theoretical tools that have been developed allowed full characterization of the multilayered HRP coatings by means of a combination between closed-form analysis of limiting behaviors and finite difference numerical computations. An analysis of the experiments in which the number of monolayers was extended to 16 allowed an approximate determination of the average thickness of each monolayer, pointing to a compact arrangement of neutravidin and biotinylated HRP. The piling up of so many monolayers on the electrode allowed an improvement of the catalytic current by a factor of ca. 10, leading to very good sensitivities in term of cosubstrate detection.

Redox enzymes immobilized on electrodes with solution cosubstrates. General procedure for simulation of time-resolved catalytic responses.

In view of the existing and potential applications of electrochemical enzymatic catalysis with redox enzymes immobilized on the electrode surface in biosensors, a numerical calculation procedure for simulating their cyclic voltammetric responses is presented. It is applicable to systems involving a redox cosubstrate in solution. The cosubstrates, substrates, products, and inhibitors are assumed to diffuse linearly (planar electrode) between the electrode and the solution. The reactions in which the various forms of the immobilized enzyme participate may be as numerous and intricate as required by the simulation with no other restriction than the computing time. They may, at will, follow or not follow Michaelis-Menten kinetics. Slow charge-transfer cosubstrates are treated in the framework of Butler-Volmer kinetic law.

The standard redox potential of the phenyl radical/anion couple.

The voltammogram of aryldiazonium tetrafluoroborates in acetonitrile (ACN), at low concentration, shows a first one-electron wave followed at a more negative potential by a small second wave; this last one corresponds to the reduction of the radical formed at the level of the first wave. Simulation of the voltammogram permits one to determine the standard redox potential of the radical/anion couple Eo(Ph./Ph-) = 0.05 V/SCE and the reduction mechanism of the diazonium cation. An electron transfer concerted with the cleavage of the C-N bond furnishes the aryl radical; this radical undergoes two competitive reactions: reduction at the electrode and H-atom transfer.

Evidence for inverted region behavior in proton transfer to carbanions.

The diphenylmethane-diphenylmethyl anion acid/base couple in N,N-dimethylformamide is taken as an example for investigating the dynamics of proton transfer at carbon in a system where the acid is not activated by an electron-withdrawing group or by removal of an electron. The laser flash electron photoinjection technique is applied to the determination of the rate constant for the protonation of diphenylmethyl anion by an extended series of acids that offers a range of driving forces encompassing over 1.2 eV. The plot of the rate constant versus the pK(a) difference between diphenylmethane and the acids or of the activation free energy versus the standard free energy of the reaction exhibits clear "inverted region" behavior (by a factor of 80 in terms of rate constants). While such behaviors have been predicted and observed for outersphere electron-transfer reactions, previous evidence for proton-transfer reactions was scarce. Entropic factors, derived from an investigation of the temperature dependence of the experimental rate constants, are also discussed.