Electric field-induced functional changes in electrode-immobilized mutant species of human cytochrome c.

Post-translational modifications and naturally occurring mutations of cytochrome c have been recognized as a regulatory mechanism to control its biology. In this work, we investigate the effect of such in vivo chemical modifications of human cytochrome c on its redox properties in the adsorbed state onto an electrode. In particular, tyrosines 48 and 97 have been replaced by the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), thus mimicking tyrosine phosphorylation. Additionally, tyrosine 48 has been replaced by a histidine producing the natural Y48H pathogenic mutant. Thermodynamics and kinetics of the interfacial electron transfer of wild-type cytochrome c and herein produced variants, adsorbed electrostatically under different local interfacial electric fields, were determined by means of variable temperature cyclic film voltammetry. It is shown that non-native cytochrome c variants immobilized under a low interfacial electric field display redox thermodynamics and kinetics similar to those of wild-type cytochrome c. However, upon increasing the strength of the electric field, the redox thermodynamics and kinetics of the modified proteins markedly differ from those of the wild-type species. The mutations promote stabilization of the oxidized form and a significant increase in the activation enthalpy values that can be ascribed to a subtle distortion of the heme cofactor and/or difference of the amino acid rearrangements rather than to a coarse protein structural change. Overall, these results point to a combined effect of the single point mutations at positions 48 and 97 and the strength of electrostatic binding on the regulatory mechanism of mitochondrial membrane activity, when acting as a redox shuttle protein.

Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants.

Post-translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylation-induced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome c with glutamine to mimic lysine acetylation and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl-mimetic mutants, but their thermal stability was significantly altered. NMR chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome c to its physiological electron donor, cytochrome c1 -which is a component of mitochondrial complex III, or cytochrome bc1 -thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome c oxidase.

Active Role of the Buffer in the Proton-Coupled Electron Transfer of Immobilized Iron Porphyrins.

Evaluation of the proton-coupled electron transfer thermodynamics of immobilized hemin is challenging due to the disparity of its electrochemical titration curves reported in the literature. Deviations from the one-electron, one-proton transfer at circumneutral pHs have been commonly ascribed to either the formation of dimeric species or the ionization of a second iron-bound water molecule. Herein, however, we report on non-idealities in the more acidic region, whose onset and extent vary with the nature and concentration of the commonly used phosphate and acetate buffers. It is shown that these deviations originate in the ligand-exchange binding between the oxidized aquo-hemin complex and the anionic components of the buffer, so that they are restricted to the pH interval where these forms coexist. A stepwise approach was developed to quantify unambiguously the apparent and intrinsic binding equilibrium constants. The apparent binding equilibrium constant exhibits a peak-shaped pH dependence, whose maximum is located at approximately the midpoint between the pKa of the iron-bound water and the first pKa of the buffer, and its magnitude is greater for the phosphate than for the acetate buffer. But strikingly, the opposite trend was found for the magnitude of the intrinsic binding equilibrium constants determined from the apparent ones, due to the different relative locations of the phosphoric and acetic pKa values with respect to that of the oxidized aquo-hemin. To probe the role of the heme propionic residues, a similar study was carried out with a propionic-free iron porphyrin containing eight ethyl residues. These substituents decrease the acidity of the iron-bound water, strengthen the iron(III)-acetate binding, weaken the iron(III)-dihydrogen phosphate binding, and enable the binding between iron(III) and monohydrogen phosphate, which was hampered in hemin by the presence of the negatively charged propionate residues. Overall, this work provides a more complete speciation of immobilized iron porphyrins under acidic conditions than previously considered, showing the substitutional lability of the aqua ligand in the oxidized state of the iron center and the reluctance of its hydroxyl counterpart to anion exchange. Knowledge of these redox- and pH-dependent bindings with the buffer components is crucial for a rigorous quantification of the proton-coupled electron transfer and the electrocatalytic activity of iron porphyrins.

Cobalt Metal-Organic Framework Based on Layered Double Nanosheets for Enhanced Electrocatalytic Water Oxidation in Neutral Media.

A new cobalt metal-organic framework (2D-Co-MOF) based on well-defined layered double cores that are strongly connected by intermolecular bonds has been developed. Its 3D structure is held together by π-π stacking interactions between the labile pyridine ligands of the nanosheets. In aqueous solution, the axial pyridine ligands are exchanged by water molecules, producing a delamination of the material, where the individual double nanosheets preserve their structure. The original 3D layered structure can be restored by a solvothermal process with pyridine, so that the material shows a "memory effect" during the delamination-pillarization process. Electrochemical activation of a 2D-Co-MOF@Nafion-modified graphite electrode in aqueous solution improves the ionic migration and electron transfer across the film and promotes the formation of the electrocatalytically active cobalt species for the oxygen evolution reaction (OER). The so-activated 2D-Co-MOF@Nafion composite exhibits an outstanding electrocatalytic performance for the OER at neutral pH, with a TOF value (0.034 s-1 at an overpotential of 400 mV) and robustness superior to those reported for similar electrocatalysts under similar conditions. The particular topology of the delaminated nanosheets, with quite distant cobalt centers, precludes the direct coupling between the electrocatalytically active centers of the same sheet. On the other hand, the increase in ionic migration across the film during the electrochemical activation stage rules out the intersheet coupling between active cobalt centers, as this scenario would impair electrolyte permeation. Altogether, the most plausible mechanism for the O-O bond formation is the water nucleophilic attack to single Co(IV)-oxo or Co(III)-oxyl centers. Its high electrochemical efficiency suggests that the presence of nitrogen-containing aromatic equatorial ligands facilitates the water nucleophilic attack, as in the case of the highly efficient cobalt porphyrins.

Physical contact between cytochrome c1 and cytochrome c increases the driving force for electron transfer.

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc1), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieske-cytochrome c1 (Cc1) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc1 switches intermittently during CIII activity. However, the electrochemical properties of Cc1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc1. Findings reveal that Cc1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc1 to Cc is thermodynamically favored in the immobilized Cc1-Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc1-Cc complex. Our data supports the Rieske-Cc1-Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior.

Cobalt Metal-Organic Framework Based on Two Dinuclear Secondary Building Units for Electrocatalytic Oxygen Evolution.

The synthesis of a new microporous metal-organic framework (MOF) based on two secondary building units, with dinuclear cobalt centers, has been developed. The employment of a well-defined cobalt cluster results in an unusual topology of the Co2-MOF, where one of the cobalt centers has three open coordination positions, which has no precedent in MOF materials based on cobalt. Adsorption isotherms have revealed that Co2-MOF is in the range of best CO2 adsorbents among the carbon materials, with very high CO2/CH4 selectivity. On the other hand, dispersion of Co2-MOF in an alcoholic solution of Nafion gives rise to a composite (Co2-MOF@Nafion) with great resistance to hydrolysis in aqueous media and good adherence to graphite electrodes. In fact, it exhibits high electrocatalytic activity and robustness for the oxygen evolution reaction (OER), with a turnover frequency number value superior to those reported for similar electrocatalysts. Overall, this work has provided the basis for the rational design of new cobalt OER catalysts and related materials employing well-defined metal clusters as directing agents of the MOF structure.

Protein crosslinking improves the thermal resistance of plastocyanin immobilized on a modified gold electrode.

Increasing the thermal stability of immobilized proteins is a motivating goal for improving the performance of electrochemical biodevices. In this work, we propose the immobilization of crosslinked plastocyanin from the thermophilic cyanobacterium Phormidium laminosum by simultaneous incubation of a mixture of plastocyanin and the coupling reagents. The thermal stability of the so built covalently immobilized protein films has been assessed by cyclic voltammetry in the 0-90 °C temperature range and has been compared to that of physisorbed films. It is shown that the protein loss along a thermal cycle is significantly reduced in the case of the crosslinked films, whose redox properties remain unaltered along a cyclic heating-cooling thermal scan, and can withstand the contact with 70 °C solutions for four hours. Comparison of thermal unfolding curves obtained by circular dichroism spectroscopy of both free and crosslinked protein confirms the improved thermic resistance of the crosslinked plastocyanin. Notably, the electron transfer thermodynamics of physisorbed and crosslinked plastocyanin films are quite similar, suggesting that the formation of intra- and inter-protein amide bonds do not affect the integrity and functionality of the copper redox centers. UV-Vis absorption and circular dichroism measurements corroborate that protein crosslinking does not alter the coordination geometry of the metal center.

Key Role of the Local Hydrophobicity in the East Patch of Plastocyanins on Their Thermal Stability and Redox Properties.

Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.

Interprotein Coupling Enhances the Electrocatalytic Efficiency of Tobacco Peroxidase Immobilized at a Graphite Electrode.

Covalent immobilization of enzymes at electrodes via amide bond formation is usually carried out by a two-step protocol, in which surface carboxylic groups are first activated with the corresponding cross-coupling reagents and then reacted with protein amine groups. Herein, it is shown that a modification of the above protocol, involving the simultaneous incubation of tobacco peroxidase and the pyrolytic graphite electrode with the cross-coupling reagents produces higher and more stable electrocatalytic currents than those obtained with either physically adsorbed enzymes or covalently immobilized enzymes according to the usual immobilization protocol. The remarkably improved electrocatalytic properties of the present peroxidase biosensor that operates in the 0.3 V ≤ E ≤ 0.8 V (vs SHE) potential range can be attributed to both an efficient electronic coupling between tobacco peroxidase and graphite and to the formation of intra- and intermolecular amide bonds that stabilize the protein structure and improve the percentage of anchoring groups that provide an adequate orientation for electron exchange with the electrode. The optimized tobacco peroxidase sensor exhibits a working concentration range of 10-900 µM, a sensitivity of 0.08 A M(-1) cm(-2) (RSD 0.05), a detection limit of 2 µM (RSD 0.09), and a good long-term stability, as long as it operates at low temperature. These parameter values are among the best reported so far for a peroxidase biosensor operating under simple direct electron transfer conditions.

Temperature-Driven Changeover in the Electron-Transfer Mechanism of a Thermophilic Plastocyanin.

Electron-transfer kinetics of the thermophilic protein Plastocyanin from Phormidium laminosum adsorbed on 1,ω-alkanedithiol self-assembled monolayers (SAMs) deposited on gold have been investigated. The standard electron-transfer rate constant has been determined as a function of electrode-protein distance and solution viscosity over a broad temperature range (0-90 °C). For either thin or thick SAMs, the electron-transfer regime remains invariant with temperature, whereas for the 1,11-undecanethiol SAM of intermediate chain length, a kinetic regime changeover from a gated or friction-controlled mechanism at low temperature (0-30 °C) to a nonadiabatic mechanism above 40 °C is observed. To the best of our knowledge, this is the first time a thermal-induced transition between these two kinetic regimes is reported for a metalloprotein.

Analytical expressions for proton transfer voltammetry: analogy to surface redox voltammetry with Frumkin interactions.

Theory for interfacial proton transfer voltammetry of a molecular film containing any acid/base loading has been developed under equilibrium conditions. Diagnostic criteria to disentangle the interplay between diffuse layer and ionization effects are outlined. Easy-to-use analytical expressions for the voltammetric features are derived for the particular case of an invariant diffuse layer effect, which turn out to be entirely analogous to those for a surface redox conversion with Frumkin interactions. It is demonstrated that, regardless of the electrolyte concentration, significant ionization of the external acid groups located nearby the diffuse layer is sufficient for the fulfillment of this relevant particular case. A strategy is outlined to determine the amount, the intrinsic pKa, and the burial depth of the voltammetrically active groups from the surface concentration dependence of the main voltammetric features. Self-assembled monolayers of 11-mercaptoundecanoic acid deposited on Au(111), containing higher amounts of buried carboxylic groups than previously reported, have been studied to assess more critically the influence of electrostatic effects on the ionization process. Preliminary evidence suggests that the protonation/deprotonation voltammetric wave involves physisorbed rather than chemisorbed thiol molecules. Application of the present theoretical approach to this system reveals that the voltammetrically active carboxylic groups are located close to the electrode surface and become more acidic upon increasing their surface concentration.

Proton transfer voltammetry at electrodes modified with acid thiol monolayers.

By combining a description of the potential profile at electrodes coated with acid thiol monolayers with a quadratic relationship between activation energy and electrode potential, a rather simple expression for proton transfer voltammograms is derived. Our electrostatic analysis shows that proton transfer can only produce narrow voltammetric peaks when the immobilized acid groups lie close to the metal substrate. Quantitative fits of experimental voltammograms obtained with an Au(111) electrode modified with a 11-mercaptoundecanoic monolayer at pH 8.5 reveal that less than 1% of the carboxylic groups in the monolayer participate in the potential induced proton transfer process and that these groups lay close to the metal surface. A preliminary analysis of the kinetic parameters suggests that the interfacial electric field facilitates an intrinsically slow proton exchange between a proton donor and acceptor pair that are not in close contact with each other at the interface.

Diffusional surface voltammetry as a probe of adsorption energetics.

Direct determination of the adsorption free energy for extremely low surface coverages (Henry limit) requires the use of a technique that must be highly sensitive to both the amount and the energetics of adsorbed molecules. Herein, we demonstrate that diffusional surface voltammetry (DSV), which embodies film and stripping voltammetries as two limiting cases, can be used to achieve this goal for electroactive adsorbates. To this end, a general analytical expression for the surface voltammetric peak potential of DSV is derived, which covers the full range of scan rates, bulk concentrations, and adsorptivity of the freely diffusing form of the redox couple, so that the surface redox conversion can be either equilibrated with or transport-isolated from the solution bulk. Strategies to get a quantitative insight into the energetics of electrosorption are outlined, and diagnostic criteria for their application are developed. In particular, it is demonstrated that DSV can be used in its stripping mode to determine group contributions to the adsorption free energy, avoiding possible interferences from intermolecular interactions or formation of oligomeric species. Application of this protocol to the reductive desorption of distinct homologous series of alkylthiolates adsorbed at mercury electrodes has allowed us to determine the contributions of the CH(n) groups (n = 0-3) to the free energy of adsorption of these molecules. These estimates are shown to correlate linearly with the corresponding group contributions to the octanol-water partition coefficient, revealing that adsorption of individual hydrocarbon groups at the mercury/solution interface scales with their hydrophobicity. Overall, the present work enlarges the capability of surface voltammetry to probe adsorption energetics down to the micromolar level, and it represents a first step toward the development of a unified treatment of stripping and film voltammetries.

Accurate analytical expressions for stripping voltammetry in the Henry adsorption limit.

A strategy is developed to derive accurate analytical expressions for low-coverage cathodic stripping voltammetry. The procedure relies on the observation that diffusion affects the location of simulated voltammetric waves but not their shape, provided that physisorption of the analyte is negligible. As a proof of the generality of the proposed approach and having in mind the stripping of thiols, analytical solutions are derived for the cathodic stripping of monomers, dimers, and a mixture of monomers and dimers, whose reliability is proved by their comparison with numerically simulated voltammograms. Application to the deposition and reductive desorption of mercaptoacetic acid at a mercury electrode demonstrates that these approximate solutions can be used to get insights into the interfacial organization of incipient films. For this particular system, a transition from monomeric to dimeric behavior is identified upon increasing the thiol surface concentration. Further generalization of the proposed methodology is achieved by deriving an approximate analytical solution for thin-layer anodic stripping voltammetry, which is satisfactorily compared to the existing summation series solution.

Chemical reactivity and electrochemistry of metal-metal-bonded zincocenes.

Attempts to prepare mixed-ligand zinc-zinc-bonded compounds that contain bulky C(5)Me(5) and terphenyl groups, [Zn(2)(C(5)Me(5))(Ar')], lead to disproportionation. The resulting half-sandwich Zn(II) complexes [(η(5)-C(5)Me(5))ZnAr'] (Ar' = 2,6-(2,6-(i)Pr(2)C(6)H(3))(2)-C(6)H(3), 2; 2,6-(2,6-Me(2)C(6)H(3))(2)-C(6)H(3), 3) can also be obtained from the reaction of [Zn(C(5)Me(5))(2)] with the corresponding LiAr'. In the presence of pyr-py (4-pyrrolidinopyridine) or DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), [Zn(2)(η(5)-C(5)Me(5))(2)] reacts with C(5)Me(5)OH to afford the tetrametallic complexes [Zn(2)(η(5)-C(5)Me(5))L(µ-OC(5)Me(5))](2) (L = pyr-py, 6; DBU, 8), respectively. The bulkier terphenyloxide Ar(Mes)O(-) group (Ar(Mes) = 2,6-(2,4,6-Me(3)C(6)H(2))(2)-C(6)H(3)) gives instead the dimetallic compound [Zn(2)(η(5)-C(5)Me(5))(OAr(Mes))(pyr-py)(2)], 7, that features a terminal Zn-OAr(Mes) bond. DFT calculations on models of 6-8 and also on the Zn-Zn-bonded complexes [Zn(2)(η(5)-C(5)H(5))(OC(5)H(5))(py)(2)] and [(η(5)-C(5)H(5))ZnZn(py)(3)](+) have been performed and reveal the nonsymmetric nature of the Zn-Zn bond with lower charge and higher participation of the s orbital of the zinc atom coordinated to the cyclopentadienyl ligand with respect to the metal within the pseudo-ZnL(3) fragment. Cyclic voltammetric studies on [Zn(2)(η(5)-C(5)Me(5))(2)] have been also carried out and the results compared with the behavior of [Zn(C(5)Me(5))(2)] and related magnesium and calcium metallocenes.

Probing interfacial solvation of incipient self-assembled monolayers.

Poor solvation of alkylthiols in a water-rich interfacial environment is shown to induce a dispersion of energetically-distinct states above a critical value of the hydrocarbon chain length, which depends on the hydrophilicity of the terminal group. Switching between single and dispersed states can be achieved by an adequate choice of the solvent composition.

Reorientation of thiols during 2D self-assembly: interplay between steric and energetic factors.

Reorientation of thiols during their 2D self-assembly is well established; however, little is known about its energetics and the factors that control its onset. We have developed a new strategy to determine the critical reorientational surface concentration (crsc) of thiols at the substrate/solution interface, which makes use of a cathodic stripping protocol. Its application to distinct homologous series of alkylthiols shows that the magnitude of the crsc and its variation with the molecular size is strongly dependent on the nature of the terminal group. Methyl-terminated alkylthiols reorient close to the saturation coverage of the lying-down phase, thus following their molecular size trend; whereas reorientation of alkylthiols bearing a negatively charged end group starts well below the monolayer coverage of the lying-down phase, with its onset being almost independent of the molecular size. Hydroxy-terminated alkylthiols show an intermediate behavior. A theoretical approach is developed to determine the reorientation equilibrium constant from the crsc value. The standard free energy of reorientation has been found to vary linearly with the alkyl chain length, and to increase upon replacing the terminal methyl group by a negatively charged one. A quantitative correlation between the reorientation equilibrium constant and the hydrophobicity of the molecule has been established. Overall, these findings have allowed us to disentangle the role of steric and energetic factors in the onset of the reorientation process of alkylthiols, demonstrating that their interplay can be finely tuned by varying either the alkyl chain length or the nature of the terminal group.

Aliphatic alcohols facilitate interfacial reorientation of thiols: correlation with alcohol adsorptivity.

The influence of a series of aliphatic alcohols on the reorientation of alkylthiols during their self-assembly has been studied by cathodic stripping voltammetry. The presence of an aliphatic alcohol in the deposition solution is shown to lower the critical reorientational surface concentration of alkylthiols, making it less sensitive to molecular size. The use of a series of alcohols differing in their molecular length or branching reveals that the onset of thiol reorientation correlates well with the extent of alcohol adsorption. A theoretical model is developed to account for this effect, whose crux is the competition between the alcohol molecule and the alkyl chain of the thiol for adsorption sites. The analytical expression derived for the critical reorientational surface concentration reveals that the effect of adding alcohol can be rationalized in terms of an apparent reorientation equilibrium constant, which embodies both the bulk concentration and the adsorption equilibrium constant of the relevant alcohol. Overall, these findings corroborate that the interfacial reorientation of n-alkanethiols is sterically controlled and demonstrate that its onset can be finely tuned by addition of a suitable coadsorbate.

Determination of the potential of zero charge of Au(111) modified with thiol monolayers.

A new method is proposed for the determination of the potential of zero charge of gold electrodes modified with thiol monolayers. It makes use of the immersion technique, in combination with a vapor deposition protocol to build the thiol monolayers. As compared to previous methods, the present approach provides more accurate results, particularly in the case of long-chain alkanethiol monolayers, and it is applicable to any monolayer irrespective of its degree of hydrophilicity. Results are presented for a series of 12 alkanethiol monolayers and for 11-mercaptoundecanol and 11-mercaptoundecanoic acid monolayers. Good agreement is found between the variation of the potential of zero charge along the alkanethiol series with the corresponding change of the surface work function. The potential of zero charge of the 11-mercaptoundecanoic acid monolayer is shown to depend on the extent of dissociation of the acid, thus opening the possibility of applying this type of measurements to the study of surface ionization processes.

Direct electron transfer kinetics in horseradish peroxidase electrocatalysis.

The study of direct electron transfer between enzymes and electrodes is frequently hampered by the small fraction of adsorbed proteins that remains electrochemically active. Here, we outline a strategy to overcome this limitation, which is based on a hierarchical analysis of steady-state electrocatalytic currents and the adoption of the "binary activity" hypothesis. The procedure is illustrated by studying the electrocatalytic response of horseradish peroxidase (HRP) adsorbed on graphite electrodes as a function of substrate (hydrogen peroxide) concentration, electrode potential, and solution pH. Individual contributions of the rates of substrate/enzyme reaction and of the electrode/enzyme electron exchange to the observed catalytic currents were disentangled by taking advantage of their distinct dependence on substrate concentration and electrode potential. In the absence of nonturnover currents, adoption of the "binary activity" hypothesis provided values of the standard electron-transfer rate constant for reduction of HRP Compound II that are similar to those reported previously for reduction of cytochrome c peroxidase Compound II. The variation of the catalytic currents with applied potential was analyzed in terms of the non-adiabatic Marcus-DOS electron transfer theory. The availability of a broad potential window, where catalytic currents could be recorded, facilitates an accurate determination of both the reorganization energy and the maximum electron-transfer rate for HRP Compound II reduction. The variation of these two kinetic parameters with solution pH provides some indication of the nature and location of the acid/base groups that control the electronic exchange between enzyme and electrode.