Complex electrochemistry of flavodoxin at carbon-based electrodes: results from a combination of direct electron transfer,flavin-mediated electron transfer and comproportionation

Staircase cyclic voltammetry (SCV) and differential pulse voltammetry on fully oxidized flavodoxin from Desulfovibrio vulgaris Hildenborough at the bare glassy carbon electrode give one redox couple at a potential of ?218 mV (standard hydrogen electrode (SHE)) at pH = 7.0 with an SCV peak current proportional to the scan rate. This response is caused by flavin mononucleotide (FMN), dissociated from the protein and adsorbed onto the electrode. The midpoint potential and the pK of 6.5 are equal to the values measured with free FMN in solution. When the cationic promoter neomycin is added, one additional and diffusion controlled response is observed. The midpoint potential is ?413 mV (SHE) at pH 7.0 with a redox-linked pK of 4.8 for the reduced form. The temperature dependence is ?1.86 mV K^?1, yielding ΔS° = ?179 J mol^?1 K^?1 and ΔH° = ?12.4 kJ mol^?1. Although the starting material was 100% quinone, no response was observed around the midpoint potential of the quinone to semiquinone reduction of ?113 mV (SHE) at pH 7.0, determined in an EPR-monitored titration with dithionite. Digital simulation shows that the peak currents of the second reduction couple approach a maximum value after a few cycles if comproportionation of fully reduced and fully oxidized flavodoxin occurs in solution and a small amount of semiquinone is either present initially or is generated by mediation of electrode-bound FMN. In the latter case the heterogeneous electron transfer rate between adsorbed FMN and flavodoxin is 6.3 × 10^?6 m s^?1. The implications of this anomalous behaviour for electrochemistry on flavin enzymes like glucose oxidase are discussed.     

Electrochemical studies of isolapachol with emphasis on oxygen interaction with its radical anions

Cyclovoltammetric studies, in aprotic medium (DMF+0.1 mol l^?1 TBAP or DMSO+0.1 mol l^?1 TEAP), on glassy carbon and/or platinum electrodes were performed with isolapachol [2-hydroxy-3-(3-methyl-1-butenyl)-1,4-naphthoquinone], in the absence and presence of oxygen, which were aimed at investigating its electrochemical reduction mechanism and possible oxygen interaction with its radical anion. The electrochemical behaviour is complex and similar to that observed for lapachol, and the first peak is related to the semiquinone formation, although complicated by the occurrence of self-protonation mechanisms and hydrogen-bonded intermediates formation. The observed positive shift in the potential of the first wave of isolapachol, in comparison to lapachol, is related to the higher acidity of the enolic group. The cyclovoltammetric results obtained in the presence of O₂ clearly indicate the consumption of the semiquinone anion-radicals of isolapachol by oxygen, in an EC type reaction, generating the deprotonated form of isolapachol and HOO^?. The observed generation of the superoxide radical, after electron transfer can be related to the mechanism of biological action and toxicity of isolapachol.     

Intra and intermolecular hydrogen bonding effects in the electrochemical reduction of α-phenolic-naphthoquinones

The electrochemical reduction of 1,4-naphthoquinone (NQ), 5-hydroxy-1,4-naphthoquinone (HNQ) and 5,8-dihydroxy-1,4-naphthoquinone (H₂NQ) has been studied in dimethylsulfoxide and acetonitrile, both in the absence and in the presence of proton donors such as methanol and acetic acid. It was established that the intervention of intramolecular hydrogen bonds in the α-phenolic-naphthoquinones (β-hydroxy-naphthoquinones) contributes significantly to the stabilization of the quinone reduced species. This effect is particularly important as a factor determining the strength of intermolecular interactions between the quinone dianion and proton donors. The intramolecular hydrogen bond diminishes the association capacity of the quinones and of their reduction products with molecules of methanol, and avoids the protonation of the semiquinone with acetic acid, favoring only association processes. The low reactivity of the reduced intermediates towards the protonation by acetic acid suggests that analogues structures of HNQ and H₂NQ will not be protonated.     

The effect of triphenylmethyl cations on the electroreduction of O2 in nitrobenzene or acetonitrile

The electroreduction of O₂ in two aprotic solvents (nitrobenzene and acetonitrile) was examined in the absence and in the presence of the very strong Lewis acid, triphenylmethyl cation, φ₃C⁺ (φ=phenyl). The addition of φ₃C⁺ causes the electroreduction of O₂ to change from the one-electron reduction to O₂^? into the two-electron reduction to φ₃CO₂Cφ₃ at a potential that is 1 V more positive than that where O₂ is reduced to O₂^?. This large positive shift in potential is used to estimate a value of the equilibrium constant for the association between O₂^2? and two φ₃C⁺ cations (K=3×10²⁶ M^?2). The mechanism of the reduction of O₂ to φ₃CO₂Cφ₃ can be regarded as an inner-sphere electron-transfer reaction between φ₃C and O₂ or, equivalently, as a radical addition to O₂.     

Cathodic reduction of 2-nitronaphthothiophen-4,9-quinone: evidence of catalysis by proton donors and its simulation

2-Nitronaphtho[2,3-b]thiophen-4,9-quinone (1) is biologically active. The reducible groups have conjugate interaction. Electrochemical experiments (cyclic voltammetry and electrolysis) were performed in order to verify possible intramolecular electron transfer or secondary redox systems and to gain insight into the redox behaviour to help in the understanding of its trypanocidal mechanism of action. Cyclic voltammograms of 1 at the Hg electrode, in DMF+TBAP or DMF+TEAP showed the presence of at least three waves, the two first related to quinone reduction and the third one relative to a catalytic process. After cathodic reduction, at potentials close to the third electron uptake, protons from adventitious water or ammonium quaternary salts can be reduced. Hydrogen formation, with the regeneration of the quinone dianion could be the cause of its catalytic nature. This effect is more pronounced with TEAP. Macroscale electrolyses reinforce the findings. This reaction can be hampered by addition of electrophiles to the medium. Simulated curves fit the experimental ones well. The fourth wave, present at fast scan rates, where the catalysis is not effective, is related to further reduction of the nitro radical anion to the hydroxylamino derivative. At the time scale of cyclic voltammetry, no intramolecular electron transfer was observed. The biological activity of 1 is, possibly, related to the very electrophilic quinone group, generating reactive radical oxygen species through redox cycling.     

Electrogenerated chemiluminescence. 75. Electrochemistry and ECL of 9,10-bis(2-naphthyl)anthracene

The spectroscopy, electrochemistry, and electrogenerated chemiluminescence (ECL) of 9,10-bis(2-naphthyl)anthracene (BNA) was investigated. BNA shows high fluorescence quantum yields (φ=0.86). Strong, intense ECL was observed on the surface of a Pt electrode in benzene + acetonitrile (1:1) while pulsing between the potentials for the first oxidization and reduction of BNA. The peak of ECL light emission was at 430 nm with an ECL efficiency about 90% of that of 9,10-diphenylanthracene (DPA). Cyclic voltammograms (CVs) of BNA show nernstian one-electron oxidations and reductions to the radical ions attributed to electron transfers to the anthracene core. The first reduction is followed by a second irreversible one-electron step and then two, closely spaced nernstian one-electron reductions assigned to electron transfer to the naphthalene moieties. The reduction CV was digitally simulated to establish the reduction mechanism.     

Electrochemical reduction of 1,4-benzoquinone. Interaction with alkylated thymine and adenine nucleobases

The electrochemical reduction of 1,4-benzoquinone (Q) in the presence of 1-octylthymine and 9-octyladenine has been performed in dimethylsulfoxide on glassy carbon electrodes. The electrochemical behaviour shows that the semiquinone (Q^?) and the quinone dianion (Q^2?) interact with 1-octylthymine (TH) following a mechanism which involves association and protonation reactions. It is demonstrated by cyclic voltammetry and NMR experiments, that the protonated dianion (QH^?) is stabilised by an excess of TH, alternatively it disproportionates into an association complex of dianion-hydroquinone [Q,QH₂]^2?. The 1-octylthymine anion (T^?) produced in the proton transfer reactions activates a homogeneous chain mechanism allowing the consumption of benzoquinone. For the experiments carried out in the presence of 9-octyladenine (AH), it was observed that only a strong hydrogen bonding association takes place between the nucleobase and Q^2?.     

Oxygen reduction mechanism on corroded zinc

The mechanism of oxygen reduction on the as-polished and corroded zinc specimens has been studied using a rotating ring disc electrode (RRDE) system. On the as-polished surface, oxygen was reduced into two distinct steps. In the first step, about 44% of O₂ was reduced to H₂O₂ in a 2-electron reaction with the rest being reduced to OH^? in a 4-electron reaction. On the other hand, in the second step, with the increase of overpotential O₂ was almost exclusively reduced to OH^? in a 4-electron reaction. The first step reduction occurred on an air-formed oxide-covered surface at more positive potential than ?1.2 V vs. Ag/AgCl and the second step reduction (E < ?1.2 V) took place on a semi-uniformly active surface. On the corroded surface, the second step was not distinctly observed on the polarization curve, because reduction of the zinc corrosion products simultaneously took place around ?1.2 V. The O₂ reduction in the first step was inhibited by deposition of the corrosion products, though the ratio of amount of O₂ reduced to OH^? in a 4-electron reaction was larger than that on the as-polished surface. The mechanism of oxygen reduction is discussed on the basis of results obtained from the RRDE experiment.     

The current for a two-electron reaction is not necessarily twice that of a one-electron reaction

Two-setp oxidation or reduction of a molecule is considered for the case where removal (or addition) of the second electron is more difficult than that of the first. In such cases, the comproportionation reaction between the final product and the original reactant to give two molecules of the one-electron intermediate is favored and can occur in the diffusion-reaction layer. When the diffusion coefficients of the three species differ, the current seen at potentials where the two-electron reaction occurs will not be twice that seen at potentials where the one-electron reaction occurs, i.e. n_app does not equal 2. For chronoamperometry, digital simulations have been employed to predict the dependence of n_app on the relative diffusion coefficients. It has been shown that equality of the diffusion coefficients of the reactant and the one-electron product is sufficient to guarantee that n_app will be 2. Diffusion coefficients of neutral tetracyanoquinodimethane (TCNQ), its radical anion and the dianion have been meaured in acetonitrile. It is predicted that n_app will be 1.98 for reduction of neutral TCNQ and 2.18 for oxidation of TCNQ^2?. Within experimental error, these values were found experimentally using normal pulse voltammetry. The effect of unequal diffusion coefficients was also observed in cyclic voltammetry and confirmed by simulation.     

Reductive activation and radical formation in a series of substituted benzoquinones: Thermodynamic and quantum chemical studies

The reduction of the quinone moiety, which is found in many anti-cancer agents, is still a poorly understood process. It is commonly assumed that the reduction of a quinone by the uptake of two electrons and two protons leads to the active hydroquinone form. For a better understanding of these reactions electrochemical data, obtained for a series of substituted benzoquinones, were analyzed. In addition quantum chemical calculations on the STO-3G level were performed to obtain data for the one- and two-electron reduction.From the electrochemical experiments, thermodynamic data can be obtained which show that the unfavourable free energy of electron uptake is overcome by the favourable binding of protons. Both reactions are influenced by the electronic properties of the substituents, as demonstrated by Hammett-type relationships between the free energy of these reactions and the sigma-para character of these substituents. In these relationships the reaction constant of the electron uptake process has an absolute value which is five times higher than that of the proton uptake.Quantum chemical calculations yielded energy values for the one-electron uptake, as expressed by U(LUMO), and for the total reduction process. Most of the results from these calculations are in accord with the thermodynamic study. The calculations also revealed a conformational change to take place upon reduction of NH₂ and N(CH₂)₂ substituted benzoquinones, which might be important for chemical and biological activity.     

Experimental evidence of formation of imines in the course of reduction of hydrazones

The reduction of hydrazones is generally suggested to proceed through a reductive cleavage of the nitrogen–nitrogen bond followed by a reduction of the carbon–nitrogen bond. This sequence of reduction processes is here supported for fluorenone (V) and benzophenone (VI) hydrazones as well as by a comparison of the reduction of fluorenone and benzophenone hydrazonium ions (I,III) with corresponding imines (II,IV). Another proof of the presence of imines as intermediates is the splitting of four-electron waves of hydrazones V and VI and hydrazonium ions I and VIII into two waves at pH < 2. This has been interpreted as due to differences in slopes dE_1/2/dpH and pK_a-values of protonated hydrazine derivatives on one side and corresponding imines on the other. In this pH-range imines formed in reductions of VI and VIII are reduced in a single two-electron wave, those of I and V in two one-electron steps. Fluorenone imine (II) is sufficiently stable to allow recording of time-independent current–voltage curves between pH 6 and 11. In this pH-range the imine (II) is reduced in two one-electron steps. Benzophenone imine (IV) has been found stable between pH 4.6 and 12. At pH 4.6–8 the reduction of the imine IV takes place in a single two-electron step, at pH 8–12 in two one-electron steps. Final proof of the initial cleavage of the N–N bond is presented by comparison with the reduction of nitrones.     

Oxidation kinetics of NADH by heteropolyanions

A series of selected Dawson-type mixed heteropolyanions readily oxidize NADH in buffered aqueous pH 7 medium. The process was monitored by UV-visible spectroscopy, which helps to establish the 2:1 stoichiometry for the oxometalate/NADH couple. The starting system for electrochemistry consists of the one-electron reduction product of the heteropolyanions in the presence of various amounts of NADH. Cyclic voltammetry confirms unequivocally that the oxidized forms of the selected heteropolyanions are capable of catalyzing efficiently the oxidation of NADH. The kinetics were studied quantitatively by double step chronocoulometry. The logarithm of the second order rate constant was a linear function of the E^o of the first redox systems of the heteropolyanions with a slope of 16.4 V^?1. This result indicates that the oxidation of NADH proceeds by a multistep mechanism involving an initial rate-limiting one-electron transfer. An estimate of the E^o value for the one-electron NADH/NADH^·+ redox couple has been obtained.     

Solvent-effect on the interconversion among one-, two- and four-electron reduction waves for the 18-molybdodiphosphate complex, [(P2O7)Mo18O54]4−

For the [(P₂O₇)Mo₁₈O₅₄]^4? complex, the presence of small cations such as H⁺, Li⁺ and Na⁺ caused one-electron waves to be converted into four- and two-electron waves in a complex manner. With the addition of a trace amount of H⁺, a four-electron reduction wave was obtained in solvents of weaker basicity like acetone, acetonitrile and propylene carbonate (PC); the relative permittivity did not affect the appearance of the four-electron wave. On the other hand, two-electron waves were obtained in solvents of stronger basicity like N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), and N-methylpyrrolidinone (NMP). With the addition of Li⁺ or Na⁺, the one-electron waves were converted into two-electron waves only in acetone, indicating that the conversion can occur in solvents of both weak basicity and low relative permittivity.     

The cathodic deprotection of the nitrobenzoyl group from phenyl nitrobenzoates in N,N-dimethylformamide

The reduction of phenyl benzoates with nitro substituents at the 2-, 3- and 4-positions of the benzoates in N, N-dimethylformamide is reported. The phenyl 4- and 3-nitrobenzoate are reduced in two cathodic steps. The first one, at about ?0.9 V vs. SCE, a reversible one-electron process, gives a rather stable anion radical. The second reduction step at potentials between ?1.5 and ?2.0 V vs. SCE leads to formation of the dianion, which decomposes giving free phenol in good yields ( > 80%). On the other hand, the phenyl 2-nitrobenzoate is reduced in one cathodic step. This step occurs at ?0.9 V with formation of an unstable anion radical which decomposes via C-O bond cleavage, giving phenol with a yield of ca. 80%. The mechanisms of the reduction of these compounds are discussed.     

Effect of pH on direct electron transfer between graphite and horseradish peroxidase

The effect of pH on the kinetics of the electroreduction of H₂O₂ catalysed by horseradish peroxidase (HRP) has been studied with LSV in the potential range from 700 to ?50 mV versus SCE (under steady-state conditions and with an RDE system) and at ?50 mV versus Ag/AgCl on HRP-modified graphite electrodes placed in a wall-jet flow-through electrochemical cell. Increasing [H₃O⁺] was shown to enhance significantly the current of the bioelectroreduction of H₂O₂ due to direct electron transfer (ET) between graphite and the enzyme over the potential range involved. It is demonstrated that at high overvoltages (E<0.2 V) H₃O⁺ does not affect the rate of the enzymatic reduction of H₂O₂, but it increases the rate of direct ET between graphite and HRP. The values of the apparent rate constant of heterogeneous ET between HRP and graphite, k_s, changed from a value of 0.54±0.05 s^?1 in phosphate buffer solution (PBS) at pH 7.9, to a value of 11.0±1.7 s^?1 in PBS at pH 6.0. Analysing the pH rate profile and the variation of the k_s with increasing [H₃O⁺] made it possible to consider the reaction mechanism as implying the participation of a proton in the limiting step of charge transfer.     

Oxygen reduction on copper in neutral NaCl solution

The reduction of oxygen on copper in neutral unbuffered 1 mol dm^?3 NaCl has been studied using rotating ring-disc electrodes at six oxygen concentrations equivalent to atmospheres of 2% O₂ + N₂ to 100% O₂. Steady-state potentiostatic measurements show that the reaction is first order with respect to [O₂] and that, following adsorption of O₂, the first electron transfer is rate determining. In 50% O₂ + N₂ and 100% O₂, a cathodic oxygen reduction peak is observed in both potentiodynamic and potentiostatic experiments at a disc potential of ?0.3 to ?0.4 V/SCE. The reaction is dominated by the overall four-electron reduction to OH^?, with only small amounts of peroxide detected by the ring electrode at disc potentials corresponding to the formation of the cathodic oxygen reduction peak. Tafel slopes increase with [O₂] and vary from ?0.13₅ V in 2% O₂ + N₂ to a limiting value of ?0.16 V to ?0.18 V in air, 50% O₂ + N₂ and 100% O₂.The results are explained by a mechanism involving oxygen reduction on two types of surface site with different reactivities. The most catalytic surface is believed to comprise Cu(0) and Cu(I) sites, where the Cu(I) species is stabilized as Cu(OH)_ads and/or submonolayer Cu₂O. The less catalytic site consists of Cu(0) only. Oxygen reduction is believed to proceed by a series pathway involving an adsorbed peroxide intermediate on both sites. Peroxide is reduced to OH^? prior to desorption at Cu(0) sites, but some is released before being reduced at Cu(0)/Cu(I) sites. Surface coverage by catalytic Cu(0)/Cu(I) species is favoured by a higher interfacial pH and more positive disc potentials.     

Effects of carbon electrode surface states for the electro-reduction of (hmc)CoOOH2+ which is an intermediate during the catalytic reduction of O2

The reduction of the cobalt(III) complex with a macrocyclic ligand C-meso-5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradecane (hmc) dissolved in solution or adsorbed on a graphite electrode in the presence of O₂ showed two cathodic peaks. As discussed in earlier reports, an intermediate (hmc)CoOOH²⁺ produced by the first two-electron reduction of (hmc)Co³⁺ in the presence of O₂ was further reduced to (hmc)Co²⁺ and HOOH. This process appeared as a second cathodic wave and represents a barrier in the overall reduction of O₂. In relation to these studies, it was found that the second cathodic reduction was greatly affected by the surface states of the carbon electrode. The reduction potential of the (hmc)CoOOH²⁺ intermediate when it was adsorbed on a pyrolytic edge plane graphite electrode (EPG) surface was more positive than the corresponding value of the dissolved species measured at a glassy carbon (GC) electrode polished with alumina. It was also shown that the reduction potential shifted in a positive direction when the solution pH was lowered or the surface of the glassy carbon electrode was heavily electro-oxidized. It is proposed that a proton transfer is involved in the electro-reduction of the (hmc)CoOOH²⁺ complex. The EPG surface which has more surface functional groups has a more acidic environment than the GC electrode and the reduction of (hmc)CoOOH²⁺ was facilitated. The oxidation of the glassy carbon electrode gives the same effect.     

Electrogenerated chemiluminescence of lucigenin enhanced by the modifications of electrodes with self-assembled monolayers and of solutions with surfactants

Electrogenerated chemiluminescence (ECL) of lucigenin (Luc²⁺·2NO^?₃, N,N′-dimethyl-9,9′-biacridinium dinitrate) in dioxygen-saturated alkaline aqueous solutions of pH 10 has been examined utilizing modifications of electrodes (i.e. self-assembled monolayer (SAM)-modified gold electrodes) and of solutions (i.e. micellar solutions containing a nonionic surfactant, Triton X-100) for the first time. In both cases of the modifications, enhanced ECL was observed, and the ECL was considered to be derived from the decomposition of a dioxetane-type intermediate formed by the radical–radical coupling reaction between an electrogenerated superoxide ion (O₂^?) and a one-electron reduced form of Luc²⁺ (i.e. a radical cation, Luc⁺). The surface modification of gold electrodes was achieved with dimercaptosuccinic acid (DMSA) and dl-thioctic acid (dl-TA) having carboxyl end groups. The amount of ECL at dl-TA-SAM-modified electrodes was about five times as great as that at the bare electrode. The enhancement of ECL at the SAM-modified electrodes would be due to the concentration effect of positively charged Luc²⁺ ions, the prevention of adsorption of the electrogenerated chemiluminescent product (i.e. N-methylacridone (NMA)) and the two-electron reduced form of Luc²⁺ (Luc⁰) on the electrode surfaces, and the effective generation of O₂^?. On the other hand, the amount of ECL in the micellar solution increased by about six times in comparison with that in the solution containing no surfactant. The enhanced ECL in micellar solutions would result mainly from the inhibition of adsorption of NMA and Luc⁰ insoluble in water on electrode surfaces.     

Substituent and solvent dependence of the one-electron reduction of 5-substituted-N-methylisatins in aprotic solvents

Potentials for the one-electron reduction of nine 5-substituted N-methylisatins were measured in ten aprotic solvents by cyclic voltammetry. Best-fit Hammett σ substituent constants were determined and used in Hammett plots to determine reaction constants, ρ, for all solvents. Correlations of solvent polarity parameters with the Hammett ρ values and with measured reduction potentials suggest that empirical solvent parameters that reflect the strength of solvent molecular cohesive forces and solvent Lewis acidity are important predictors of the ability of solvent to facilitate the one-electron reduction of isatins in aprotic media by stabilizing the radical anion reduction products.     

Mechanism of the electrochemical reduction of benzyl chlorides catalysed by Co(salen)

The electrochemical reduction of benzyl and 4-(trifluoromethyl)benzyl chlorides catalysed by Co(salen) (H₂salen, N,N′-bis(salicylidene)-ethane-1,2-diamine) was studied in acetonitrile. Electrogenerated (Co¹(salen))^? reacts with the halide to give an organocobalt(III) complex. Further one-electron reduction of the latter yields an unstable intermediate that undergoes rapid decomposition by cleavage of the CoC bond. The mechanism of bond breaking in the one-electron-reduced organocobalt(II) complex was investigated. The results of preparative-scale electrolysis on solutions containing Co(salen) and benzyl chloride, performed under different experimental conditions, in particular in the presence of radical or carbanion scavengers, indicate homolytic cleavage of the CoC bond.