Electrochemical studies of cytochrome c on gold electrodes with promotor of humic acid and 4-aminothiophenol

p-Aminothiophenol (PATP) and humic acids (HA or HAs) were applied jointly as the electron transfer accelerants of redox reactions of cytochrome c (Cyt c) on gold electrodes. The electrochemical properties of the modified electrodes were studied by field emission scanning electron microscope, ultraviolet-visible spectroscopy, electrochemical impedance spectroscopy, Raman spectroscopy and cyclic voltammetry. The immobilized Cyt c displayed a couple of stable and well-defined redox peaks with a formal potential of −0.101 V (vs. SCE) in pH 7.0 phosphate buffer solution. Cyt c adsorption is in the form of a monolayer with average surface coverage of 5.28 pmol cm⁻². The electron transfer rate constant was calculated to be 2.14 s⁻¹. It indicate that the HA film acted as a good adsorption matrix for Cyt c and an excellent accelerant for the redox of Cyt c. The Cyt c-HA modified gold electrode showed a new couple of well-marked redox peaks when 2,4-dichlorophenol was added to the test solution.     

Direct electrochemistry of bovine heart cytochrome c facilitated by cysteine derivatives and analogues. Some effects of facilitator structure

Bovine heart cytochrome c electrochemistry was investigated by cyclic voltammetry using gold electrodes modified by self-assembled monolayers of cysteine and some of its derivatives, including glutathione. Glutathione shows a peculiar dependence of the facilitating ability on the oxidation state of the sulfur functionality. Whereas the disulfide form acts as an efficient facilitator, the thiol form is completely inactive. This behavior was accounted for by the lower stability of adsorbed thiol layers as compared to disulfide layers. Apparently, small molecules of cysteine derivative form rather stable adsorbed layers with a high degree of dimer formation. Conversely, reduced glutathione is not able to turn into the disulfide form in the adsorbed layer. Consequently, only the layer produced by the adsorption of the disulfide form itself is stable enough for promoting direct electron transfer reactions of cyt c. Electronic Publication     

Interfacial electrochemistry of cytochrome c at tin oxide,indium oxide,gold, and platinum electrodes

Cyclic voltammetry has been used to study the heterogeneous electron transfer kinetics of horse heart cytochrome c in pH 7 tris/cacodylate media at several electrode surfaces. Reversible voltammetric responses (formal heterogeneous electron transfer rate constant>10^?2 cm/s) were observed at bare gold electrodes and at tin-doped indium oxide semiconductor electrodes for certain experimental conditions. Quasireversible voltammetric responses were more typically observed at fluorine-doped tin oxide semiconductor electrodes, bare platinum electrodes, and at the indium oxide electrodes. Reaction rates at bare metal electrodes were strongly dependent on pretreatment procedures and experimental protocol. Reaction rates at metal oxide electrodes were strongly dependent on solution conditions, pretreatment procedures, and on the hydration state of the electrode surface. A general mechanistic scheme involving both interfacial electrostatic and chemical interactions is proposed for cytochrome c electrode reactions. The asymmetric distribution of surface charges on cytochrome c appears to play a dominant role in controlling electron transfer rates by its interaction with the electric field at the electrode surface. Electron transfer distances are also considered, and it is concluded that electron transfer between an electrode surface and the exposed heme edge of properly oriented cytochrome c molecules involves maximum distances of ca. 0.6–0.9 nm.     

Direct and Cytochrome c Mediated Electrochemistry of Bilirubin Oxidase on Gold

Direct electron transfer (DET) of bilirubin oxidase from Myrothecium verrucaria (BOD) was established on promoter‐modified gold electrodes. The electrochemical behavior of the enzyme in solution was studied by means of cyclic voltammetry evaluating the biocatalytic reduction of dioxygen. The reaction of BOD at Au electrodes was shown to be efficient only at low pH. In addition, a novel interaction between BOD and cytochrome c (cyt.c) was found. It was shown that BOD efficiently accepts cyt.c as an electron donor in both cases when cyt.c is in solution and electrostatically adsorbed. The results suggest that cyt.c can play the role of a mediator facilitating electron transfer in a pH range where no DET could be observed between the enzyme and the electrode. For the interaction between cyt.c and BOD in solution the reaction kinetics has been studied electrochemically and spectrophotometrically.     

Direct Electrochemistry of Heme Proteins on Electrodes Modified with Didodecyldimethyl Ammonium Bromide and Carbon Black

A novel matrix based on commercially available carbon black (CB) N220 and didodecyldimethyl ammonium bromide (DDAB) was shown to be a reliable support for direct electron transfer reactions between screen printed electrode (SPE) and Fe(III)‐heme proteins. Cytochrome c (cyt c), myoglobin (Mb), horseradish peroxidase (HRP) and cytochromes P450 (CYP 51A1, CYP 3A4, CYP 2B4) generated well‐shaped cyclic voltammograms on SPE/CB/DDAB electrodes (both in solution and in immobilized state). The attractive performance characteristics of CB modified electrodes are advantageous over single‐walled carbon nanotubes (SW CNT) based ones. The achieved direct electrochemistry of heme proteins on CB/DDAB‐modified electrodes provided successful elaboration of the immunosensor for cardiac Mb. The immunosensor showed applicability for diagnostics of myocardial infarction displaying significant difference in cardiac Mb content of human blood plasma samples taken from the corresponding patients.     

Electron transfer of horse spleen ferritin at gold electrodes modified by self-assembled monolayers

Electron transfer is known to be an important step in the sequestering of iron by cellular ferritin. In this work, direct electron transfer between ferritin and a gold electrode was performed in order to probe its electron transfer kinetics. Gold electrodes were modified by the formation of self-assembled monolayers of 3-mercapto-propionic acid on the gold surface. Cyclic voltammetry using these electrodes shows that ferritin exhibits slow electron transfer kinetics at low potentials, yet fairly well-defined current—potential curves. In addition, the voltammetry indicates that adsorption of ferritin precedes the electron transfer step. Controlled potential electrolysis measurements yielded an n-value of 1910 electrons transferred per mole of ferritin. Cyclic voltammetry of a solution containing ferritin as well as nitrilotriacetate yields no electrolytic currents at potentials where the iron—nitrilotriacetate complex undergoes redox reactions, indicating that the currents observed in the voltammetry of ferritin were not due to free iron in the ferritin sample. In addition, the voltammetry of iron-free ferritin (apoferritin) did not yield appreciable currents, providing additional support to the suggestion that the observed voltammetric currents were due to the redox reactions of ferritin iron. Self-assembled monolayers containing carboxylate end groups effectively promoted the direct electron transfer of ferritin at a gold electrode, thus demonstrating that the electron transfer mechanisms of ferritin can now be probed electrochemically.     

A Simple Fabrication Method for Three‐Dimensional Gold Nanoparticle Electrodes and Their Application to the Study of the Direct Electrochemistry of Cytochrome c

A simple method for constructing gold nanoparticle‐modified electrodes with three‐dimensional nanostructures is demonstrated. The electrodes were prepared by casting citrate‐reduced AuNPs onto polycrystalline gold electrodes. The resultant electrodes had a large surface area‐to‐volume ratio, adequate for high protein loading and conferring high stability. The gold nanoparticle electrodes were covered with a self‐assembled monolayer of 11‐mercaptoundecanoic acid for electrostatic immobilization of cytochrome c (cyt c). At the electrode, direct, reversible electron transfer from cyt c was observed with remarkable stability. Moreover, an extremely high surface coverage of electrochemically active cyt c, 167 fully packed monolayers, was obtained through use of the electrode.     

Microscale Crystals of Cytochrome c and Calixarene on Electrodes: Interprotein Electron Transfer between Defined Sites

The assembly of redox proteins on electrodes is an important step in biosensor development. Recently, p‐sulfonato‐calix[4]arene was shown to act as “molecular glue” for the assembly and crystallization of cytochrome c (cyt c). Electrochemical data are presented for microscale cyt c–calixarene crystals grown on self‐assembled monolayers (SAM)‐modified Au electrodes. The crystals were characterized by cyclic voltammetry and exceptionally high concentrations of electroactive cyt c were obtained. The peak currents were found to increase linearly with the square root of the scan rate, thus allowing an evaluation of the rate constant for electron self‐exchange. This study revealed high electroactivity accompanied by fast interprotein electron transfer in crystals, which may have implications for the construction of novel bioelectronic devices.     

Surface-enhanced resonance Raman spectroscopic study of yeast iso-1-cytochrome c and its mutant

The structural stability and redox properties of yeast iso-1-cytochrome c and its mutant, F82H, were studied by surface-enhanced resonance Raman scattering (SERRS) spectroscopy. Phenylalanine, which exists at the position-82 in yeast iso-1-cytochrome c, is replaced by histidine in the mutant. The SERRS spectra of the proteins on the bare silver electrodes indicate that the mutant possesses a more stable global structure with regard to the adsorption-induced conformational alteration. The redox potential of the mutant negatively shifts by about 400 mV, relative to that of yeast iso-1-cytochrome c. This is ascribed to axial ligand switching and higher solvent accessibility of the heme iron in the mutant during the redox reactions.     

GldA overexpressing-engineered E. coli as superior electrocatalyst for microbial fuel cells

Faster electron transfer between bacteria and electrodes in microbial fuel cells can significantly improve the power density of MFCs for practical applications. A recombinant Escherichia coli (E. coli) strain overexpressing glycerol dehydrogenase (GldA) was engineered as the MFC biocatalyst instead of the natural bacteria. Efficient mediators were produced in the fuel cell with this engineered E. coli resulting in lower polarization and much higher power density than with natural E. coli and E. coli with electro-evolved mediators. For the first time, we demonstrate that engineering E. coli by introduction of appropriate oxidoreductase via gene manipulation can greatly improve the rate of electron transfer. This work provides an efficient and economic approach to biologically engineering bacteria for improving MFC performance.     

Bis(1,4-di-tert-butyl-1,4-diazabutadiene)copper(i) [(3,6-di-tert-butyl-o-benzosemiquinono)(3,6-di-tert-butyl-catecholato)cuprate(ii)]. The molecular structure and intramolecular electron transfer

Bis(1,4-di-tert-butyl-1,4-diazabutadiene)copper(i) [(3,6-di-tert-butyl-o-benzosemiquinono)(3,6-di-tert-butylcatecholato)cuprate(ii)] (1) was synthesized. Complex 1 contains the 1,4-di-tert-butyl-1,4-diazabutadiene and 3,6-di-tert-butyl-o-benzoquinone ligands in the reduced form. The structure of 1 was established by X-ray diffraction analysis. The ESR spectra indicate that dissolution of complex 1 in organic solvents (toluene, THF, CH₂Cl₂, etc.) leads to its symmetrization to give neutral complex 2, which occurs in solutions as an equilibrium mixture of two redox isomers, viz., catecholate (Cat) complex 2c and semiquinone (SQ) complex 2s. In the coordination sphere of the copper atom, the reversible intramolecular metal—ligand electron transfer can proceed as successive steps as exemplified by the reactions of 2 with CO and 2,6-dimethylphenylisonitrile. Copper(i) o-semiquinone complex 2s can be reversibly transformed into copper(ii) catecholate complex 2c through electron transfer from the copper(i) atom to the SQ ligand. The subsequent addition of the neutral ligand (CO or CNAr) to 2c induces, in turn, electron transfer from the Cat ligand to the copper(ii) atom accompanied by the transformation of the catecholate complex into the o-semiquinone complex. In the case of CO, this transformation is also reversible and is efficiently controlled by the temperature.     

UV-ozone dry-cleaning process for indium oxide electrodes for protein electrochemistry

The present study reports, for the first time, on electrochemical responses of cytochrome c at a UV-ozone treated indium oxide electrode. Results from surface tension measurements indicate that UV-ozone treatment is an efficient cleaning procedure to remove organic species contamination on surfaces. Well-defined redox responses for cytochrome c were observed at a UV-ozone treated fully hydrophilic indium oxide electrode. Electrochemical parameters, including the diffusion coefficient, the heterogeneous electron transfer rate constant and the redox potential, were in good agreement with those previously reported. However, decrease in peak current for cytochrome c and [Fe(CN)₆]⁴⁻ were observed at a UV-ozone treated electrode. From XPS results, this behavior would be understood to indicate a decrease in homogeneous active electrode surface area by a decrease in conductivity of the indium oxide surface by UV-ozone treatment. Simple and effective UV-ozone treatment methods are useful for surface contamination sensitive electrochemistry.     

Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins

Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b₅ (Cyt b₅) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand.     

Nanobiomolecular Multiprotein Clusters on Electrodes for the Formation of a Switchable Cascadic Reaction Scheme

A supramolecular multicomponent protein architecture on electrodes is developed that allows the establishment of bidirectional electron transfer cascades based on interprotein electron exchange. The architecture is formed by embedding two different enzymes (laccase and cellobiose dehydrogenase) and a redox protein (cytochrome c) by means of carboxy‐modified silica nanoparticles in a multiple layer format. The construct is designed as a switchable dual analyte detection device allowing the measurement of lactose and oxygen, respectively. As the switching force we apply the electrode potential, which ensures control of the redox state of cytochrome c. The two signal chains are operating in a non‐separated matrix and are not disturbed by the other biocatalyst.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current—voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

The electrochemical behavior ofortho-nitroanilides in an aprotic medium

Electrochemical reduction ofortho-nitroanilides in an aprotic medium has been studied by polarography and ESR. It has been shown that nitroanilide anion-radicals are formed at the first reduction wave potentials. The transfer of a second electron is accompanied by subsequent reactions (autoprotonation and homogeneous electron transfer), which occur in the bulk of the solution and lead to fairly stable intermediate anion-radicals ofortho-compounds.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1067–1070, June, 1993.     

ELECTRON TRANSFER REACTIONS OCCURRING UPON LASER FLASH PHOTOLYSIS OF PHOSPHOLIPID BILAYER SYSTEMS CONTAINING CHLOROPHYLL,BENZOQUINONE AND CYTOCHROME c-II. ELECTRICALLY NEUTRAL AND POSITIVELY-CHARGED VESICLES*

Abstract— The primary and secondary electron transfer reactions which occurred upon laser flash photolysis of electrically neutral and positively-charged lipid bilayer vesicles containing chlorophyll, benzoquinone and cytochrome c were determined by time-resolved difference spectral and kinetic measurements, and compared with previous results obtained with negatively-charged vesicles (Y. Fang and G. Tollin, Photochem. Photobiol. 1988). The extent to which oxidized cytochrome c could function as an electron acceptor from triplet state chlorophyll, and reduced cytochrome c could act as an electron donor to chlorophyll cation radical, decreased from negatively-charged to electrically neutral to positively-charged vesicles, in agreement with expectations based on changes in the ability of cytochrome c to bind to the bilayer. In all three types of vesicles, cytochrome c reduction by benzoquinone anion radical occurred in the aqueous phase.     

Voltammetry of immobilized cytochrome c on novel binary self-assembled monolayers of thioctic acid and thioctic amide modified gold electrodes

Horse heart cytochrome c (cyt c) was adsorbed on the binary self-assembled monolayers (SAMs) composed of thioctic acid (T-COOH) and thioctic amide (T-NH₂) at gold electrodes via electrostatic interaction. The cyt c adsorbed on the modified gold electrode exhibited well-defined reversible electrochemical behavior in 10 mM phosphate buffer solution (PBS, pH 7.0). The surface concentration (Γ) of electroactive species, cyt c, on the binary SAMs was higher than that in single-component SAMs of T-COOH, and reached a maximum value of 9.2 × 10⁻¹² mol cm⁻² when the ratio of T-COOH to T-NH₂ in adsorption solution was of 3:2, and the formal potential (E^0′=(E_pa+E_pc)/2) of cyt c was −0.032 V (vs. Ag|AgCl (3 M NaCl)) in a 10 mM PBS. The interaction between cyt c and the binary SAMs made the E^0′ shift negatively when compared with that of cyt c in solution (+0.258 V vs. NHE, i.e., +0.058 V vs. Ag|AgCl (3 M NaCl)). The fractional coverage of bound cyt c was a 0.64 theoretical monolayer. The standard electron transfer rate constant of cyt c immobilized on the binary SAMs was also higher than that on single-component SAMs of T-COOH, and the maximum value of 15.8 ± 0.6 s⁻¹ was obtained when the ratio of T-COOH to T-NH₂ in adsorption solution was at 3:2. The results suggest that the electrode modified with the binary SAMs functions better than the electrode modified with single-component SAMs of T-COOH.     

Characterization of a Novel Cytochrome Involved in Geobacter sulfurreducens’ Electron Harvesting Pathways

Electron harvesting bacteria are key targets to develop microbial electrosynthesis technologies, which are valid alternatives for the production of value-added compounds without utilization of fossil fuels. Geobacter sulfurreducens, that is capable of donating and accepting electrons from electrodes, is one of the most promising electroactive bacteria. Its electron transfer mechanisms to electrodes have been progressively elucidated, however the electron harvesting pathways are still poorly understood. Previous studies showed that the periplasmic cytochromes PccH and GSU2515 are overexpressed in current-consuming G. sulfurreducens biofilms. PccH was characterized, though no putative partners have been identified. In this work, GSU2515 was characterized by complementary biophysical techniques and in silico simulations using the AlphaFold neural network. GSU2515 is a low-spin monoheme cytochrome with a disordered N-terminal region and an α-helical C-terminal domain harboring the heme group. The cytochrome undergoes a redox-linked heme axial ligand switch, with Met⁹¹ and His⁹⁴ as distal axial ligands in the reduced and oxidized states, respectively. The reduction potential of the cytochrome is negative and modulated by the pH in the physiological range: −78 mV at pH 6 and −113 mV at pH 7. Such pH-dependence coupled to the redox-linked switch of the axial ligand allows the cytochrome to drive a proton-coupled electron transfer step that is crucial to confer directionality to the respiratory chain. Biomolecular interactions and electron transfer experiments indicated that GSU2515 and PccH form a redox complex. Overall, the data obtained highlight for the first time how periplasmic proteins bridge the electron transfer between the outer and inner membrane in the electron harvesting pathways of G. sulfurreducens.