Electron Transfer In Cytochrome Research Articles

Preparation a novel 1-pyreneacetic acid functionalized graphene/self-assembled monolayer modified gold electrode to immobilize and study interfacial electron transfer of cytochrome c by electrochemical approaches

A new hybrid of favorable orientated cytochrome c (cyt c) and a 1-Pyreneacetic acid (PA) functionalized graphene (Gr) on Au electrode (AuE) was prepared. In the hybrid system, Gr was functionalized with (PA) molecule through π-π interaction (PA-Gr). Afterward the PA-Gr attached to the AuE via Thionalide self-assembled monolayer (SAM), then conjugated with cyt c. The AuE/SAM/PA-Gr/cyt c hybrid system was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and scanning electrochemical microscopy (SECM) techniques. SECM approach curves were applied to measure the standard tunneling electron transfer (ET) and bimolecular rate constants which were 56.6 s−1 and 1 × 109cm3mol−1 s−1, respectively.

Fermi level-tuned optics of graphene for attocoulomb-scale quantification of electron transfer at single gold nanoparticles

Measurement of electron transfer at single-molecule level is normally restricted by the detection limit of faraday current, currently in a picoampere to nanoampere range. Here we demonstrate a unique graphene-based electrochemical microscopy technique to make an advance in the detection limit. The optical signal of electron transfer arises from the Fermi level-tuned Rayleigh scattering of graphene, which is further enhanced by immobilized gold nanostars. Owing to the specific response to surface charged carriers, graphene-based electrochemical microscopy enables an attoampere-scale detection limit of faraday current at multiple individual gold nanoelectrodes simultaneously. Using the graphene-based electrochemical microscopy, we show the capability to quantitatively measure the attocoulomb-scale electron transfer in cytochrome c adsorbed at a single nanoelectrode. We anticipate the graphene-based electrochemical microscopy to be a potential electrochemical tool for in situ study of biological electron transfer process in organelles, for example the mitochondrial electron transfer, in consideration of the anti-interference ability to chemicals and organisms.

Quantum biological tunnel junction for electron transfer imaging in live cells

Quantum biological electron transfer (ET) essentially involves in virtually all important biological processes such as photosynthesis, cellular respiration, DNA repair, cellular homeostasis, and cell death. However, there is no real-time imaging method to capture biological electron tunnelling in live cells to date. Here, we report a quantum biological electron tunnelling (QBET) junction and its application in real-time optical detection of QBET and the dynamics of ET in mitochondrial cytochrome c during cell life and death process. QBET junctions permit to see the behaviours of electron tunnelling through barrier molecules with different barrier widths. Using QBET spectroscopy, we optically capture real-time ET in cytochrome c redox dynamics during cellular apoptosis and necrosis in living cells. The non-invasive real-time QBET spectroscopic imaging of ET in live cell open a new era in life sciences and medicine by providing a way to capture spatiotemporal ET dynamics and to reveal the quantum biological mechanisms.

Electrochemical biointerface based on electrodeposition AuNPs on 3D graphene aerogel: Direct electron transfer of Cytochrome c and hydrogen peroxide sensing

An electrochemical biointerface of 3D graphene aerogel (3DGA) decorated with Au nanoparticles (AuNPs) was fabricated to immobilize Cytochrome c (Cyt c) for H2O2 detection. Morphology and surface characterization of 3DGA-AuNPs reveal a successful formation of 3D-networked structure that can enhance conductivity and effectively immobilize enzyme. The immobilized Cyt c retained its bioactivity and realized the direct electron transfer on electrode owing to the biocompatibility of AuNPs and large surface area of 3DGA. The as-prepared 3DGA-AuNPs/Cyt c/GCE exhibited a pair of well-defined redox peaks of FeIII/II redox couple of Cyt c and revealed excellent catalytic activity toward H2O2 with high sensitivity of 351.57μA·mM−1·cm−2. This biosensor was further successfully used to real-time detection of trace amount of H2O2 released from living cells, indicating the promise for practical application.

Hydrogen bond-linked pathways of peptide units and polar groups of amino acid residues suitable for electron transfer in cytochrome c proteins

Electron transfer occurs through heme-Fe across the cytochrome c protein. The current models of long range electron transfer pathways in proteins include covalent σ-bonds, van der Waals forces, and through space jump. Hydrogen-bond-linked pathways of delocalized electron units in peptide units and polar side chains of amino acid residues in proteins and internal water molecules are better suited for intramolecular atom-to-atom electron transfer in proteins. Crystal structures of cytochrome c proteins from horse (1HRC), tuna (3CYT), rice (1CCR), and yeast (3CX5) were analyzed using pymol software for 'Hydrogen Bonds' marking the polar atoms within the distance of 2.6-3.3Å and tracing the atom-to-atom pathways linked by hydrogen bonds. Pathways of hydrogen-bond-linked peptide units, polar side chains of the amino acid residues, and buried water molecules connect heme-Fe through axially coordinated Met80-S and His18-N have been traced in cytochrome c proteins obtained from horse, tuna, rice and yeast with an identical hydrogen-bonded sequence around the heme-Fe: Asn-N-water-O-Tyr-O-Met-S-heme-Fe-His (HN-C=N)-Pro-Asn-Pro-Gly (peptide unit, HN-C=O)-water-O. More than half of the amino acid residues in these pathways are among the conserved list and delocalized electron units, internal water molecules and hydrogen bonds are conspicuous by their presence.

Electron Transfer of Cytochrome c on Surface-Enhanced Raman Scattering-Active Substrates: Material Dependence and Biocompatibility.

Surface-enhanced Raman spectroscopy (SERS) represents a powerful approach for studying the structure and reaction of proteins in fundamental and applied sciences. The surface properties of SERS-active materials determine important parameters such as Raman enhancement ability, biocompatibility, and electronic communication between supports and proteins. Here, electron transfer (ET) of Cyt c on noble metals and transition metals is investigated by SERS spectroscopy. The results here indicate that the ET occurs from the reduced state of Cyt c to silver substrate, depending on the laser wavelengths. Nickel and cobalt can directly transfer electrons to the oxidized state of Cyt c, which enables a reductive activity of these transition metal nanoparticles (NPs). This study demonstrates the role of transition metals as electron donors for Cyt c and has proved that the charge transfer theory for SERS is applicable for explanation of the ET between Cyt c and Ag NPs.

Gold nanoparticle-mediated electron transfer of cytochrome c on a self-assembled surface

Gold nanoparticles provide short-range electron transfer between cytochrome c and gold electrode.

Optically transparent carbon nanotube film electrode for thin layer spectroelectrochemistry.

Carbon nanotube (CNT) film was evaluated as an optically transparent electrode (OTE) for thin layer spectroelectrochemistry. Chemically inert CNT arrays were synthesized by chemical vapor deposition (CVD) using thin films of Fe and Co as catalysts. Vertically aligned CNT arrays were drawn onto a quartz slide to form CNT films that constituted the OTE. Adequate conductivity and transparency make this material a good OTE for spectroelectrochemistry. These properties could be varied by the number of layers of CNTs used to form the OTE. Detection in the UV/near UV region down to 200 nm can be achieved using these transparent CNT films on quartz. The OTE was characterized by transmission electron microscopy, scanning electron microscopy, Raman spectroscopy, UV-visible spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy, and thin layer spectroelectrochemistry. Ferricyanide, tris(2,2'-bipyridine) ruthenium(II) chloride, and cytochrome c were used as representative redox probes for thin layer spectroelectrochemistry using the CNT film OTE, and the results correlated well with their known properties. Direct electron transfer of cytochrome c was achieved on the CNT film electrode.

Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (R RMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.

In silico spectroscopy of tryptophan and tyrosine radicals involved in the long-range electron transfer of cytochrome c peroxidase.

Cytochrome c peroxidase (CcP) is a heme-containing enzyme that catalyzes the oxidation of the ferrocytochrome c to ferricytochrome c with concomitant reduction of H2O2 to H2O. Its catalytic cycle involves the formation of a double oxidized species (compound I) consisting of an oxoferryl center (Fe(IV)═O) and an amino acid radical (R(•)). Here we use a quantum-mechanics/molecular-mechanics (QM/MM) computational protocol based on density functional theory (DFT) and multiconfigurational perturbation theory (CASPT2) methods to reproduce specific features of compound I EPR and UV-vis spectra. The results show that the employed QM/MM models can correctly predict the magnetic, electronic and vibrational properties of the observed amino acid radicals of compound I. Furthermore, we have been able to confirm that the principal radical species of compound I is a tryptophan cationic radical located on residue 191 (Trp191(•+)) and that three tyrosine residues (Tyr203, Tyr236, and Tyr251), located along two possible ET pathways involving Trp191(•+), are possible candidates to host the secondary radical species.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

Cytochrome (cyt) c is an important electron transfer protein. The ruffling deformation of its heme cofactor has been suggested to relate to its electron transfer rate. However, there is no direct experimental evidence demonstrating this correlation. In this work, we studied Pseudomonas aeruginosa cytochrome c551 and its F7A mutant. These two proteins, although similar in their X-ray crystal structure, display a significant difference in their heme out-of-plane deformations, mainly along the ruffling coordinate. Resonance Raman and vibrational coherence measurements also indicate significant differences in ruffling-sensitive modes, particularly the low-frequency γa mode found between ∼50-60 cm(-1). This supports previous assignments of γa as having a large ruffling content. Measurement of the photoreduction kinetics finds an order of magnitude decrease of the photoreduction cross-section in the F7A mutant, which has nearly twice the ruffling deformation as the WT. Additional measurements on cytochrome c demonstrate that heme ruffling is correlated exponentially with the electron transfer rates and suggest that ruffling could play an important role in redox control. A major relaxation of heme ruffling in cytochrome c, upon binding to the mitochondrial membrane, is discussed in this context.

Screen-printed graphite macroelectrodes for the direct electron transfer of cytochrome c: a deeper study of the effect of pH on the conformational states, immobilization and peroxidase activity

The direct electron transfer of cytochrome c has been studied at screen-printed graphite macroelectrodes without recourse to mediators or the need for any electrode pre-treatment as is commonly employed within the literature. A wide range of pH values from 2.0 to 11.0 have been explored upon the electrochemical response of cytochrome c and different voltammetric signatures have been observed. The direct electron transfer of the alkaline transition of cytochrome c was found impeded within alkaline media leading to either an irreversible redox process or even no voltammetric responses. In acidic aqueous media the electrochemical process is observed to undergo a mixed diffusion and adsorption controlled process rather than a purely diffusional process of the native conformation as observed at pH 7.0. Interestingly, at pH 3.5 a new conformational state is revealed in cooperation with the native conformation. The immobilization of the protein was satisfactorily obtained using a simple method by cycling the protein at specific solution pH values allowing amperometric responses to be obtained and gives rise to useful pseudo-peroxidase activity for sensing H2O2. Apparent Michaelis-Menten constant values (Km) were calculated via the Lineweaver-Burk method with deduced values of 25 ± 4, 98 ± 12 and 230 ± 30 mM, respectively for pH values of 2.0, 3.0 and 7.0. Such work is important for those utilising cytochrome c in bio-electrochemical and related applications.

Phase-Sensitive Detection in Modulation Excitation Spectroscopy Applied to Potential Induced Electron Transfer in Cytochrome c Oxidase

Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides was investigated by modulated excitation surface-enhanced infrared-absorption spectroscopy (SEIRAS). Sequential electron transfer (ET) within CcO was initiated by electrochemical excitation. During modulated excitation by periodic potential pulses with frequencies between 20 and 500 Hz, time-resolved infrared spectra were measured by the step-scan technique, with time resolution in the millisecond range. Conformational changes of the protein structure as a result of ET lead to rather complex SEIRA spectra with many overlapping bands embedded in a broad background signal. Phase-sensitive detection (PSD) was used to separate single components within the broad band of overlapping structural bands in the amide I region. PSD is able to extract the periodic response of single components with the same frequency as the excitation from noise or from static background and therefore enhances the signal-to-noise ratio. Moreover, PSD enables validation of the fit model used for the deconvolution of overlapping bands by analyzing phase lags of single components acquired at different stimulation frequencies. Phase lags between the evaluated vibrational components and the modulated excitation increase with increasing excitation frequencies, an inherent prerequisite of this evaluation method.

Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film

We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm(-2). In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30% of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA-SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA-MIP-modified electrode occurred with an affinity constant of 100,000 mol(-1) L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins.

Phosphate mediated adsorption and electron transfer of cytochrome c. A time-resolved SERR spectroelectrochemical study

The study of proteins immobilized on biomimetic or biocompatible electrodes represents an active field of research as it pursues both fundamental and technological interests. In this context, adsorption and redox properties of cytochrome c (Cyt) on different electrode surfaces have been extensively reported, although in some cases with contradictory results. Here we report a SERR spectroelectrochemical study of the adsorption and electron transfer behaviour of the basic protein Cyt on electrodes coated with amino-terminated monolayers. The obtained results show that inorganic phosphate (Pi) and ATP anions are able to mediate high affinity binding of the protein with preservation of the native structure and rendering an average orientation that guarantees efficient pathways for direct electron transfer. These findings aid the design of Cyt-based bioelectronic devices and understanding the modulation by Pi and ATP of physiological functions of Cyt.

Native CuA redox sites are largely resilient to pH variations within a physiological range

Previous studies on engineered CuA centres have shown that one of the histidine ligands is protonated and dissociated from the metal site at physiological pH values, thus suggesting a role in regulating proton-coupled electron transfer of cytochrome c oxidases in vivo. Here we report that for native CuA such protonation does not take place at physiologically relevant pH values and, furthermore, no significant changes in the spectroscopic and redox properties of the metal site occur at low pH.

Direct electron transfer of cytochrome c at mono-dispersed and negatively charged perylene–graphene matrix

Mono-dispersed 3,4,9,10-perylene tetracarboxylic acid (PTCA) functionalized graphene sheets (PTCA-graphene) were fabricated by a chemical route and dispersed well in aqueous solution. PTCA-graphene with plenty of –COOH groups as electrostatic absorbing sites were beneficial to the loading of Cytochrome c (Cyt c). Cyt c, which was tightly immobilized on the PTCA-graphene modified glassy carbon electrode, maintained its natural conformation. Direct electron transfer of Cyt c and the electro-catalytic activity towards the reduction of H2O2 were also achieved. It has been substantiated that PTCA-graphene is a preferable biocompatible matrix for Cyt c.

Mechanisms for the Direct Electron Transfer of Cytochrome c Induced by Multi-Walled Carbon Nanotubes

Multi-walled carbon nanotube (MWCNT)-modified electrodes can promote the direct electron transfer (DET) of cytochrome c (Cyt c). There are several possible mechanisms that explain the DET of Cyt c. In this study, several experimental methods, including Fourier transform infrared spectroscopy, circular dichroism, ultraviolet-visible absorption spectroscopy, and electron paramagnetic resonance spectroscopy were utilized to investigate the conformational changes of Cyt c induced by MWCNTs. The DET mechanism was demonstrated at various nano-levels: secondary structure, spatial orientation, and spin state. In the presence of MWCNTs, the secondary structure of Cyt c changes, which exposes the active site, then, the orientation of the heme is optimized, revolving the exposed active center to the optimum spatial orientation for DET; and finally, a transition of spin states is induced, providing relatively high energy and a more open microenvironment for electron transfer. These changes at different nano-levels are closely connected and form a complex process that promotes the electron transfer of Cyt c.

On the Molecular Mechanism of Electron Transfer of Cytochrome c Modulated by Gold Nanoparticles in Nano-Sandwich Architecture

On the Molecular Mechanism of Electron Transfer of Cytochrome c Modulated by Gold Nanoparticles in Nano-Sandwich Architecture

Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV–vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (ks) of 5.9 ± 0.2 s−1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H2O2, and the amperometric signal increase steadily with the concentration of H2O2 in the range from 5 μM to 1.5 mM. The detection limit is 1 μM at pH 7.4. The apparent Michaelis-Menten constant (Km) of the sensor is 0.53 mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H2O2.