Proton-coupled electron transfer of cytochrome c.

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by omega-carboxyl alkanethiols of different chain lengths (C(x)). The dynamics of the electron-transfer (ET) reaction of the adsorbed heme protein, initiated by a rapid potential jump to the redox potential, was monitored by time-resolved surface enhanced resonance Raman (SERR) spectroscopy. Under conditions of the present experiments, only the reduced and oxidized forms of the native protein state contribute to the SERR spectra. Thus, the data obtained from the spectra were described by a one-step relaxation process yielding the rate constants of the ET between the adsorbed Cyt-c and the electrode for a driving force of zero electronvolts. For C(16)- and C(11)-SAMs, the respective rate constants of 0.073 and 43 s(-1) correspond to an exponential distance dependence of the ET (beta = 1.28 A(-1)), very similar to that observed for long-range intramolecular ET of redox proteins. Upon further decreasing the chain length, the rate constant only slightly increases to 134 s(-1) at C(6)- and remains essentially unchanged at C(3)- and C(2)-SAMs. The onset of the nonexponential distance dependence is paralleled by a kinetic H/D effect that increases from 1.2 at C(6)- to 4.0 at C(2)-coatings, indicating a coupling of the redox reaction with proton-transfer (PT) steps. These PT processes are attributed to the rearrangement of the hydrogen-bonding network of the protein associated with the transition between the oxidized and reduced state of Cyt-c. Since this unusual kinetic behavior has not been observed for electron-transferring proteins in solution, it is concluded that at the Ag/SAM interface the energy barrier for the PT processes of the adsorbed Cyt-c is raised by the electric field. This effect increases upon reducing the distance to the electrode, until nuclear tunneling becomes the rate-limiting step of the redox process. The electric field dependence of the proton-coupled ET may represent a possible mechanism for controlling biological redox reactions via changes of the transmembrane potential.     

Oligonucleotide-modified electrodes for fast electron transfer to cytochrome c

Gold electrodes were modified with short ds-oligonucleotides via thiol binding to form a thin and stable surface layer. The modification was characterised by impedance measurements and used as a promoter for fast electron transfer to cytochrome c. The protein was investigated both immobilised and in solution showing reversible electrochemical behaviour in each case. The modification proved to have a good adsorption capability for the redox protein which was also found to be reversible. In the immobilised state at the electrode cytochrome c reacted with superoxide radicals in solution, exemplified by cyclic voltammetric measurements.     

Reversible immobilization and direct electron transfer of cytochrome c on a pH-sensitive polymer interface

A pH-sensitive polymer interface has been used as a matrix for reversible immobilization of cytochrome c (Cyt c) on an Au surface through a dip-coating process. The pH-sensitive behavior of the polymer brush interface has been demonstrated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The reversible immobilization and electron-transfer properties of Cyt c have been investigated by in situ UV/Vis spectrophotometry and CV. The results have shown that the poly(acrylic acid) (PAA) brush acted as an excellent adsorption matrix and a good accelerant for the direct electron transfer of Cyt c, which gave redox peaks with a formal potential of 40 mV versus Ag/AgCl in pH 7.6 phosphate buffer solution. The average surface coverage of Cyt c on the PAA film was about 1.7 x 10(-10) mol cm(-2), indicating a multilayer of Cyt c. The electron-transfer rate constant was calculated to be around 0.19 s(-1) according to the CV experiments. The interface was subjected to in situ attenuated total internal reflection Fourier-transform infrared (ATR-FTIR) spectroscopic analysis, in order to further confirm the immobilization of Cyt c on the surface. This polymer-protein system may have potential applications in the design of biosensors, protein separation, interfacial engineering, biomimetics, and so on.     

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.     

Oriented immobilization and electron transfer to the cytochrome c oxidase

Direct electron transfer to cytochrome c oxidase (CcO) is investigated as a function of packing density of the surface layer. This is varied by the surface concentration of chelator molecules when the enzyme is immobilized on the electrode using the his-tag technology. Chelator molecules with a terminal nitrilotriacetic acid group are synthesized ex situ in contrast to in situ synthesis used in a previous work. Self-assembled monolayers of the chelator mixed at different mole fractions with a dilution molecule are prepared to bind the CcO after complex formation with Ni²⁺ ions. The CcO, which is immobilized in the solubilized form, is then reconstituted into a protein-tethered bilayer lipid membrane (ptBLM). Varying the mixing ratio of chelator to dilution molecules enabled us to control the packing density of CcO residing in the ptBLM. Subtle differences in the architecture of the protein/lipid layers revealed by surface-enhanced IR absorption spectroscopy are considered to be essential for an effective electron transfer. Cyclic voltammograms are measured under anaerobic conditions at different scan rates and analyzed by means of a model which describes the transfer of four electrons to CcO in the ptBLM. The rate constants thus obtained show a marked dependence on the packing density.     

Graphene-based modified electrode for the direct electron transfer of Cytochrome c and biosensing

Chitosan-dispersed graphene nano-flakes were prepared by a chemical route to reduce graphene oxide and dispersed fully in water to form a stable black aqueous solution. The as-prepared graphene nano-flakes were successfully immobilized on glassy carbon electrode to construct a graphene modified electrode. Cytochrome c was adsorbed tightly on the surface of the modified electrode and the direct electron transfer of Cytochrome c was achieved. Cytochrome c on the surface of electrode maintains its bioactivity and shows an enzyme-like activity for the reduction of nitric oxide, displaying a potential application for the fabrication of novel biosensors to sense nitric oxide. This research will enlarge the applications of graphene-based materials in biosensor field.     

A voltammetric study of direct electron transfer to cytochrome c using a very large assembly of carbon microelectrodes

A very large assembly of more than 8000 carbon fibre microdisk electrodes was used to study direct electron transfer to cytochrome c. Near steady-state cyclic voltammograms were observed, which exhibited excellent signal-to-noise ratios despite the low concentrations of cytochrome c employed (1-50 microM). The high resolution of the voltammograms allowed the formal potential of the native form of cytochrome c to be determined over a range of solution pH.     

Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.     

Electrocatalytic investigation of light-induced electron transfer between cytochrome c6 and photosystem I

A light-activated electron-transfer chain was assembled using solubilized cyanobacterial photosystem I as photoactive enzyme, cytochrome c(6) (also from cyanobacteria) as electron donor, and methyl viologen as electron acceptor. The photocatalytic activity of the ensemble was measured by direct and reversible electrochemistry of cytochrome c(6) at a surface-modified gold electrode. Analysis of the electrochemical response with an appropriate model for the reaction mechanism allowed the relation of the overall catalytic reaction rate to the individual steps of the catalytic cycle. Second-order rate constants were determined for the first time under steady-state conditions. The results validate this approach as an efficient method for the study of electron transfer between photoactive enzymes and their redox partners.     

Functional modification of cytochrome c by peroxynitrite in an electron transfer reaction.

The redox reaction of cytochrome c after modification with peroxynitrite under physiological conditions was investigated. Cytochrome c was treated with a bolus of synthetic peroxynitrite at a sub-millimolar concentration, and then subjected to reduction by superoxide and oxidation by hydrogen peroxide. The ability for the membrane potential formation in the mitochondrial respiratory chain was also evaluated. After the treatment with peroxynitrite, the cytochrome c molecule was mono-nitrated mainly at a tyrosine residue, using liquid chromatography-electrospray ionizing mass spectrometry (LC-ESI-MS) and HPLC. Although the redox capacity of cytochrome c was not affected by the peroxynitrite treatment, the oxidation of ferrocytochrome c to ferricytochrome c by hydrogen peroxide was accelerated. When cytochrome c was treated with peroxynitrite in the presence of 5-methoxytryptamine, an inhibitor for the tyrosine nitration by peroxynitrite, the acceleration of hydrogen peroxide-mediated oxidation was suppressed. It was also found that the formation of membrane potential in the rat liver mitochondria was suppressed when peroxynitrite-treated cytochrome c was used instead of the intact cytochrome c in vitro. From these results, we concluded that the peroxynitrite-treated cytochrome c was nitrated at a tyrosine residue and became more susceptible to oxidation by hydrogen peroxide, concomitantly losing the ability to transfer electrons in the mitochondrial respiratory chain. It is suggested that the peroxynitrite-induced modification of cytochrome c increases the susceptibility to non-physiological oxidants, and may cause dysfunction of mitochondria by suppressing of membrane potential.     

Effect of deuterium substitution on electron transfer at cytochrome c/SAM interfaces

The involvement of protons in the heterogeneous electron transfer between cytochrome c and a gold electrode to which it is attached was studied by comparing the electron transfer rate constants for H2O and D2O solutions. Rate constants were measured as a function of the electrochemical cell solution and the protein incubant solution, i.e., k (0)(incubant, cell). Two separate isotope effects exist: a cell "isotope effect", KIE cell = k (0)(H2O, H2O): k (0)(H2O, D2O), which is manifest at short time scales (<30 s) and arises from the viscosity difference between H2O and D2O, and an incubant isotope effect, KIE inc= k (0)(H2O, H2O): k (0)(D2O, H2O), which is manifest at longer times (>2 h) and results from H/D exchange. The two isotope effects are approximately equal ( approximately 1.2) and a total isotope effect KIE total = k (0)(H2O, H2O): k (0)(D2O, D2O) can be constructed that is the product of KIE cell and KIE inc. The nature of the electron transfer process, possible coupling to a proton transfer process, and the involvement of specific hydrogens in the transfer mechanism are discussed.     

Mechanism of intramolecular electron transfer in the photoexcited Zn-substituted cytochrome c: theoretical and experimental perspective

Photoinduced electron transfer (ET) in zinc-substituted cytochrome c (Zn-cyt c) has been utilized in many studies on the long-range ET in protein. Attempting to understand its ET mechanism in terms of electronic structure of the molecule, we have calculated an all-electron wave function for the ground-state of Zn-cyt c on the basis of density functional theory (DFT). The four molecular orbitals (MOs) responsible for excitation by UV-vis light (Gouterman's 4-orbitals) are assigned on the basis of the excited states of chromophore model for Zn-porphine complex calculated with the time-dependent DFT method. ET rates between each Gouterman's 4-orbitals and other MOs were estimated using Fermi's golden rule. It appeared that the two occupied MOs of the 4-orbitals show exclusively higher ET rate from/to particular MOs that localize on outermost amino acid residues (Lys 7 or Asn 54), respectively, whereas ET rates involving the two unoccupied MOs of the 4-orbitals are much slower. These results imply that the intramolecular ET in photoexcited Zn-cyt c is governed by the hole transfer through occupied MOs. The couplings of MOs between zinc porphyrin core and specific amino acid residues on the protein surface have been demonstrated in Zn-cyt c immobilized on an Au electrode via carboxylic acid group-terminated self-assembled monolayer. The Zn-cyt c-modified electrode showed photocurrents responsible for photoillumination. The action spectrum of the photocurrent was identical with the absorption spectrum of Zn-cyt c, indicating photoinduced electron conduction via occupied MOs. The voltage dependence of the photocurrent appeared to be linear and bidirectional like a photoconductor, which strongly supports the intramolecular ET mechanism in Zn-cyt c proposed on the basis of the theoretical calculations.     

Gold sputtered electrode surfaces enhance direct electron transfer reactions of human cytochrome P450s

We immobilized human cytochrome P450 (CYP), a membrane-bound enzyme, onto both smooth and nanostructured surfaces of gold electrodes via a naphthalene thiolate monolayer film. Rapid electron transfer of CYP with an electrode as a redox partner took place when the enzyme was immobilized onto an electrode surface with nanostructures. This structure was easily prepared by conventional sputtering techniques. A well-defined pair of peaks was observed at ? 0.175 V (vs. SHE) with the largest heterogeneous electron transfer rate constant of 340 s^? 1 for human CYP. The positive redox potential shift of 45 mV upon drug (testosterone) binding was clearly detected, which corresponded to a change in the spin states of heme iron in CYP. The present study showed that gold sputtered surfaces are very useful for direct electron transfer reactions of human CYP isoforms.     

Complex kinetics of the electron transfer between the photoactive redox label TUPS and the heme of cytochrome c

The photoinduced covalent redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) has been attached to two lysine residues (K8 and K39) at opposite sides of horse heart cytochrome c, as well as to cysteines, at the same positions, introduced by site-directed mutagenesis. Electron transfer between TUPS and the heme of cytochrome c deviates from the expected monoexponential kinetic behavior. Neither the overall rate nor the individual exponential components of electron transfer, as followed by kinetic absorption spectroscopy, correlate with the length of the covalent link connecting the dye with the protein. Molecular dynamics calculations show that TUPS can approach the protein surface and occupy several such positions. This heterogeneity may explain the multiexponential electron-transfer kinetics. The calculated optimal electron-transfer pathways do not follow the covalent link but involve through space jumps from the dye to the protein moiety, effectively decoupling the length of the covalent link and the electron-transfer rates.     

Direct evidence of electron flow via the heme c group for the direct electron transfer reaction of fructose dehydrogenase using a silver nanoparticle-modified electrode

The direct electron transfer reaction of fructose dehydrogenase (FDH) from Gluconobacter sp. on alkanethiol-modified silver nanoparticles (AgNPs) was examined using cyclic voltammetry and surface-enhanced resonance Raman scattering (SERRS). Using cyclic voltammetry, catalytic oxidation currents (based on the direct electron transfer reaction of FDH) were observed from a potential of approximately −100 mV (vs. Ag/AgCl, 3 M NaCl) in the presence of d-fructose, without a mediator. A comparison of the SERRS spectra and the resonance Raman spectra of FDH in solution indicated that the heme c site retained its six-coordinated low-spin heme after immobilization. Moreover, SERRS also demonstrated that the heme c of the adsorbed FDH was the electron transfer site within the enzyme.     

Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome c(4)

The di-heme protein Pseudomonas stutzeri cytochrome c(4) (cyt c(4)) has emerged as a useful model for studying long-range protein electron transfer (ET). Recent experimental observations have shown a dramatically different pattern of intramolecular ET between the two heme groups in different local environments. Intramolecular ET in homogeneous solution is too slow (>10 s) to be detected but fast (ms-μs) intramolecular ET in an electrochemical environment has recently been achieved by controlling the molecular orientation of the protein assembled on a gold electrode surface. In this work we have performed computational modeling of the intramolecular ET process by a combination of density functional theory (DFT) and quantum mechanical charge transfer theory to disclose reasons for this difference. We first address the electronic structures of the model heme core with histidine and methionine axial ligands in both low- and high-spin states by structure-optimized DFT. The computations enable estimating the intramolecular reorganization energy of the ET process for different combinations of low- and high-spin heme couples. Environmental reorganization free energies, work terms ("gating") and driving force were determined using dielectric continuum models. We then calculated the electronic transmission coefficient of the intramolecular ET rate using perturbation theory combined with the electronic wave functions determined by the DFT calculations for different heme group orientations and Fe-Fe separations. The reactivity of low- and high-spin heme groups was notably different. The ET rate is exceedingly low for the crystallographic equilibrium orientation but increases by several orders of magnitude for thermally accessible non-equilibrium configurations. Deprotonation of the propionate carboxyl group was also found to enhance the ET rate significantly. The results are discussed in relation to the observed surface immobilization effect and support the notion of conformationally gated ET.     

Photoinduced electron transfer between cytochrome c and a novel 1,4,5,8-naphthalenetetracarboxylic diimide with amphiphilic character

N-dodecyl-N'-(2-phosphonoethyl)-1,4,5,8-naphthalenetetracarboxylic diimide (DNDI) is a novel naphthalenic diimide with amphiphilic character. DNDI was synthesized through the sequential reaction of 1,4,5,8-naphthalenetetracarboxylic dianhydride, first with dodecylamine and then with 2-aminoethylphosphonic acid. Fluorescence measurements showed that DNDI forms excimers in water at sufficiently high concentrations. The fluorescence quantum yield of DNDI in diluted solutions is sensitive to the polarity of the microenvironment, decreasing as going from water to less polar solvents. This property allowed to monitor the incorporation of DNDI into cetyl trimethyl ammonium bromide (CTAB) micelles, with a binding constant of 1.2x10(4) M-1. UV irradiation (365 nm) of solutions containing DNDI and the redox protein cytochrome c (cyt c) resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) state, a reaction that was inhibited by the incorporation of DNDI into CTAB micelles. DNDI formed host-guest complexes with alpha-cyclodextrin (alpha-CD) through the inclusion of the dodecyl group, resulting in an increased aqueous solubility of the compound.     

Electrostatic docking of a supramolecular host-guest assembly to cytochrome c probed by bidirectional photoinduced electron transfer

A water-soluble octacarboxyhemicarcerand was used as a shuttle to transport redox-active substrates across the aqueous medium and deliver them to the target protein. The results show that weak multivalent interactions and conformational flexibility can be exploited to reversibly bind complex supramolecular assemblies to biological molecules. Hydrophobic electron donors and acceptors were encapsulated within the hemicarcerand, and photoinduced electron transfer (ET) between the Zn-substituted cytochrome c (MW = 12.3 kD) and the host-guest complexes (MW = 2.2 kD) was used to probe the association between the negatively charged hemicarceplex and the positively charged protein. The behavior of the resulting ternary protein-hemicarcerand-guest assembly was investigated in two binding limits: (1) when K(encaps) ? K(assoc), the hemicarcerand transports the ligand to the protein while protecting it from the aqueous medium; and (2) when K(assoc) > K(encaps), the hemicarcerand-protein complex is formed first, and the hemicarcerand acts as an artificial receptor site that intercepts ligands from solution and positions them close to the active site of the metalloenzyme. In both cases, ET mediated by the protein-bound hemicarcerand is much faster than that due to diffusional encounters with the respective free donor or acceptor in solution. The measured ET rates suggest that the dominant binding region of the host-guest complex on the surface of the protein is consistent with the docking area of the native redox partner of cytochrome c. The strong association with the protein is attributed to the flexible conformation and adaptable charge distribution of the hemicarcerand, which allow for surface-matching with the cytochrome.