Specific vectorial immobilization of oligonucleotide-modified yeast cytochrome C on carbon nanotubes.

Iso-1-cytochrome c from the yeast Saccharomyces cerevisiae (YCC) contains a surface cysteine residue, Cys102, that is located opposite to the lysine-rich side containing the exposed heme edge, which is the docking site for enzymes. Site-specific vectorial immobilization of YCC via Cys102 on single-walled carbon nanotubes (SWNT) thus provides a selective interface between nanoscopic electronic devices and complex enzymes. We have achieved this by modification of Cys102 with an oligonucleotide (dT(18)). Atomic force microscopy, fluorescence imaging, and cyclic voltammetry show the specific adsorption of YCC, modified with dT(18), on the SWNT sidewall with retention of its native properties. Pretreatment of the SWNT with Triton-X405 blocks the nonspecific binding of untreated YCC but does not interfere with binding of the oligonucleotide-modified YCC.     

Topological and Electron‐Transfer Properties of Yeast Cytochrome c Adsorbed on Bare Gold Electrodes

The redox metalloprotein yeast cytochrome c was directly self‐chemisorbed on “bare” gold electrodes through the free sulfur‐containing group Cys102. Topological, spectroscopic, and electron transfer properties of the immobilised molecules were investigated by in situ scanning probe microscopy and cyclic voltammetry. Atomic force and scanning tunnelling microscopy revealed individual protein molecules adsorbed on the gold substrate, with no evidence of aggregates. The adsorbed proteins appear to be firmly bound to gold and display dimensions in good agreement with crystallographic data. Cyclic voltammetric analysis showed that up to 84 % of the electrode surface is functionalised with electroactive proteins whose measured redox midpoint potential is in good agreement with the formal potential. Our results clearly indicate that this variant of cytochrome c is adsorbed on bare gold electrodes with preservation of morphological properties and redox functionality.     

Electrochemical control of protein monolayers at indium tin oxide surfaces for the reagentless optical biosensing of nitric oxide

Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.     

A scanning tunnelling study of immobilised cytochrome P450cam

A site-specifically engineered surface cysteine residue, located in a region where the haem moiety is closest to the surface, is used to anchor cytochrome P450cam enzyme molecules covalently to a gold electrode. More reproducibly ordered adsorption, at high coverage, occurs with this K344C mutant than with the wild-type enzyme. The subsequently formed close-packed monolayer arrays have been probed by scanning tunnelling microscopy under ambient conditions and under aqueous (buffered) solution at high resolution. Initial indications suggest that the immobilised enzyme is both electrochemically addressable and catalytically active.     

The redox chemistry of the covalently immobilized native and low-pH forms of yeast iso-1-cytochrome c

Cyclic voltammetry experiments were carried out on native Saccharomyces cerevisiae iso-1-cytochrome c and its C102T/N62C variant immobilized on bare polycrystalline gold electrode through the S-Au bond formed by a surface cysteine. Experiments were carried out at different temperatures (5-65 degrees C) and pH values (1.5-7). The E degrees ' value at pH 7 (+370 mV vs SHE) is approximately 100 mV higher than that for the protein in solution. This difference is enthalpic in origin and is proposed to be the result of the electrostatic repulsion among the densely packed molecules onto the electrode surface. Two additional electrochemical waves are observed upon lowering the pH below 5 (E degrees ' = +182 mV) and 3 (E degrees ' = +71 mV), which are attributed to two conformers (referred to as "intermediate" and "acidic", respectively) featuring an altered heme axial ligation. This is the first determination of the reduction potential for low-pH conformers of cytochrome c in the absence of denaturants. Since the native form of cytochrome c can be restored, bringing back the pH to neutrality, the possibility offered by this transition to reversibly modulate the redox potential of cytochrome c is appealing for bioelectronic applications. The immobilized C102T/N62C variant, which differs from the native protein in the orientation of the heme group with respect to the electrode, shows very similar reduction thermodynamics. For both species, the rate constant for electron transfer between the heme and the electrode increases for the acidic conformer, which is also found to act as a biocatalytic interface for dioxygen reduction.     

Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction

We demonstrate for the first time, by a combined mass spectrometric and computational approach, that G- and F-actin can be covalently modified by the lipid-derived aldehyde, 4-hydroxy-trans-2-nonenal, providing information on the molecular mass of modified protein and the mechanism and site of adduction.ESI-MS analysis of actin treated with different molar ratios of HNE (1 : 1 to 1 : 20) showed the formation of a protein derivative in which there was an increase of 156 Da (42028 Da) over native actin (41872 Da), consistent with the adduction of one HNE residue through Michael addition. To identify the site of HNE adduction, G- and F-actin were stabilized by NaBH(4) reduction and digested with trypsin. LC-ESI-MS/MS analysis in data-dependent scan mode of the resulting peptides unequivocally indicated that Cys374 is the site of HNE adduction. Computational studies showed that the reactivity of Cys374 residue is due to a significant accessible surface and substantial thiol acidity due to the particular microenvironment surrounding Cys374.     

Direct immobilization of native yeast iso-1 cytochrome C on bare gold: fast electron relay to redox enzymes and zeptomole protein-film voltammetry

Cyclic voltammetry shows that yeast iso-1-cytochrome c (YCC), chemisorbed on a bare gold electrode via Cys102, exhibits fast, reversible interfacial electron transfer (k(0) = 1.8 x 10(3) s(-1)) and retains its native functionality. Vectorially immobilized YCC relays electrons to yeast cytochrome c peroxidase, and to both cytochrome cd(1) nitrite reductase (NIR) and nitric oxide reductase from Paracoccus denitrificans, thereby revealing the mechanistic properties of these enzymes. On a microelectrode, we measured nitrite turnover by approximately 80 zmol (49 000 molecules) of NIR, coadsorbed on 0.65 amol (390 000 molecules) of YCC.     

Silicon surface modification with a mixed silanes layer to immobilize proteins for biosensor with imaging ellipsometry

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (MTES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.     

Nanohybrid Materials Consisting of Poly[(3‐aminobenzoic acid)‐co‐(3‐aminobenzenesulfonic acid)‐co‐aniline] and Multiwalled Carbon Nanotubes for Immobilization of Redox Active Cytochrome c

The development of a new surface architecture for the efficient direct electron transfer of positively charged redox proteins is presented. For this reason different kinds of polyaniline terpolymers consisting of aminobenzoic acid (AB), aminobenzenesulfonic acid (ABS) and aniline (A) with different monomer ratios were synthesized. The P(AB‐ABS‐A) were grafted to the surface of multiwalled carbon nanotubes (MWCNTs). FTIR measurements prove the covalent binding to the carboxylic groups of the MWCNTs while conductivity tests show an increase in the conductivity of the nanohybrid in comparison to the polymers. The [MWCNT‐P(AB‐ABS‐A)] nanohybrids were used for the immobilization of redox active cytochrome c (cyt.c). The positively charged protein can electrostatically interact with the negatively charged nanohybrid. Cyclic voltammetry (CV) shows an increase in the protein loading on [MWCNT‐P(AB‐ABS‐A)] coupled to cysteamine modified gold electrodes in comparison to non‐grafted MWCNTs. A further increase in the sulfonation degree of P(AB‐ABS‐A) leads to an enhanced current output of the modified electrodes. The redox activity of the polymer decreases after the immobilization of the cyt.c on the nanohybrid. For the first time polymers covalently grafted to the surface of MWCNTs are used in a biosensor.     

Cytochrome c self-assembly on alkanethiol monolayer electrodes as characterized by AFM, IR, QCM, and direct electrochemistry

With the advantage of carbodiimide coupling chemistry, horse heart cytochrome c (cyt c) has been covalently immobilized onto self-assembled monolayers (SAMs) from 11-mercaptoundecanoic acid (MUDA) developed on single-crystal or polycrystalline gold substrate surfaces. The cyt c immobilized substrates thus prepared have been characterized by atomic force microscopy (AFM); we have succeeded in obtaining surface topographical images down to single-protein resolution. AFM imaging has also shown densely packed, uniform protein monolayer formation that is highly suggestive of self-assembly of cyt c molecules on MUDA SAMs. Covalent attachment of cyt c has been further evidenced by reflection-absorption FT-IR as well as microgravimetric analysis using a quartz crystal microbalance (QCM). In the latter, the specific MUDA and cyt c surface concentrations were determined to be 0.86 +/- 0.11 nmol cm-2 (n = 5) and 28 +/- 12 pmol cm-2 (n = 5), both of which agree fairly well with their theoretical counterparts. The obtained QCM chips having the cyt c/MUDA/Au interfacial structure were found to be capable of the direct electrochemistry of the surface-attached cyt c molecules. Cyclic voltammetric measurements on the chips gave particular redox waves showing characteristics of surface process. The electroactive protein surface concentration was determined to be 7.2 +/- 4.8 pmol cm-2 (n = 6); it was almost consistent with values found in literature, while it was limited to 26% in magnitude for the QCM data. This was deemed to have arisen from the orientation variation of the surface-confined cyt c molecules and is discussed briefly.     

Direct Electrochemistry of Cytochrome c Covalently Immobilized on ω-Carboxyalkanethiol Monolayer Electrode and its Biosensor Applications

The study of direct electron transfer (ET) between solid electrodes and proteins or enzymes has been attracting considerable research interest for several decades since it represents a basic feature for the application of biocatalysts in chemical sensors and other electrical devices. We have been focusing our research interest on the use of SAMs for the study of diffusionless, direct electrochemistry of cytochromes. In the present paper, we report electrochemistry of cytochrome c covalently immobilized on ω-carboxyalkanethiol monolayer electrodes. A carboxylic acid terminated monolayer was utilized to provide an uniform surface for attaching cytochrome c, and characterization of the redox reaction of the protein was made with using cyclic voltammetric and electrochemical impedance measurements.     

Structures of Cytochrome b_5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

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Stabilization of yeast cytochrome C covalently immobilized on fused silica surfaces

The surface-enhanced conformational stability of yeast cytochrome c (YCC) covalently immobilized on a fused silica prism with heterobifunctional cross-linkers has been studied by attenuated total reflection absorption spectroscopy using the Soret band of the heme prosthetic group as a probe. Comparison of the results to those of horse cytochrome c physisorbed on the same substrate as well as to the corresponding proteins in solution indicates that the surface plays a significant role in stabilizing the native conformation of the surface-bound YCC. Unfolding to extended configurations was so hindered that the native conformation of the covalently immobilized protein is essentially unaffected by the presence of denaturants such as methanol and 1-propanol.     

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.     

Characterization of a novel microperoxidase from Marinobacter hydrocarbonoclasticus by electrospray ionization tandem mass spectrometry

Microperoxidases are small heme-peptides obtained by proteolytic digestion of cytochrome c, exhibiting peroxidase activity. They consist of a short- or medium-length polypeptide chain, covalently linked to an iron protoporphyrin IX moiety via two thioether bonds involving Cys residues at the c-porphyrin A and B pyrrole rings. These small molecules are interesting for a wide range of possible applications. We have structurally characterized, by means of electrospray ionization (ESI) mass and tandem mass spectrometric experiments, a novel microperoxidase called MMP-5 (Marinobacter MicroPeroxidase-5), obtained by proteolytic digestion of cytochrome c552, a monoheminic electron-transfer protein isolated from Marinobacter hydrocarbonoclasticus. This microperoxidase, which still maintains the functional peptide moieties for peroxidase activity, is devoid of the two amino acids intercalating the Cys residues linked to the c-porphyrin, thus increasing its water solubility. Once submitted to the ESI source potential, MMP-5 showed an interesting tendency for the reduction of the iron protoporphyrin substructure. This behaviour was clearly evidenced by the mass shift exhibited by the reduced form.     

Nucleic Acid–Promoted Electron Transfer to Cytochrome c

Gold electrodes were modified with short oligonucleotides in order to facilitate the electron transfer to the small redox protein cytochrome c. DNA immobilization was followed by impedance spectroscopy. The electron exchange was found to be quasi-reversible for both the protein in solution or adsorbed at the electrode surface. Variations of base sequence or structure (DNA-PNA hybrids) of the nucleic acid promoter layer did not significantly change the electron transfer rate constant which was in the range of 0.2–1×10⁻² cm/s. Cytochrome c adsorbed at low ionic strength was coupled to molecules in solution such as superoxide or laccase. Aspects of the DNA conductivity and possibilities for DNA detection are also discussed.     

Engineering artificial redox chains by molecular 'Lego'

This work reports on a novel approach for building artificial redox chains: the molecular 'Lego' approach. This exploits the scaffold of natural redox proteins by fusing together functional protein modules with the desired properties. The molecular 'Lego' mimics the natural molecular evolution that proceeded by modular assembly of genes/DNA segments. Non-physiological electron transfer partners, flavodoxin (fld) and cytochrome c553 (c553) from Desulfovibrio vulgaris and the haem domain of P450 BM3 (BMP) from Bacillus megaterium have been used as building blocks in different combinations to build artificial redox chains. The kinetic characterization of the electron transfer (ET) between the separate building blocks has been carried out. Under pseudo-first order conditions, a limiting ET rate, klim, of 0.48 +/- 0.05 s-1 and 43.77 +/- 2.18 s-1 and an apparent binding constant, Kapp, of 21 +/- 6 microM and 1.23 +/- 0.32 microM have been found for the fld/c553 and fld/BMP redox pairs, respectively. These results show that fld can be used as a module for transferring electrons to c553 and BMP. A 3D model of the fld/c553 and fld/BMP complexes was used to guide the construction of covalently linked assemblies via engineered disulfide bridges or by fusion of the relevant genes via an engineered loop. The first approach led to the construction, expression and characterization of the S35C and S64C mutants of fld and M23C and G51C mutants of c553. Although the redox potentials of the separate mutants were found to be the same as those of recombinant wild type proteins (-408 mV for the semiquinone/hydroquinone couple of fld and +32 mV for the c553), the c553 homo-dimers M23C-M23C and G51C-G51C were found to have redox potentials of +88 and +105 mV, respectively. These differences have been analysed in terms of exposure of the haem cofactors to the solvent, and these lead to some interesting questions on the redox potentials of the transient redox complexes in physiological systems. The fld-c553 S64C-M23C and S35C-M23C chimeras were constructed, expressed and purified but the FMN was found to be destabilised resulting in the apo-form of these proteins. The gene fusion strategy was used to produce covalently linked assemblies of both fld-c553 and fld-BMP. The former was expressed using a seven amino acid (GPGPGPG) loop linking the C-terminus of fld to the N-terminus of c553. The fld-BMP fusion protein was successfully expressed by using the naturally occurring loop of the P450 BM3 (residues 471-479) to link the BMP domain at the N-terminus with fld domain at the C-terminus. This fusion was found to be correctly folded and functional. Efficient ET from the FMN to the haem domain (370 s-1) was also found to be in the same region of the physiological redox partners (250 s-1). This work demonstrates the feasibility of the molecular 'Lego' approach in generating functional multi-domain proteins with designed properties, beyond the restrictions imposed by the naturally occurring protein domains.     

pH-Induced changes in adsorbed cytochrome c. voltammetric and surface-enhanced resonance Raman characterization performed simultaneously at chemically modified silver electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.     

Gold nanoparticle-cytochrome C complexes: the effect of nanoparticle ligand charge on protein structure

We report the effect of nanoparticle ligand charge on the structure of a covalently, site-specifically linked protein. Au nanoparticles with positive, negative, and neutral ligands were appended to a specific cysteine, C102, of Saccharomyces cerevisiae cytochrome c. Conjugates were purified by HPLC or gel electrophoresis. Circular dichroism spectroscopy shows that changing the nanoparticle ligand dramatically influences the attached cytochrome c structure. The protein retains its structure with neutral ligands but denatures in the presence of charged species. This is rationalized by the electrostatic interaction of amino acids in the local vicinity of C102 with the endgroups of the ligand.     

Photoactive, covalent attachment of deoxyribonucleic acid on gold with double-strand specificity using self-assembled monolayers containing psoralen

Taking advantages of psoralen photochemistry, we have developed a new method of immobilizing DNA on gold substrate surfaces. A psoralen derivative having an alkylamine function was synthesized, and was self-assembled on gold substrate surfaces in a combined use of a thiol-derivatized molecule, 3,3′-dithiobis(succinimidyl propionate) forming amide bonds on the surface. We found that by irradiating with long wavelength ultraviolet light (320-400 nm), DNA molecules added in the solution phase were covalently immobilized on the monolayer surface through the photoadduct formation of the psoralen molecules with the DNA nucleobases. The present method has its advantage that is applicable to native DNAs, no chemically modifying DNAs, in spite of its covalent immobilization principle. We have examined 12 mer synthetic oligonucleotide immobilizations and have found that the surface concentration thus attained was to be 20 pmol cm⁻², which is consistent with saturated surface coverage. Interestingly, the immobilization occurred double-stranded-DNA-preferentially; no immobilization for single-stranded DNAs. Characterization of the immobilization chemistry has been achieved using atomic force microscopic imaging, infrared absorption, X-ray photoelectron spectroscopy, electrochemistry, and quartz-crystal microbalance and their results were described.