Self-assembly of mono- and bidentate oligoarylene thiols onto polycrystalline Au.

Four thiolated oligoarylene molecules (i) 4-methoxy-terphenyl-4″-methanethiol (MTM), (ii) 4-methoxy-terphenyl-3″,5″-dimethanethiol (MTD), (iii) 4-nitro-terphenyl-4″-methanethiol (NTM), and (iv) 4-nitro-terphenyl-3″,5″-dimethanethiol (NTD) were synthesized and self-assembled as monolayers (SAMs) on polycrystalline Au electrodes of organic field-effect transistors (OFETs). SAMs were characterized by contact angle and AC/DC electrochemical measurements, whereas atomic force microscopy was used for imaging the pentacene films grown on the coated electrodes. The electrical properties of functionalized OFETs, the electrochemical SAMs features and the morphology of pentacene films were correlated to the molecular organization of the thiolated oligoarylenes on Au, as calculated by means of the density functional theory. This multi-methodological approach allows us to associate the systematic replacement of the SAM anchoring head group (viz. methanethiol and dimethanethiol) and/or terminal tail group (viz. nitro-, -NO2, and methoxy, -OCH3) with the change of the electrical features. The dimethanethiol head group endows SAMs with higher resistive features along with higher surface tensions compared with methanethiol. Furthermore, the different number of thiolated heads affects the kinetics of Au passivation as well as the pentacene morphology. On the other hand, the nitro group confers further distinctive properties, such as the positive shift of both threshold and critical voltages of OFETs with respect to the methoxy one. The latter experimental evidence arise from its electron-withdrawing capability, which has been verified by both DFT calculations and DC electrochemical measurements.

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha.

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.

The role of a conserved tyrosine residue in high-potential iron sulfur proteins.

Conserved tyrosine-12 of Ectothiorhodospira halophila high-potential iron sulphur protein (HiPIP) iso-I was substituted with phenylalanine (Y12F), histidine (Y12H), tryptophan (Y12W), isoleucine (Y12I), and alanine (Y12A). Variants Y12A and Y12I were expressed to reasonable levels in cells grown at lower temperatures, but decomposed during purification. Variants Y12F, Y12H, and Y12W were substantially destabilized with respect to the recombinant wild-type HiPIP (rcWT) as determined by differential scanning calorimetry over a pH range of 7.0-11.0. Characterization of the Y12F variant by NMR indicates that the principal structural differences between this variant and the rcWT HiPIP result from the loss of the two hydrogen bonds of the Tyr-12 hydroxyl group with Asn-14 O delta 1 and Lys-59 NH, respectively. The effect of the loss of the latter interaction is propagated through the Lys-59/Val-58 peptide bond, thereby perturbing Gly-46. The delta delta GDapp of Y12F of 2.3 kcal/mol with respect to rcWT HiPIP (25 degrees C, pH 7.0) is entirely consistent with the contribution of these two hydrogen bonds to the stability of the latter. CD measurements show that Tyr-12 influences several electronic transitions within the cluster. The midpoint reduction potentials of variants Y12F, Y12H, and Y12W were 17, 19, and 22 mV (20 mM MOPS, 0.2 M sodium chloride, pH 6.98, 25 degrees C), respectively, higher than that of rcWT HiPIP. The current results indicate that, although conserved Tyr-12 modulates the properties of the cluster, its principle function is to stabilize the HiPIP through hydrogen bonds involving its hydroxyl group and electrostatic interactions involving its aromatic ring.

Redox properties of cytochrome c.

The redox properties of cytochromes (cyt) c, a ubiquitous class of heme-containing electron transport proteins, have been extensively investigated over the last two decades. The reduction potential (E degrees') is central to the chemistry of cyt c for two main reasons. First, E degrees' influences both the thermodynamic and kinetic aspects of the electron exchange reaction with redox partners. Second, this thermodynamic parameter is remarkably sensitive to changes in the properties of the heme and the protein matrix, and hence can be profitably used for the investigation of the solution chemistry of cyt c. This research area owes much to the exploitation of voltammetric techniques for the determination of E degrees' for metalloproteins, which dates back to the late 1970s. Since then, much effort has been devoted to the comprehension of the molecular factors that control E degrees' in cyt c, which include first coordination sphere effects on the heme iron, the interactions of the heme group with the surrounding polypeptide chain and the solvent, and also include medium effects related to the nature and ionic composition of the solvent, pH, the presence of potential protein ligands, and the temperature. This article provides an overview of the most significant advances made in this field recently.

Redox properties and acid-base equilibria of zucchini mavicyanin.

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.

Silybin, a new iron-chelating agent.

Silybin, a natural occurring flavolignan isolated from the fruits of Silibum marianum, has been reported to exert antioxidant and free radical scavenging abilities. It was suggested to act also as an iron chelator. The complexation and protonation equilibria of the ferric complex of this compound have been studied by potentiometric, spectrophotometric and electrochemical techniques. The formation of the complex silybin-Ga(III) in anhydrous DMSO-d6 has been studied by 1H NMR spectroscopy. Mass spectrometry and infrared spectroscopy on silybin-Fe(III) complex confirm all data obtained by 1H NMR spectroscopy. The experimental results show that silybin binds Fe(III) even at acidic pH. Different ternary complexes were observed at increasing methoxide ion concentration and their stability constants have been calculated. The results show the possible role of silybin in relation to the chelation therapy of chronic iron overload, as occurs in the treatment of Cooley's anemia.

Voltammetric and surface-enhanced resonance Raman spectroscopic characterization of cytochrome C adsorbed on a 4-mercaptopyridine monolayer on silver electrodes.

To combine voltammetric techniques with surface-enhanced resonance Raman scattering (SERRS), cytochrome c (cyt c) was immobilized on a roughened silver electrode chemically modified with a self-assembled monolayer (SAM) of 4-mercaptopyridine (PySH). All measurements were performed on the same electrode in a homemade spectroelectrochemical cell suitable for such applications. Cyt c on a PySH-SAM shows a quasi-reversible, monoelectronic, adsorption-controlled CV response with a formal reduction potential of -0.061 V (vs SCE), which is comparable to the values found for native cyt c adsorbed on different SAMs. SERRS spectra proved that cyt c adsorbed on a PySH monolayer is present in the native conformer (the B1 state). Voltammetric and SERRS experiments at high ionic strength revealed that the interaction between the SAM and the protein is electrostatic in nature. In conclusion, PySH was found to be suitable for adsorption of cyt c at SERRS-active silver surfaces. In comparison with other SAMs, PySH requires less time (10 min vs 12-18 h) to form a long-time durable and reproducible coating on the roughened electrode surface.

Axial ligation and polypeptide matrix effects on the reduction potential of heme proteins probed on their cyanide adducts.

The enthalpic and entropic changes accompanying the reduction reaction of the six-coordinate cyanide adducts of cytochrome c, microperoxidase-11 and a few plant peroxidases were measured electrochemically. Once the compensating changes in reduction enthalpy and entropy due to solvent reorganization effects are factorized out, it is found that cyanide binding stabilizes enthalpically the ferriheme following the order: cyochrome c > peroxidase > microperoxidase-11. The effect is inversely correlated to the solvent accessibility of the heme. Comparison of the reduction thermodynamics for the cyanide adducts of cytochrome c and plant peroxidases with those for microperoxidase-11 and myoglobin, respectively, yielded an estimate of the consequences of protein encapsulation and of the anionic character of the proximal histidine on the reduction potential of the heme-cyanide group. Insertion of the heme-CN group into the folded peptide chain of cyt c induces an enthalpy-based decrease in E degrees ' of approximately 100 mV, consistent with the lower net charge of the oxidized as compared to the reduced iron center, whereas a full imidazolate character of the proximal histidine stabilizes enthalpically the ferriheme by approximately 400 mV. The latter value should be best considered as an upper limit since it also includes some solvation effects arising from the nature of the protein systems being compared.

Anion binding to cytochrome c2: implications on protein-Ion interactions in class I cytochromes c.

The binding of several inorganic and carboxylate anions to cytochrome c2 from Rhodopseudomonas palustris has been investigated by monitoring the salt-induced changes in the redox potential of the heme, using an interpretative model based on the extended Debye-Hückel equation. Most anions were found to interact specifically with the protein at one or multiple sites. Binding constants to the oxidized protein in the range 10(1)-10(2) m-1 were determined from the anion concentration dependence of the chemical shift of the isotropically shifted heme methyl resonances. For several anions the stoichiometry and strength of the binding to cytochrome c2 were found comparable with those determined for mitochondrial cytochromes c, in spite of the limited sequence similarity (less than 40%) and the lower positive charge of the bacterial protein. These analogies were interpreted as indicative of the existence of common binding sites which are proposed to be located in the conserved lysine-rich domain around the solvent-exposed heme edge, which is also the surface area likely involved in the interaction with redox partners. The changes in E degrees due to partial neutralization of the positive charge of cytochrome c2 due to specific anion binding were found comparable with those for the mitochondrial species.

Redox thermodynamics of the native and alkaline forms of eukaryotic and bacterial class I cytochromes c.

The reduction potentials of beef heart cytochrome c and cytochromes c2 from Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Rhodobacter capsulatus were measured through direct electrochemistry at a surface-modified gold electrode as a function of temperature in nonisothermal experiments carried out at neutral and alkaline pH values. The thermodynamic parameters for protein reduction (DeltaS degrees rc and DeltaH degrees rc) were determined for the native and alkaline conformers. Enthalpy and entropy terms underlying species-dependent differences in E degrees and pH- and temperature-induced E degrees changes for a given cytochrome were analyzed. The difference of about +0.1 V in E degrees between cytochromes c2 and the eukaryotic species can be separated into an enthalpic term (-DeltaDeltaH degrees rc/F) of +0.130 V and an entropic term (TDeltaDeltaS degrees rc/F) of -0.040 V. Hence, the higher potential of the bacterial species appears to be determined entirely by a greater enthalpic stabilization of the reduced state. Analogously, the much lower potential of the alkaline conformer(s) as compared to the native species is by far enthalpic in origin for both protein families, and is largely determined by the substitution of Met for Lys in axial heme ligation. Instead, the biphasic E degrees /temperature profile for the native cytochromes is due to a difference in reduction entropy between the conformers at low and high temperatures. Temperature-dependent 1H NMR experiments suggest that the temperature-induced transition also involves a change in orientation of the axial methionine ligand with respect to the heme plane.

Solvent-based deuterium isotope effects on the redox thermodynamics of cytochrome c.

The reduction thermodynamics of cytochrome c (cytc), determined electrochemically, are found to be sensitive to solvent H/D isotope effects. Reduction of cytochrome c is enthalpically more favored in D(2)O with respect to H(2)O, but is disfavored on entropic grounds. This is consistent with a reduction-induced strengthening of the H-bonding network within the hydration sphere of the protein. No significant changes in E degrees ' occur, since the above variations are compensative. As a main result, this work shows that the oxidation-state-dependent differences in protein solvation, including electrostatics and solvent reorganization effects, play an important role in determining the individual enthalpy and entropy changes of the reduction process. It is conceivable that this is a common thermodynamic feature of all electron transport metalloproteins. The isotope effects turn out to be sensitive to buffer anions which specifically bind to cytc. Evidence is gained that the solvation thermodynamics of both redox forms of cytc are sensibly affected by strongly hydrated anions.

Effects of specific anion-protein binding on the alkaline transition of cytochrome c.

The thermodynamic parameters of the alkaline transition of beef heart ferricytochrome c have been measured through direct electrochemistry experiments carried out at variable pH and temperature in the presence of different sulfate concentrations. Sulfate is known to bind specifically to cytochrome c in a sequential manner at two surface sites. The effects of such a specific binding reflect on the thermodynamics of the transition and can be satisfactorily interpreted within the frame of the Debye-Hückel theory with simple electrostatic considerations. In particular, the increase in the thermodynamic pKa values (extrapolated to I = 0) upon sulfate binding turns out to be a fully enthalpic effect which can be accounted for by considering the coulombic effects of the formation of ionic couple(s) on the protein surface. This study also shows that the apparent pKa values at finite ionic strength are only moderately affected by the nature of the anion in solution, and differences tend to vanish at high ionic strength.

Effects of nonspecific ion-protein interactions on the redox chemistry of cytochrome c.

The effects of the ionic atmosphere on the enthalpic and entropic contributions to the reduction potential of native (state III) beef heart cytochrome c have been determined through variable-temperature direct electrochemistry experiments. At neutral or slightly alkaline pH values, from 5 to 50 degrees C, the reduction enthalpy and entropy become less negative with decreasing ionic strength. The reduction entropy extrapolated at null ionic strength is approximately zero, indicating that, in the absence of the screening effects of the salt ions on the network of the electrostatic interactions at the protein-solvent interface, the solvation properties and the conformational flexibility of the two redox states are comparable. The moderate decrease in E degrees' observed with increasing ionic strength [DeltaE degrees'IS = (E degrees')I = 0.1 M - (E degrees')I = (0)M = -0.035 V at 25 degrees C], once the compensating enthalpic and entropic effects of the salt-induced changes in the hydrogen bonding within the hydration sphere of the molecule in the two redox states are factored out, results in being ultimately determined by the stabilizing enthalpic effect of the negatively charged ionic atmosphere on the ferri form. At pH 9, the ionic strength dependence of the reduction termodynamics of cytochrome c follows distinctive patterns, possibly as a result of specific binding of the hydroxide ion to the protein. A decrease in ionic strength at constant pH, as well as a pH increase at constant ionic strength, induces a depression of the temperature of the transition from the low-T to high-T conformer of cytochrome c, which suggests that a temperature-induced decrease in the pK(a) for a residue deprotonation is the key event of this conformational change.

Influence of surface charges on redox properties in high potential iron-sulfur proteins.

The pH-dependence of the reduction potential determined through differential pulse voltammetry for the high potential iron sulfur proteins (HiPIP) from R. globiformis, C. vinosum, R. gelatinosus, E. vacuolata (I and II), E. halophila (I and II) is reported. A decrease in reduction potential with pH is invariably observed in the pH range where deprotonation of the imidazolium nitrogen of histidine residue(s) occurs. No pH dependence is observed for the only protein lacking histidines. It appears that surface charges like the His imidazolium groups are capable of influencing the reduction potential despite the known quencing of the electrostatic interactions due to solvent effects.

Thermodynamics of the alkaline transition of cytochrome c.

The apparent equilibrium constant (Kapp) of the alkaline transition (AT) of beef heart cytochrome c, obtained from pH titrations of the current intensities in cyclic voltammetry experiments, has been measured as a function of the temperature from 5 to 65 degrees C, at different ionic strength (I = 0.01-0.2 M). The temperature profile of the pKapp values is biphasic and yields two distinct sets of DeltaH degrees 'AT and DeltaS degrees 'AT values below and above approximately 40 degrees C. In the low-temperature range, the process is endothermic and is accompanied by a small positive entropy change, while at higher temperatures it becomes less endothermic and involves a pronounced entropy loss. The temperature dependence of the transition thermodynamics is most likely the result of the thermal transition of native ferricytochrome c from a low-T to an high-T conformer which occurs at alkaline pH values at a temperature comparable with above (Ikeshoji, T., Taniguchi, I., and Hawkridge, F. M. (1989) J. Electroanal. Chem. 270, 297-308; Battistuzzi, G., Borsari, M., Sola, M., and Francia, F. (1997) Biochemistry 36, 16247-16258). Thus, it is apparent that the transitions of the two native conformers to the corresponding alkaline form(s) are thermodynamically distinct processes. It is suggested that this difference arises from either peculiar transition-induced changes in the hydration sphere of the protein or to the preferential binding of different lysines to the heme iron in the two temperature ranges. Extrapolation of the Kapp values at null ionic strength allowed the determination of the thermodynamic equilibrium constants (Ka) at each temperature, hence of the "true" standard thermodynamic parameters of the transition. The pKa value at 25 degrees C was found to be 8.0. A pKapp value of 14.4 was calculated for the alkaline transition of ferrocytochrome c at 25 degrees C and I = 0.1 M. The much greater relative stabilization of the native state in the reduced as compared to the oxidized form turns out to be almost entirely enthalpic in origin, and is most likely due to the greater affinity of the methionine sulfur for the Fe(II) ion. Finally, it is found that the Debye-Hückel theory fits the ionic strength dependence of the pKapp values, at least qualitatively, as observed previously for the ionic strength dependence of the reduction potential of this protein class. It is apparent that the increase in the pKapp values with increasing ionic strength is for the most part an entropic effect.

Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2. Probing surface charges through anion-binding studies.

The effects of increasing concentrations of Cl-, ClO4-, and HCO3- on the redox potential of Rhodopseudomonas palustris cytochrome c2 indicate that the two polyatomic anions bind specifically to the protein at one site, while chloride simply exerts an ionic atmosphere effect. The change in E degree upon specific anion binding allows us to probe for the influence of surface charges on the redox potential of cytochromes c. The decrease in redox potential at null ionic strength (delta E degree I = 0) due to anion neutralization of one positive surface charge was found to be 23 mV with perchlorate and 33 mV with bicarbonate. These values compare reasonably well with previous theoretical predictions and estimates of the effect of charge alteration on the E degree values in cytochromes c chemically modified or mutated at surface lysines. These delta E degree values, determined on the unmodified protein, are unprecedented for c-type cytochromes. The anion-induced chemical shift changes of the hyperfine-shifted heme 1H-NMR resonances of the oxidized protein yield lower limit values of 53 M-1 and 18 M-1 for the affinity constant for specific HCO3- and ClO4- binding, respectively.

Cyclic voltammetry and 1H-NMR of Rhodopseudomonas palustris cytochrome c2 pH-dependent conformational states.

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c2 from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E degree values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 1H-NMR resonances of the protein are sensitive to a number of acid/base equilibria. In particular, four equilibria have been determined for the oxidized protein with pKa values of 2.5, 5.5, 6.6 and 9.0. The lowest pKa most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pKa is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pKa of 6.6 modulates the E degree values without determining any detectable spectral change and most likely involves the acid/base equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s-1 at pH 9.2.

Mutation of the metal-bridging proton-donor His63 residue in human Cu, Zn superoxide dismutase. Biochemical and biophysical analysis of the His63-->Cys mutant.

The bridging His63 residue in human Cu, Zn superoxide dismutase, which binds both metals, has been replaced by a Cys residue. The mutant protein has been purified from Escherichia coli and appears to be a normal dimer. Spectroscopic techniques (electronic spectroscopies, EPR, nuclear magnetic relaxation dispersion) show that Cys63 binds the zinc ion, but not the copper ion, and that the latter is probably five co-ordinated with three histidine ligands and two water molecules. The reduction potential of the copper ion in the Cu2+/Cu+ pair decreases from 0.41 V to 0.27 V at neutral pH but still remains intermediate between those of the O2/O2- and O2-/H2O2 pairs so that copper can both oxidize and reduce the O2- substrate, a requirement for dismutase activity. The enzyme binds the substrate-analogue azide (N3-), which displaces one water molecule, with near normal affinity, whereas the enzyme activity with the O2- substrate is reduced to less than 1% of wild-type levels at pH 7.8. The properties of the mutant enzyme are discussed in relation to the superoxide-copper electron transfer process and to the catalytic mechanism.

Anion binding to mitochondrial cytochromes c studied through electrochemistry. Effects of the neutralization of surface charges on the redox potential.

The redox potential of horse and bovine heart cytochromes c determined through cyclic voltammetry is exploited to probe for anion-protein interactions, using a Debye-Hückel-based model. In parallel, protein charge neutralization resulting from specific anion binding allows monitoring for surface-charge/E(o) relationships. This approach shows that a number of anions, most of which are of biological relevance, namely CI-, HPO(2-)4, HCO3-, NO3, SO(2-)4, CIO4-, citrate3- and oxalate2-, bind specifically to the protein surface, often in a sequential manner as a result of the presence of multiple sites with different affinities. The binding stoichiometries of the various anions toward a given cytochrome are in general different. Chloride and phosphate appear to bind to a greater extent to both proteins as compared to the other anions. Differences in binding specificity toward the two cytochromes, although highly sequence-related, are observed for a few anions. The data are discussed comparatively in terms of electrostatic and geometric properties of the anions and by reference to the proposed location and amino acid composition of the anion binding sites, when available. Specific binding of this large set of anions bearing different charges allows the electrostatic effect on Eo due to neutralization of net positive protein surface charge(s) to be monitored. (J)H NMR indeed indicates the absence of significant salt-induced structural perturbations, hence the above change in Eo is predominantly electrostatic in origin. A systematic study of protein surface-charge/Eo relationships using this approach is unprecedented. Values of 15-25 mV (extrapolated at zero ionic strength) are obtained for the decrease in Eo due to neutralization of one positive surface charge, which are of the same order of magnitude as previous estimates obtained with either mutation or chemical modification of surface lysines. The effects of the anion-induced decrease of net positive charge on Eo persist also at a relatively high ionic strength and add to the general effects related to the charge shielding of the protein as a whole due to the surrounding ionic atmosphere: hence the ionic strength dependence of the rate of electron transfer between cytochromes c and redox partners could also involve salt-induced changes in the driving force.