Daniel Murgida - Academia.edu (original) (raw)
Papers by Daniel Murgida
Free Radical Biology and Medicine
Faraday Discussions
We have operando detected the formation of singlet oxygen in a real Li–O2 battery by DMA fluoresc... more We have operando detected the formation of singlet oxygen in a real Li–O2 battery by DMA fluorescence decay and its suppression by using physical quenchers, reaching an extended battery cycle life due to mitigation of spurious reactions
Chemical reviews, Jan 8, 2017
Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible struc... more Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phos...
Free Radical Biology and Medicine, 2016
Biochemistry, 2015
We report a resonance Raman and UV-vis characterization of the active site structure of oxidative... more We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to the wild type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increased of enzymatic activity observed for all protein variants. For Cyt-c nitrated in Tyr74 and sulfoxidized in Met80 the measured apparent binding affinities towards CL are about four times larger than for WT Cyt-c. Based on the present results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.
The Journal of Physical Chemistry B, 2006
The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver ele... more The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver electrode, was studied by stationary and time-resolved surface-enhanced resonance Raman (SERR) spectroscopy to probe the redox site structure and the mechanism and dynamics of the potential-dependent interfacial processes. The layers were built up by sequential adsorption of polycations (poly[ethylene imine] (PEI); polyallylamine hydrochloride (PAH)) and polyanions (poly[styrene sulfonate] (PSS)). All multilayers terminated by PSS electrostatically bind Cyt-c. On PEI/PSS coatings, Cyt-c is peripherally bound and fully redox-active. Due to the interfacial potential drop, the apparent redox potential is lowered by 40 mV compared to that in solution. The rate constant for the heterogeneous electron transfer (ET) of ca. 0.1 s(-1) is consistent with electron tunneling through largely ordered PEI/PSS layers. ET is coupled to a reversible conformational transition of Cyt-c that involves a change of the coordination pattern of the heme. Additional (PAH/PSS) double layers cause a broadening of the redox transition and a drastic negative shift of the redox potential, which is attributed to the formation of PSS/Cyt-c complexes. It is concluded that Cyt-c can effectively compete with PAH for binding of PSS, resulting in a rearrangement of the layered structure and a penetration of the PSS-bound Cyt-c into the PAH/PSS double layers. This conclusion is consistent with SERR intensity and quartz microbalance measurements. ET was found to be overpotential-independent and faster than that for PEI/PSS coatings, which is interpreted in terms of specific PSS/Cyt-c complexes serving as gates for the heterogeneous ET.
The Journal of Physical Chemistry B, 2002
Cytochrome c was electrostatically bound on Ag electrodes coated with self-assembled monolayers o... more Cytochrome c was electrostatically bound on Ag electrodes coated with self-assembled monolayers of carboxylterminated alkylthiols. Employing stationary and time-resolved surface enhanced resonance Raman spectroscopy, activation energies of the interfacial redox process were determined as a function of the electric field strength that was controlled by varying protein-electrode distance via the thiol alkyl chain length. At weak electric fields (long chain lengths), temperature-and overpotential-dependent measurements consistently yield a reorganization energy of 0.26 and 0.22 eV, respectively, which is distinctly lower than for cytochrome c in solution. This decrease is attributed to the lowering of the contribution of solvent reorganization for the reaction of the immobilized protein. At short alkyl chain length, high electric fields strongly raise the activation barrier for the structural reorganization of the protein and the rearrangement of the hydrogen bond network becomes rate limiting for the interfacial redox process as indicated by the H/D kinetic isotope effect that increases with the electric field strength (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). Thus, rate constants measured as a function of the temperature provide the activation enthalpy for the underlying proton-transfer steps. The values of 24.2 and 34.3 kJ mol -1 determined in H 2 O and D 2 O, respectively, as well as the ratio of the preexponential factors A(H 2 O)/A(D 2 O) of ca. 0.8 cannot be reconciled within the semiclassical description of proton transfer but indicate thermally activated nuclear tunneling. The electric-field-induced alteration of the activation barrier that controls the dynamics of the interfacial electron transfer of cytochrome c may represent a general mechanism for modulating biological charge-transfer dynamics at membranes.
The Journal of Physical Chemistry B, 2008
Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayer... more Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) consisting either of 11-mercaptoundecanoic acid (MUA) or of 1:1 mixtures of MUA and either 11-mercaptoundecanol (MU) or 7-mercaptoheptanol (MH). The redox potentials and the apparent rate constants for the interfacial redox process as well as for the protein reorientation were determined by stationary surfaceenhanced resonance Raman (SERR) and time-resolved SERR spectroscopy, respectively. For YCC immobilized on MUA and MUA/MU at pH 7.0 and 6.0, the negative shifts of the redox potentials with respect to that for the protein in solution can be rationalized in terms of the potential of the zero-charge determined by impedance measurements. The apparent electron transfer rate constants of YCC on MUA/MU and MU/MH at pH 6.0 were determined to be 8 and 18 s -1 , respectively. A decrease of the relaxations constants by a factor of ca. 2 was found for pH 7.0, and a comparable low value was determined for a pure MUA even at pH 6.0. In each system, the rate constant for protein reorientation was found to be the same as that for the electron transfer, implying that protein reorientation is the rate limiting step for the interfacial redox process. This gating step is distinctly slower than that for horse heart cytochrome c (HHCC) observed previously under similar conditions (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). The different rate constants of protein reorientation for both proteins and the variations of the rate constants for the different SAMs and pH are attributed to the electric field dependence of the free energy of activation which is assumed to be proportional to the product of the electric field strength and the molecular dipole moment of the protein. The latter quantity is determined by molecular dynamics simulations and electrostatic calculations to be more than 2 times larger for YCC than for HHCC. Moreover, the dipole moment vector and the heme plane constitute an angle of ca. 10 and 45°in YCC and HHCC, respectively. The different magnitudes and directions of the dipole moments as well as the different electric field strengths at the various SAM/protein interfaces allow for a qualitative description of the protein-, SAM-, and electrode-specific kinetics of the interfacial redox processes studied in this and previous works.
The Journal of Physical Chemistry B, 2008
The cbb3 oxygen reductase from Bradyrhizobium japonicum was immobilized on nanostructured silver ... more The cbb3 oxygen reductase from Bradyrhizobium japonicum was immobilized on nanostructured silver electrodes by anchoring the enzyme via a His-tag to a Ni-NTA coating, followed by reconstitution of a lipid bilayer. The immobilized enzyme retains the native structure and catalytic activity as judged by in situ surface-enhanced vibrational spectroscopy and cyclic voltammetry, respectively. Spectroelectrochemical titrations followed by SERR spectroscopy of the integral enzyme and its monohemic (fixO) and dihemic subunits (fixP), allowed the determination of the reduction potentials for the different heme c groups. Both in the isolated subunits and in the integral enzyme the Met/His-coordinated hemes from the two subunits present identical reduction potentials of 180 mV, whereas for the bis-His heme from fixP the value is ca. 400 mV. The determination of reduction potentials of the individual hemes c reported in this work provides the basis for further exploring the mechanism of electroprotonic energy transduction of this complex enzyme.
The Journal of Physical Chemistry B, 2006
Samples of a nematic mixture of ZLI1132 and of a twisted nematic mixture composed of ZLI1132 and ... more Samples of a nematic mixture of ZLI1132 and of a twisted nematic mixture composed of ZLI1132 and chiral inductor S811, including 1%-10% (w/w) 4-N,N-dimethylaminoazobenzene (DAB), (4′-nitro)-4-N,Ndimethylaminoazobenzene (NDAB), spiropyran (SP), or spirooxazine (SO) were irradiated to produce the photochromic transformation of the dopant. The changes in the system were monitored by time-resolved transmission spectroscopy, time-resolved birefringence, or polarized Raman scattering. The medium sensitivity of the kinetics and spectroscopy of some of the probes was used to derive information on polarity of the medium. In the systems studied, apart from the changes in absorption spectrum, great changes in birefringence can be photoinduced and the order of the nematic phase can be changed in either direction, depending on the dopant. The open form of SP can discriminate orientation polarity. Although the polarity parallel to the mesogenic director is similar to that for acetone, the perpendicular orientation has a polarity similar to acetonitrile. In agreement with this observation, the kinetics of the Z f E isomerization of NDAB, oriented parallel to the mesogenic director, also experiences a polarity similar to that for acetone. The decay rate constant of the open form of SP displays a linear relationship between its Arrhenius parameters, which is universal in a great variety of homogeneous solvents, solvent mixtures, and liquid crystals, therefore validating the hypothesis that the same type of transformation is observed in all these cases, namely, the decay of the open form monomer. The dopants used have been proven to be adequate probes of bulklike properties in locally heterogeneous systems as liquid crystals.
Physical Chemistry Chemical Physics, 2011
The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to ... more The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.
Physical Chemistry Chemical Physics, 2005
Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on e... more Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.
Physical Chemistry Chemical Physics, 2009
Photochemistry and Photobiology, 1998
Photosensitized reaction of xanthinic compounds (XH) as caffeine (CF), theobromine (TB) and theop... more Photosensitized reaction of xanthinic compounds (XH) as caffeine (CF), theobromine (TB) and theophylline (TF) by benzophenone (BZ) in ethanol solution was investigated. In the three cases four main reaction products (benzopinacol; diphenylcarbinol; 171-diphenyl-1,2-propanediol and S-[l-(l-hydroxyethyl)] xanthine) were identified and then characterized by melting point, 'H NMR, 13C NMR and mass spectrometry. The quenching of triplet BZ by the three XH was detected and a thorough kinetic analysis was performed. Caffeine produces mainly physical quenching, while TF reacts by N-H hydrogen abstraction. For TB both mechanisms are operative. Heats of reactions were calculated for chosen reactive steps of the mechanism by the PM3 method. They provide additional support to the proposed reaction scheme. We demonstrate that the mechanism leading to XP formation does not proceed through the X* radical directly obtained by H abstraction. An alternative reaction path through an intermediate radical originated on the addition of ethanol radical to XH is proposed. Redox potentials for the oxidation of XH were estimated by cyclic voltametry and by using the Rehm-Weller equation the redox quenching of triplet BZ by XH was discarded.
Journal of the American Chemical Society, 2008
Heterogeneous electron transfer of proteins at biomimetic interfaces is characterized by unusual ... more Heterogeneous electron transfer of proteins at biomimetic interfaces is characterized by unusual distance dependences of the electron-transfer rates, whose origin has been elusive and controversial. Using a two-color, time-resolved, surface-enhanced resonance Raman spectroelectrochemical approach, we have been able to monitor simultaneously and in real time the structure, electron-transfer kinetics, and configurational fluctuations of cytochrome c electrostatically adsorbed to electrodes coated with selfassembled monolayers. Our results show that the overall electron-transfer kinetics is determined by protein dynamics rather than by tunnelling probabilities and that the protein dynamics in turn is controlled by the interfacial electric field. Implications for interprotein electron transfer at biological membranes are discussed.
Journal of the American Chemical Society, 2005
The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immo... more The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.
Journal of the American Chemical Society, 2001
Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag elec... more Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by ω-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 (β ) 1.28 Å -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.
Journal of Luminescence, 1999
Xanthines (ca!eine, theophylline and theobromine) and purine have very low quantum yields of #uor... more Xanthines (ca!eine, theophylline and theobromine) and purine have very low quantum yields of #uorescence in ethanol and acetonitrile solutions at room temperature ( D (10\; naphthalene as #uorescence reference). At 77 K xanthines show good #uorescence and phosphorescence spectra, while purine only shows phosphorescence. Working at low temperature, the e!ect of the properties of the solid matrix (solvent composition and the presence of inorganic salts) on the emission spectra were studied. It was observed that the addition of inorganic salts changes the optical properties of the matrix (snowed matrix, cracked matrix, etc.) as well as produces heavy atom e!ect. These e!ects are opposite in the case of #uorescence, but are co-operative for phosphorescence.
Journal of Electroanalytical Chemistry, 2011
Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectrosc... more Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectroscopy have been employed to study the interfacial redox process of cytochrome c (Cyt-c) immobilised on various metal electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated mercaptanes. The experiments, carried out with Ag, Au and layered Au-SAM-Ag electrodes, afford apparent heterogeneous electron transfer constants (k relax ) that reflect the interplay between electron tunnelling, redox-linked protein structural changes, protein re-orientation, and hydrogen bond re-arrangements in the protein and in the protein/SAM interface. It is shown that the individual processes are affected by the interfacial electric field strength that increases with decreasing thickness of the SAM and increasing difference between the actual potential and the potential of zero-charge. At thick SAMs of mercaptanes including 15 methylene groups, electron tunnelling (k ET ) is the rate-limiting step. Pronounced differences for k ET and its overpotential-dependence are observed for the three metal electrodes and can be attributed to the different electric-field effects on the free-energy term controlling the tunnelling rate. With decreasing SAM thickness, electron tunnelling increases whereas protein dynamics is slowed down such that for SAMs including less than 10 methylene groups, protein re-orientation becomes rate-limiting, as reflected by the viscosity dependence of k relax . Upon decreasing the SAM thickness from 5 to 1 methylene group, an additional H/D kinetic isotope effect is detected indicating that at very high electric fields rearrangements of the interfacial or intra-protein hydrogen bond networks limit the rate of the overall redox process.
JBIC Journal of Biological Inorganic Chemistry, 2007
Free Radical Biology and Medicine
Faraday Discussions
We have operando detected the formation of singlet oxygen in a real Li–O2 battery by DMA fluoresc... more We have operando detected the formation of singlet oxygen in a real Li–O2 battery by DMA fluorescence decay and its suppression by using physical quenchers, reaching an extended battery cycle life due to mitigation of spurious reactions
Chemical reviews, Jan 8, 2017
Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible struc... more Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phos...
Free Radical Biology and Medicine, 2016
Biochemistry, 2015
We report a resonance Raman and UV-vis characterization of the active site structure of oxidative... more We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to the wild type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increased of enzymatic activity observed for all protein variants. For Cyt-c nitrated in Tyr74 and sulfoxidized in Met80 the measured apparent binding affinities towards CL are about four times larger than for WT Cyt-c. Based on the present results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein.
The Journal of Physical Chemistry B, 2006
The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver ele... more The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver electrode, was studied by stationary and time-resolved surface-enhanced resonance Raman (SERR) spectroscopy to probe the redox site structure and the mechanism and dynamics of the potential-dependent interfacial processes. The layers were built up by sequential adsorption of polycations (poly[ethylene imine] (PEI); polyallylamine hydrochloride (PAH)) and polyanions (poly[styrene sulfonate] (PSS)). All multilayers terminated by PSS electrostatically bind Cyt-c. On PEI/PSS coatings, Cyt-c is peripherally bound and fully redox-active. Due to the interfacial potential drop, the apparent redox potential is lowered by 40 mV compared to that in solution. The rate constant for the heterogeneous electron transfer (ET) of ca. 0.1 s(-1) is consistent with electron tunneling through largely ordered PEI/PSS layers. ET is coupled to a reversible conformational transition of Cyt-c that involves a change of the coordination pattern of the heme. Additional (PAH/PSS) double layers cause a broadening of the redox transition and a drastic negative shift of the redox potential, which is attributed to the formation of PSS/Cyt-c complexes. It is concluded that Cyt-c can effectively compete with PAH for binding of PSS, resulting in a rearrangement of the layered structure and a penetration of the PSS-bound Cyt-c into the PAH/PSS double layers. This conclusion is consistent with SERR intensity and quartz microbalance measurements. ET was found to be overpotential-independent and faster than that for PEI/PSS coatings, which is interpreted in terms of specific PSS/Cyt-c complexes serving as gates for the heterogeneous ET.
The Journal of Physical Chemistry B, 2002
Cytochrome c was electrostatically bound on Ag electrodes coated with self-assembled monolayers o... more Cytochrome c was electrostatically bound on Ag electrodes coated with self-assembled monolayers of carboxylterminated alkylthiols. Employing stationary and time-resolved surface enhanced resonance Raman spectroscopy, activation energies of the interfacial redox process were determined as a function of the electric field strength that was controlled by varying protein-electrode distance via the thiol alkyl chain length. At weak electric fields (long chain lengths), temperature-and overpotential-dependent measurements consistently yield a reorganization energy of 0.26 and 0.22 eV, respectively, which is distinctly lower than for cytochrome c in solution. This decrease is attributed to the lowering of the contribution of solvent reorganization for the reaction of the immobilized protein. At short alkyl chain length, high electric fields strongly raise the activation barrier for the structural reorganization of the protein and the rearrangement of the hydrogen bond network becomes rate limiting for the interfacial redox process as indicated by the H/D kinetic isotope effect that increases with the electric field strength (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). Thus, rate constants measured as a function of the temperature provide the activation enthalpy for the underlying proton-transfer steps. The values of 24.2 and 34.3 kJ mol -1 determined in H 2 O and D 2 O, respectively, as well as the ratio of the preexponential factors A(H 2 O)/A(D 2 O) of ca. 0.8 cannot be reconciled within the semiclassical description of proton transfer but indicate thermally activated nuclear tunneling. The electric-field-induced alteration of the activation barrier that controls the dynamics of the interfacial electron transfer of cytochrome c may represent a general mechanism for modulating biological charge-transfer dynamics at membranes.
The Journal of Physical Chemistry B, 2008
Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayer... more Iso-1 yeast cytochrome c (YCC) was adsorbed on Ag electrodes coated with self-assembled monolayers (SAMs) consisting either of 11-mercaptoundecanoic acid (MUA) or of 1:1 mixtures of MUA and either 11-mercaptoundecanol (MU) or 7-mercaptoheptanol (MH). The redox potentials and the apparent rate constants for the interfacial redox process as well as for the protein reorientation were determined by stationary surfaceenhanced resonance Raman (SERR) and time-resolved SERR spectroscopy, respectively. For YCC immobilized on MUA and MUA/MU at pH 7.0 and 6.0, the negative shifts of the redox potentials with respect to that for the protein in solution can be rationalized in terms of the potential of the zero-charge determined by impedance measurements. The apparent electron transfer rate constants of YCC on MUA/MU and MU/MH at pH 6.0 were determined to be 8 and 18 s -1 , respectively. A decrease of the relaxations constants by a factor of ca. 2 was found for pH 7.0, and a comparable low value was determined for a pure MUA even at pH 6.0. In each system, the rate constant for protein reorientation was found to be the same as that for the electron transfer, implying that protein reorientation is the rate limiting step for the interfacial redox process. This gating step is distinctly slower than that for horse heart cytochrome c (HHCC) observed previously under similar conditions (Murgida, D. H.; Hildebrandt, P. J. Am. Chem. Soc. 2001, 123, 4062-4068). The different rate constants of protein reorientation for both proteins and the variations of the rate constants for the different SAMs and pH are attributed to the electric field dependence of the free energy of activation which is assumed to be proportional to the product of the electric field strength and the molecular dipole moment of the protein. The latter quantity is determined by molecular dynamics simulations and electrostatic calculations to be more than 2 times larger for YCC than for HHCC. Moreover, the dipole moment vector and the heme plane constitute an angle of ca. 10 and 45°in YCC and HHCC, respectively. The different magnitudes and directions of the dipole moments as well as the different electric field strengths at the various SAM/protein interfaces allow for a qualitative description of the protein-, SAM-, and electrode-specific kinetics of the interfacial redox processes studied in this and previous works.
The Journal of Physical Chemistry B, 2008
The cbb3 oxygen reductase from Bradyrhizobium japonicum was immobilized on nanostructured silver ... more The cbb3 oxygen reductase from Bradyrhizobium japonicum was immobilized on nanostructured silver electrodes by anchoring the enzyme via a His-tag to a Ni-NTA coating, followed by reconstitution of a lipid bilayer. The immobilized enzyme retains the native structure and catalytic activity as judged by in situ surface-enhanced vibrational spectroscopy and cyclic voltammetry, respectively. Spectroelectrochemical titrations followed by SERR spectroscopy of the integral enzyme and its monohemic (fixO) and dihemic subunits (fixP), allowed the determination of the reduction potentials for the different heme c groups. Both in the isolated subunits and in the integral enzyme the Met/His-coordinated hemes from the two subunits present identical reduction potentials of 180 mV, whereas for the bis-His heme from fixP the value is ca. 400 mV. The determination of reduction potentials of the individual hemes c reported in this work provides the basis for further exploring the mechanism of electroprotonic energy transduction of this complex enzyme.
The Journal of Physical Chemistry B, 2006
Samples of a nematic mixture of ZLI1132 and of a twisted nematic mixture composed of ZLI1132 and ... more Samples of a nematic mixture of ZLI1132 and of a twisted nematic mixture composed of ZLI1132 and chiral inductor S811, including 1%-10% (w/w) 4-N,N-dimethylaminoazobenzene (DAB), (4′-nitro)-4-N,Ndimethylaminoazobenzene (NDAB), spiropyran (SP), or spirooxazine (SO) were irradiated to produce the photochromic transformation of the dopant. The changes in the system were monitored by time-resolved transmission spectroscopy, time-resolved birefringence, or polarized Raman scattering. The medium sensitivity of the kinetics and spectroscopy of some of the probes was used to derive information on polarity of the medium. In the systems studied, apart from the changes in absorption spectrum, great changes in birefringence can be photoinduced and the order of the nematic phase can be changed in either direction, depending on the dopant. The open form of SP can discriminate orientation polarity. Although the polarity parallel to the mesogenic director is similar to that for acetone, the perpendicular orientation has a polarity similar to acetonitrile. In agreement with this observation, the kinetics of the Z f E isomerization of NDAB, oriented parallel to the mesogenic director, also experiences a polarity similar to that for acetone. The decay rate constant of the open form of SP displays a linear relationship between its Arrhenius parameters, which is universal in a great variety of homogeneous solvents, solvent mixtures, and liquid crystals, therefore validating the hypothesis that the same type of transformation is observed in all these cases, namely, the decay of the open form monomer. The dopants used have been proven to be adequate probes of bulklike properties in locally heterogeneous systems as liquid crystals.
Physical Chemistry Chemical Physics, 2011
The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to ... more The subunit II of the caa(3) oxygen reductase from Rhodothermus marinus contains, in addition to the Cu(A) center, a c-type heme group in the cytochrome c domain (Cyt-D) that is the putative primary electron acceptor of the enzyme. In this work we have combined surface-enhanced resonance Raman (SERR) spectroelectrochemistry, molecular dynamics (MD) simulations and electron pathway calculations to assess the most likely interaction domains and electron entry/exit points of the truncated Cyt-D of subunit II in the reactions with its electron donor, HiPIP and electron acceptor, Cu(A). The results indicate that the transient interaction between Cyt-D and HiPIP relies upon a delicate balance of hydrophobic and polar contacts for establishing an optimized electron transfer pathway that involves the exposed edge of the heme group and guaranties efficient inter-protein electron transfer on the nanosecond time scale. The reorganization energy of ca. 0.7 eV was determined by time-resolved SERR spectroelectrochemistry. The intramolecular electron transfer pathway in integral subunit II from Cyt-D to the Cu(A) redox center most likely involves the iron ligand histidine 20 as an electron exit point in Cyt-D.
Physical Chemistry Chemical Physics, 2005
Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on e... more Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.
Physical Chemistry Chemical Physics, 2009
Photochemistry and Photobiology, 1998
Photosensitized reaction of xanthinic compounds (XH) as caffeine (CF), theobromine (TB) and theop... more Photosensitized reaction of xanthinic compounds (XH) as caffeine (CF), theobromine (TB) and theophylline (TF) by benzophenone (BZ) in ethanol solution was investigated. In the three cases four main reaction products (benzopinacol; diphenylcarbinol; 171-diphenyl-1,2-propanediol and S-[l-(l-hydroxyethyl)] xanthine) were identified and then characterized by melting point, 'H NMR, 13C NMR and mass spectrometry. The quenching of triplet BZ by the three XH was detected and a thorough kinetic analysis was performed. Caffeine produces mainly physical quenching, while TF reacts by N-H hydrogen abstraction. For TB both mechanisms are operative. Heats of reactions were calculated for chosen reactive steps of the mechanism by the PM3 method. They provide additional support to the proposed reaction scheme. We demonstrate that the mechanism leading to XP formation does not proceed through the X* radical directly obtained by H abstraction. An alternative reaction path through an intermediate radical originated on the addition of ethanol radical to XH is proposed. Redox potentials for the oxidation of XH were estimated by cyclic voltametry and by using the Rehm-Weller equation the redox quenching of triplet BZ by XH was discarded.
Journal of the American Chemical Society, 2008
Heterogeneous electron transfer of proteins at biomimetic interfaces is characterized by unusual ... more Heterogeneous electron transfer of proteins at biomimetic interfaces is characterized by unusual distance dependences of the electron-transfer rates, whose origin has been elusive and controversial. Using a two-color, time-resolved, surface-enhanced resonance Raman spectroelectrochemical approach, we have been able to monitor simultaneously and in real time the structure, electron-transfer kinetics, and configurational fluctuations of cytochrome c electrostatically adsorbed to electrodes coated with selfassembled monolayers. Our results show that the overall electron-transfer kinetics is determined by protein dynamics rather than by tunnelling probabilities and that the protein dynamics in turn is controlled by the interfacial electric field. Implications for interprotein electron transfer at biological membranes are discussed.
Journal of the American Chemical Society, 2005
The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immo... more The aa3 type B oxygen reductase from the thermophilic archaeon Acidianus ambivalens (QO) was immobilized on silver electrodes and studied by potential-dependent surface-enhanced resonance Raman (SERR) spectroscopy. The immobilized enzyme retains the native structure at the level of the heme pockets and exhibits reversible electrochemistry. From the potential dependence of specific spectral marker bands, the midpoint potentials of hemes a and a3 were unambiguously determined for the first time, being 320 +/- 20 mV for the former and 390 +/- 20 mV for the latter. Both hemes could be treated as independent one-electron Nernstian redox couples, indicating that the interaction potential is smaller than 50 mV. The reversed order of the midpoint potentials compared to those of type A (mitochondrial-like) oxidases, as well as the lack of substantial Coulombic interactions, suggests a different mechanism of electroprotonic energy transduction. In contrast to type A enzymes, a-a3 intraprotein electron transfer in QO is already guaranteed by the order of the midpoint potentials at the onset of enzyme reduction and, therefore, does not require a complex network of cooperativities to ensure exergonicity. In the immobilized state, conformational transitions of the QO a3-CuB active site, which are believed to be essential for proton translocation, are drastically slowed compared to those in solution. We ascribe this finding to the effect of the interfacial electric field, which is of the same order of magnitude as in biological membranes. These results suggest that the membrane potential may play an active role in the regulation of the enzymatic activity of QO.
Journal of the American Chemical Society, 2001
Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag elec... more Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by ω-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 (β ) 1.28 Å -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.
Journal of Luminescence, 1999
Xanthines (ca!eine, theophylline and theobromine) and purine have very low quantum yields of #uor... more Xanthines (ca!eine, theophylline and theobromine) and purine have very low quantum yields of #uorescence in ethanol and acetonitrile solutions at room temperature ( D (10\; naphthalene as #uorescence reference). At 77 K xanthines show good #uorescence and phosphorescence spectra, while purine only shows phosphorescence. Working at low temperature, the e!ect of the properties of the solid matrix (solvent composition and the presence of inorganic salts) on the emission spectra were studied. It was observed that the addition of inorganic salts changes the optical properties of the matrix (snowed matrix, cracked matrix, etc.) as well as produces heavy atom e!ect. These e!ects are opposite in the case of #uorescence, but are co-operative for phosphorescence.
Journal of Electroanalytical Chemistry, 2011
Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectrosc... more Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectroscopy have been employed to study the interfacial redox process of cytochrome c (Cyt-c) immobilised on various metal electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated mercaptanes. The experiments, carried out with Ag, Au and layered Au-SAM-Ag electrodes, afford apparent heterogeneous electron transfer constants (k relax ) that reflect the interplay between electron tunnelling, redox-linked protein structural changes, protein re-orientation, and hydrogen bond re-arrangements in the protein and in the protein/SAM interface. It is shown that the individual processes are affected by the interfacial electric field strength that increases with decreasing thickness of the SAM and increasing difference between the actual potential and the potential of zero-charge. At thick SAMs of mercaptanes including 15 methylene groups, electron tunnelling (k ET ) is the rate-limiting step. Pronounced differences for k ET and its overpotential-dependence are observed for the three metal electrodes and can be attributed to the different electric-field effects on the free-energy term controlling the tunnelling rate. With decreasing SAM thickness, electron tunnelling increases whereas protein dynamics is slowed down such that for SAMs including less than 10 methylene groups, protein re-orientation becomes rate-limiting, as reflected by the viscosity dependence of k relax . Upon decreasing the SAM thickness from 5 to 1 methylene group, an additional H/D kinetic isotope effect is detected indicating that at very high electric fields rearrangements of the interfacial or intra-protein hydrogen bond networks limit the rate of the overall redox process.
JBIC Journal of Biological Inorganic Chemistry, 2007