Time-resolved Surface-enhanced Resonance Raman Spectroscopy Research Articles

2nd coordination sphere controlled electron transfer of iron hangman complexes on electrodes probed by surface enhanced vibrational spectroscopy.

Iron hangman complexes exhibit improved catalytic properties regarding O2 and H2O2 reduction, which are attributed to the presence of a proton donating group in defined vicinity of the catalytic metal centre. Surface enhanced resonance Raman (SERR) and IR (SEIRA) spectro-electrochemistry has been applied concomitantly for the first time to analyse such iron hangman porphyrin complexes attached to electrodes in aqueous solution. While the SERR spectra yield information about the redox state of the central iron, the SEIRA spectra show protonation and deprotonation events of the 2nd coordination sphere. To investigate the influence of a proton active hanging group on the heterogeneous electron transfer between the iron porphyrin and the electrode, two hangman complexes with either an acid or ester functional group were compared. Using time resolved SERR spectroscopy the electron transfer rates of both complexes were determined. Complexes with an acid group showed a slow electron transfer rate at neutral pH that increased significantly at pH 4, while complexes with an ester group exhibited a much faster, but pH independent rate. SEIRA measurements were able to determine directly for the first time a pKa value of 3.4 of a carboxylic hanging group in the immobilized state that shifted to 5.2 in D2O buffer solution. The kinetic data showed an increase of the heterogeneous electron transfer rate with the protonation degree of the acid groups. From these results, we propose a PCET which is strongly modulated by the protonation state of the acid hanging group via hydrogen bond interactions.

Multi-layer electron transfer across nanostructured Ag-SAM-Au-SAM junctions probed by surface enhanced Raman spectroscopy

We have developed a new layered Au-Ag electrode for studying interfacial electron transfer processes by surface enhanced resonance Raman (SERR) spectroscopy. The device consists of a nanostructured Ag support which is separated from a Au film via a thin self-assembled monolayer (SAM) of amino-terminated mercaptanes (C(y)-NH(2), with y = 6, 8, 11). The Au film is biocompatibly coated to allow for binding of redox-active proteins. We have explored the performance of this device for analysing interfacial electron transfer processes by stationary and time-resolved SERR spectroscopy, using the heme protein cytochrome c (Cyt-c) as a benchmark protein. The SERRS intensity of Cyt-c on Ag-(C(y)-NH(2))-Au electrodes and Ag electrodes was comparable when the protein was electrostatically attached to the metal coated by a SAM of carboxyl-terminated mercaptanes (C(x)-COOH) surface but 25 times higher upon covalent attachment via Cys102 to the bare Au surface. In the case of electrostatic adsorption the protein remained exclusively in its native state. Electron transfer between the protein and the Ag electrode occurred in an almost ideal Nernstian behaviour with a number of transferred electrons close to one (n = 0.8-0.9). Conversely, the covalent attached Cyt c showed two broad redox transitions (n = 0.3) and a partial conversion to a non-native species which remained redox inactive in the potential range from +0.1 to -0.3 V. For the electrostatically immobilised Cyt, apparent electron transfer rates of 0.8 and 49 s(-1) were obtained for y = 11 and x = 15 and 10, respectively, indicating a fast long-distance electron transfer through the multilayer with the electron tunneling through the C(x)-COOH SAM being the rate limiting step.

Characterization of the Electron Transfer of a Ferrocene Redox Probe and a Histidine-Tagged Hemoprotein Specifically Bound to a Nitrilotriacetic-Terminated Self-Assembled Monolayer

We report the selective, controlled binding of a model redox probe, 1,1'-bis(N-imidazolylmethyl)ferrocene (Fc-Im2), and a small redox hemoprotein, histidine-tagged recombinant human neuroglobin (hNb), at the surface of metal electrodes (gold and SER-active silver) modified by a self-assembled monolayer (SAM) of a nitrilotriacetic (NTA)-terminated thiol. The resulting SAMs were characterized by cyclic voltammetry and surface-enhanced resonance Raman (SERR) spectroscopy coupled to electrochemistry. Once specifically bounded to the Ni(II)-NTA-modified gold electrode, nearly ideal cyclic voltammetric behavior with relatively fast electron-transfer (ET) communication through the SAM was determined for the Fc-Im2 redox probe. However, no direct electron transfer could be evidenced for the hNb redox protein under the same conditions. This outcome was different from the result obtained during SERR experiments coupled to electrochemistry in which a direct electrochemical conversion of hNb immobilized on a Ni(II)-NTA-modified SER-active Ag electrode was observed. The SERR spectra of the immobilized hNb was the same as the resonance Raman spectra of the protein in homogeneous solution, allowing us to conclude that the native structure of hNb was retained upon immobilization and that the direct ET was not the result of some partial or complete protein denaturation. The long-range ET rate constant (kET) through the SAM was determined by time-resolved SERR spectroscopy. A value of kET=0.12 s(-1) was obtained, which is within the predicted range of a fully nonadiabatic ET through a SAM thickness of approximately 26 A and close to the values previously determined for analogous small redox proteins at similar long-range ET distances. A SERR spectroelectrochemical titration of the immobilized hNb was also carried out, showing both an apparent standard potential (E0') negatively shifted by 100 mV compared with hNb in solution and a gentle slope in the titration curve. These results suggest a range of chemical environments in the surroundings of the redox protein and a variety of interactions with the NTA-terminated SAM. The influence of protein immobilization on E0' is discussed together with the long-range ET rate constant and molecular orientation of the surface-immobilized hNb.

Electron Transfer Kinetics of Cytochrome c Probed by Time-Resolved Surface-Enhanced Resonance Raman Spectroscopy

An improved setup including a measuring cell was designed for time-resolved surface-enhanced resonance Raman (SERR) spectroscopy. The cell is based on a rotating disk electrode (RDE) made from electrochemically roughened Ag. Cytochrome c (cc) adsorbed on a monolayer of mercaptoethanol is investigated with respect to heterogeneous electron transfer. Cyclic voltammograms and potential-dependent static SERR spectra indicate cc to be electroactive on the Ag electrode. The standard redox potential was found to be 234 mV. Time-resolved SERR spectra were then measured triggered by periodic potential pulses changing the protein between the oxidized and reduced state at a frequency of 10 Hz. Monoexponential functions obtained from the intensity of the band at 1361 cm-1 plotted versus time yielded the rate constants of heterogeneous electron transfer to be k(ox) = 46 +/- 7 s(-1) and k(red) = 84 +/- 20 s(-1). These relatively low rates are in line with the orientation of cc on the mercaptoethanol-modified Ag electrode. In this case the heme cleft pointed away from the surface thus hampering electron transfer.

Kinetics of the electron transfer reaction of Cytochrome c 552 adsorbed on biomimetic electrode studied by time-resolved surface-enhanced resonance Raman spectroscopy and electrochemistry

Cytochrome c (552) (Cyt-c (552)) and its redox partner ba ( 3 )-oxidase from Thermus thermophilus possess structural differences compared with Horse heart cytochrome c (cyt-c)/cytochrome c oxidase (CcO) system, where the recognition between partners and the electron transfer (ET) process is initiated via electrostatic interactions. We demonstrated in a previous study by surface-enhanced resonance Raman (SERR) spectroscopy that roughened silver electrodes coated with uncharged mixed self-assembled monolayers HS-(CH(2))( n )-CH(3)/HS-(CH(2))( n + 1)-OH 50/50, n = 5, 10 or 15, was a good model to mimic the Cyt-c (552) redox partner. All the adsorbed molecules are well oriented on such biomimetic electrodes and transfer one electron during the redox process. The present work focuses on the kinetic part of the heterogeneous ET process of Cyt-c (552) adsorbed onto electrodes coated with such mixed SAMs of different alkyl chain length. For that purpose, two complementary methods were combined. Firstly cyclic voltammetry shows that the ET between the adsorbed Cyt-c (552) and the biomimetic electrode is direct and reversible. Furthermore, it allows the estimation of both the density surface coverage of adsorbed Cyt-c (552) and the kinetic constants values. Secondly, time-resolved SERR (TR-SERR) spectroscopy showed that the ET process occurs without conformational change of the Cyt-c (552) heme group and allows the determination of kinetic constants. Results show that the kinetic constant values obtained by TR-SERR spectroscopy could be compared to those obtained from cyclic voltammetry. They are estimated at 200, 150 and 40 s(-1) for the ET of Cyt-c (552) adsorbed onto electrodes coated with mixed SAMs HS-(CH(2))( n )-CH(3)/HS-(CH(2))( n + 1)-OH 50/50, n = 5, 10 or 15, respectively.

Electrochemical and Spectroscopic Investigations of Immobilized De Novo Designed Heme Proteins on Metal Electrodes

On the basis of rational design principles, template-assisted four-helix-bundle proteins that include two histidines for coordinative binding of a heme were synthesized. Spectroscopic and thermodynamic characterization of the proteins in solution reveals the expected bis-histidine coordinated heme configuration. The proteins possess different binding domains on the top surfaces of the bundles to allow for electrostatic, covalent, and hydrophobic binding to metal electrodes. Electrostatic immobilization was achieved for proteins with lysine-rich binding domains (MOP-P) that adsorb to electrodes covered by self-assembled monolayers of mercaptopropionic acid, whereas cysteamine-based monolayers were employed for covalent attachment of proteins with cysteine residues in the binding domain (MOP-C). Immobilized proteins were studied by surface-enhanced resonance Raman (SERR) spectroscopy and electrochemical methods. For all proteins, immobilization causes a decrease in protein stability and a loosening of the helix packing, as reflected by a partial dissociation of a histidine ligand in the ferrous state and very low redox potentials. For the covalently attached MOP-C, the overall interfacial redox process involves the coupling of electron transfer and heme ligand dissociation, which was analyzed by time-resolved SERR spectroscopy. Electron transfer was found to be significantly slower for the mono-histidine-coordinated than for the bis-histidine-coordinated heme. For the latter, the formal heterogeneous electron-transfer rate constant of 13 s(-1) is similar to those reported for natural heme proteins with comparable electron-transfer distances, which indicates that covalently bound synthetic heme proteins provide efficient electronic communication with a metal electrode as a prerequisite for potential biotechnological applications.

Redox and conformational equilibria and dynamics of cytochrome c at high electric fields.

Cytochrome c (Cyt-c) adsorbed in the electrical double layer of the Ag electrode/electrolyte interface has been studied by stationary and time-resolved surface-enhanced resonance Raman spectroscopy to analyse the effect of strong electric fields on structure and reaction equilibria and dynamics of the protein. In the potential range between +0.1 and -0.55 V (versus saturated calomel electrode), the adsorbed Cyt-c forms a potential-dependent reversible equilibrium between the native state B1 and a conformational state B2. The redox potentials of the bis-histidine-coordinated six-coordinated low-spin and five-coordinated high-spin substates of B2 were determined to be -0.425 and -0.385 V, respectively, whereas the additional six-coordinated aquo-histidine-coordinated high-spin substate was found to be redox-inactive. The redox potential for the conformational state B1 was found to be the same as in solution in agreement with the structural identity of the adsorbed B1 and the native Cyt-c. For all three redox-active species, the formal heterogeneous electron transfer rate constants are small and of the same order of magnitude (3-13 s-1), which implies that the rate-limiting step is largely independent of the redox-site structure. These findings, as well as the slow and potential-dependent transitions between the various conformational (sub-)states, can be rationalized in terms of an electric field-induced increase of the activation energy for proton-transfer steps linked to protein structural reorganisation. Further increasing the electric field strength by shifting the electrode potential above +0.1 V leads to irreversible structural changes that are attributed to an unfolding of the polypeptide chain.

Conformational and Redox Equilibria and Dynamics of Cytochrome c Immobilized on Electrodes via Hydrophobic Interactions

Surface enhanced resonance Raman (SERR) spectroscopy was employed to study structure and dynamics of cytochrome c (Cyt-c) immobilized on Ag electrodes that were coated with self-assembled monolayers (SAM) of n-alkanethiols HS-(CH 2 ) n -CH 3 . Cyt-c is bound to these electrodes by hydrophobic interactions most likely via the peptide segment 81-85 that may partially penetrate into the monolayers. The immobilized proteins are partially converted to the conformational state B2 that exhibits a different heme pocket structure and coordination configuration than the native state B1. The B2/B 1 conformational equilibrium, which can also be induced by binding to electrodes coated with ω-carboxyl alkanethiols (Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2001, 105, 1578), is potential-dependent with the Bl state prevailing at potentials < -0.3 V. On hydrophobic coatings, the equilibrium constant is largely independent of the thickness of the SAM except for HS-CH 2 CH 3 coatings, which do not allow integration of the apolar peptide segment. Using time-resolved SERR spectroscopy, the dynamics of the conversion from the ferric B2 state to the ferrous B I state were analyzed. The underlying conformational transitions are potential-dependent and involve a fast step (ca. 10 4 -10 5 s - 1 ) that may either be the formation of the ferric B2 state or the back-conversion to the ferrous B I state. The conformational transitions are substantially faster than those observed for electrostatically bound Cyt-c where rearrangements of the hydrogen-bonding network in the protein are associated with high electric field induced activation barriers. For the hydrophobically bound Cyt-c, the fast conformational transitions proceed on the same time scale as the intermolecular electron transfer between Cyt-c and its natural partner cytochrome c oxidase and may play a role in the biological process.

Electron transfer dynamics of cytochrome c bound to self-assembled monolayers on silver electrodes

Cytochrome c (Cyt- c) was electrostatically immobilised on Ag electrodes coated with self-assembled monolayers (SAM) that are formed by ω-carboxyl alkanethiols with different alkyl chain lengths (C x ). Surface enhanced resonance Raman (SERR) spectroscopy demonstrated that electrostatic binding does not lead to conformational changes of the heme protein under the conditions of the present experiments. Employing time-resolved SERR spectroscopy, the rate constants of the heterogeneous electron transfer (ET) between the adsorbed Cyt- c and the Ag electrode were determined for a driving force of zero electronvolts. For SAMs with long alkyl chains (C 16, C 11), the rate constants display a normal exponential distance dependence, whereas for shorter chain lengths (C 6, C 3, C 3), the ET rate constant approaches a constant value (ca. 130 s −1). The onset of the non-exponential distance-dependence is paralleled by an increasing kinetic H/D effect, indicating a coupling of the redox reaction with proton transfer (PT) steps. This unusual kinetic behaviour is attributed to the effect of the electric field at the Ag/SAM interface that increasingly raises the energy barrier for the PT processes with decreasing distance of the adsorbed Cyt- c from the electrode. The distance-dependence of the electric field strength is estimated on the basis of a simple electrostatic model that can consistently describe the redox potential shifts of Cyt- c as determined by stationary SERR spectroscopy for the various SAMs. At low electric fields, PT is sufficiently fast so that rate constants, determined as a function of the driving force, yield the reorganisation energy (0.217 electronvolts) of the heterogeneous ET.

Active-Site Structure and Dynamics of Cytochrome c Immobilized on Self-Assembled Monolayers—A Time-Resolved Surface Enhanced Resonance Raman Spectroscopic Study

Time-resolved surface-enhanced resonance Raman spectroscopy can selectively probe the molecular structure and dynamics of the active sites of redox proteins that are immobilized on self-assembled monolayers on Ag electrodes (see picture). As shown for the protein cytochrome c, this method is capable of analyzing complex interfacial processes in terms of electron transfer and non-Faradaic reactions.

Dynamics of the Heterogeneous Electron-Transfer Reaction of Cytochrome c552 from Thermus thermophilus. A Time-Resolved Surface-Enhanced Resonance Raman Spectroscopic Study

The heterogeneous electron-transfer process of cytochrome c552 (Cyt-c552) adsorbed on an Ag electrode was studied by surface-enhanced resonance Raman (SERR) spectroscopy. Upon adsorption the heme protein Cyt-c552, which acts as an electron carrier in the respiratory chain of Thermus thermophilus, exists in a potential-dependent equilibrium between two conformational states (B1, B2) similar to cytochrome c (Cyt-c) (Wackerbarth et al. Appl. Spectrosc. 1999, 53, 283). From the stationary SERR spectra measured as a function of the potential, the apparent redox potential of state B1 was determined to be −0.044 V (versus saturated calomel electrode) which is 31 mV more negative than the redox potential of Cyt-c552 in solution. On the basis of a model for the interfacial potential distribution that accounts for the potential drop at the redox site of the adsorbed protein, it is concluded that the true redox potential of the adsorbed state B1 is the same as that for the heme protein in solution. This conclusion is consistent with the structural identity of state B1 and Cyt-c552 in solution that is derived from the comparison of the SERR and resonance Raman spectra. On the other hand, the formation of B2 is associated with substantial structural changes of the heme pocket and a large negative shift of the redox potential. The dynamics of the heterogeneous reduction of B1 was studied by time-resolved SERR spectroscopy which combines the spectroscopic measurements with the potential jump technique. In contrast to Cyt-c, the electron transfer was found to be much faster than the conformational transition to B2 so that the data could be analyzed on the basis of a one-step relaxation process. For the formal unimolecular electron-transfer rate constant a value of 4.6 s-1 was obtained. On the basis of the rate constants measured as a function of the overpotential, the reorganization energy was determined to be 0.15 eV. This unusually low value may be due to a strongly diminished contribution of the solvent reorientation for the adsorbed species and the specific heme pocket structure of Cyt-c552 optimized for the electron-transfer reaction. The differences in the electron-transfer mechanism and dynamics compared to Cyt-c are obviously related to the unique structural properties of Cyt-c552 which may be the consequence of the adaptation to the extreme living conditions of the thermophilic bacterium.

Time-Resolved Surface-Enhanced Resonance Raman Spectroscopy for Studying Electron-Transfer Dynamics of Heme Proteins

Time-Resolved Surface-Enhanced Resonance Raman Spectroscopy for Studying Electron-Transfer Dynamics of Heme Proteins