Surface-enhanced Resonance Raman Research Articles

Vibrational Spectro-Electrochemistry of Heme Proteins

Heme proteins perform a plethora of distinct cellular functions, including e.g. electron transport (ET), energy conversion, detoxification, catalysis, signaling, and gene regulation, and as such inspire a myriad of biotechnological applications. We use resonance Raman (RR) and Surface enhanced RR (SERR) spectroscopies to probe the architecture of the heme pocket in diverse heme proteins and enzymes, which is essential for the understanding of their physiological properties as well as for the evaluation of their potential for development of the 3rd generation bioelectronic devices (1,2). Moreover, plasmonic metal that gives origin to the surface enhancement of the Raman signal of the molecules found in its close proximity can serve as an electrode, thus driving electrochemical processes. In the case of heme proteins attached to plasmonic Ag electrodes, SERR spectra selectively show vibrational bands originating from the heme moiety only, which are sensitive to spin, coordination and redox state and of the heme iron. These properties that govern the catalytic performance of heme enzymes can be monitored in potential dependent manner by SERR spectro-electrochemistry.We have demonstrated that SERR spectro-electrochemistry possesses unique capacity of to i) disentangle ET processes in multi hemic proteins, such as 28 heme containing nitrite reductase, which represent a challenge for all other experimental approaches and ii) detect subtle immobilization induced structural changes in enzymes of biotechnological interest, which e.g. in the case of cytochrome P450 may prevent their successful applications (1-4). Here we show that SERRS monitoring of electrocatalytic processes by immobilized heme peroxidases, can provide information on catalytically relevant species in situ. Several members of a recently discovered family of heme dye-decolorizing peroxidases (DyPs) that possess remarkable catalytic properties in solution and high biotechnological potential, have been immobilized on biocompatible Ag electrodes. Their structural and electrocatalytic properties studied by RR, SERR spectro-electrochemistry and electrochemistry (2). The immobilized DyP from Pseudomonas putida (PpDyP), in particular, shows native structure and outstanding analytical and catalytic parameters, and hence an exceptional potential for development of 3rd generation biosensors for H2O2 detection. In terms of sensitivity, the bioelectrodes carrying immobilized PpDyP outperform HRP based counterparts reported in the literature (2,4). The biosensor based on a PpDyP variant that harbors mutations at the second shell of the heme cavity reveals further improved storage. Our work highlights the importance of integrated, multidisciplinary approach to simultaneously evaluate the structure and catalytic properties of the enzymes, which ensures faster identification and optimization of the promising candidates for biotechnological applications.

SERR Spectroelectrochemistry as a Guide for Rational Design of DyP-Based Bioelectronics Devices.

Immobilised dye-decolorizing peroxidases (DyPs) are promising biocatalysts for the development of biotechnological devices such as biosensors for the detection of H2O2. To this end, these enzymes have to preserve native, solution properties upon immobilisation on the electrode surface. In this work, DyPs from Cellulomonas bogoriensis (CboDyP), Streptomyces coelicolor (ScoDyP) and Thermobifida fusca (TfuDyP) are immobilised on biocompatible silver electrodes functionalized with alkanethiols. Their structural, redox and catalytic properties upon immobilisation are evaluated by surface-enhanced resonance Raman (SERR) spectroelectrochemistry and cyclic voltammetry. Among the studied electrode/DyP constructs, only CboDyP shows preserved native structure upon attachment to the electrode. However, a comparison of the redox potentials of the enzyme in solution and immobilised states reveals a large discrepancy, and the enzyme shows no electrocatalytic activity in the presence of H2O2. While some immobilised DyPs outperform existing peroxidase-based biosensors, others fail to fulfil the essential requirements that guarantee their applicability in the immobilised state. The capacity of SERR spectroelectrochemistry for fast screening of the performance of immobilised heme enzymes places it in the front-line of experimental approaches that can advance the search for promising DyP candidates.

A Resonance Raman Marker Band Characterizes the Slow and Fast Form of Cytochrome c Oxidase.

Cytochrome c oxidase (CcO) in its as-isolated form is known to exist in a slow and fast form, which differ drastically in their ability to bind oxygen and other ligands. While preparation methods have been established that yield either the fast or the slow form of the protein, the underlying structural differences have not been identified yet. Here, we have performed surface enhanced resonance Raman (SERR) spectroscopy of CcO immobilized on electrodes in both forms. SERR spectra obtained in resonance with the heme a3 metal-to-ligand charge transfer (MLCT) transition at 650 nm displayed a sharp vibrational band at 748 or 750 cm-1 when the protein was in its slow or fast form, respectively. DFT calculations identified the band as a mode of the His-419 ligand that is sensitive to the oxygen ligand and the protonation state of Tyr-288 within the binuclear complex. Potential-dependent SERR spectroscopy showed a redox-induced change of this band around 525 mV versus Ag/AgCl exclusively for the fast form, which coincides with the redox potential of the Tyr-O/Tyr-O- transition. Our data points to a peroxide ligand in the resting state of CcO for both forms. The observed frequencies and redox sensitivities of the Raman marker band suggest that a radical Tyr-288 is present in the fast form and a protonated Tyr-288 in the slow form.

In Situ Spectroelectrochemical Investigations of Electrode-Confined Electron-Transferring Proteins and Redox Enzymes.

This perspective analyzes recent advances in the spectroelectrochemical investigation of redox proteins and enzymes immobilized on biocompatible or biomimetic electrode surfaces. Specifically, the article highlights new insights obtained by surface-enhanced resonance Raman (SERR), surface-enhanced infrared absorption (SEIRA), protein film infrared electrochemistry (PFIRE), polarization modulation infrared reflection–absorption spectroscopy (PMIRRAS), Förster resonance energy transfer (FRET), X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), and differential electrochemical mass spectrometry (DMES)-based spectroelectrochemical methods on the structure, orientation, dynamics, and reaction mechanisms for a variety of immobilized species. This includes small heme and copper electron shuttling proteins, large respiratory complexes, hydrogenases, multicopper oxidases, alcohol dehydrogenases, endonucleases, NO-reductases, and dye decolorizing peroxidases, among other enzymes. Finally, I discuss the challenges and foreseeable future developments toward a better understanding of the functioning of these complex macromolecules and their exploitation in technological devices.

Characterisation of the Cyanate Inhibited State of Cytochrome c Oxidase

Heme-copper oxygen reductases are terminal respiratory enzymes, catalyzing the reduction of dioxygen to water and the translocation of protons across the membrane. Oxygen consumption is inhibited by various substances. Here we tested the relatively unknown inhibition of cytochrome c oxidase (CcO) with isocyanate. In contrast to other more common inhibitors like cyanide, inhibition with cyanate was accompanied with the rise of a metal to ligand charge transfer (MLCT) band around 638 nm. Increasing the cyanate concentration furthermore caused selective reduction of heme a. The presence of the CT band allowed for the first time to directly monitor the nature of the ligand via surface-enhanced resonance Raman (SERR) spectroscopy. Analysis of isotope sensitive SERR spectra in comparison with Density Functional Theory (DFT) calculations identified not only the cyanate monomer as an inhibiting ligand but suggested also presence of an uretdion ligand formed upon dimerization of two cyanate ions. It is therefore proposed that under high cyanate concentrations the catalytic site of CcO promotes cyanate dimerization. The two excess electrons that are supplied from the uretdion ligand lead to the observed physiologically inverse electron transfer from heme a3 to heme a.

DNA-Driven Nanoparticle Assemblies for Biosensing and Bioimaging

DNA molecules with superior flexibility, affinity and programmability have garnered considerable attention for the controllable assembly of nanoparticles (NPs). By controlling the density, length and sequences of DNA on NPs, the configuration of NP assemblies can be rationally designed. The specific recognition of DNA enables changes to be made to the spatial structures of NP assemblies, resulting in differences in tailorable optical signals. Comprehensive information on the fabrication of DNA-driven NP assemblies would be beneficial for their application in biosensing and bioimaging. This review analyzes the progress of DNA-driven NP assemblies, and discusses the tunable configurations determined by the structural parameters of DNA skeletons. The collective optical properties, such as chirality, fluorescence and surface enhanced Raman resonance (SERS), etc., of DNA-driven NP assemblies are explored, and engineered tailorable optical properties of these spatial structures are achieved. We discuss the development of DNA-directed NP assemblies for the quantification of DNA, toxins, and heavy metal ions, and demonstrate their potential application in the biosensing and bioimaging of tumor markers, RNA, living metal ions and phototherapeutics. We hihghlight possible challenges in the development of DNA-driven NP assemblies, and further direct potential prospects in the practical applications of macroscopical materials and photonic devices.

Immobilized dye-decolorizing peroxidase (DyP) and directed evolution variants for hydrogen peroxide biosensing

Immobilized dye-decolorizing peroxidase from Pseudomonas putida MET94 (PpDyP) and three variants generated by directed evolution (DE) are studied aiming at the design of a biosensor for H2O2 detection. Structural properties of the enzymes in solution and immobilized state are addressed by resonance Raman (RR) and surface enhanced RR (SERR) spectroscopy, and the electrocatalytic properties are analyzed by electrochemistry. The wild-type (wt) and 29E4 variant (with E188K and H125Y mutations) represent excellent candidates for development of H2O2 biosensors, since they exhibit a good dynamic response range (1–200 μM H2O2), short response times (2 s) and a superior sensitivity (1.3–1.4 A⋅M−1⋅cm−2) for H2O2, as well as selectivity and long term stability. In contrast to the solution state, 6E10 (with E188K, A142V and H125Y mutations) and 25F6 (with E188K, A142V, H125Y and G129D mutations) variants display much lower activity and are inhibited by high concentrations of H2O2 upon adsorption on an electrode. In terms of sensitivity, the bioelectrodes employing wt PpDyP and 29E4 variant outperform HRP based counterparts reported in the literature by 1–4 orders of magnitude. We propose the development of wt or 29E4 PpDyP based biosensor as a valuable alternative to devices that rely on peroxidases.

Redox induced protonation of heme propionates in cytochrome c oxidase: Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations

Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3 have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a3 propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3 only protonation of one heme a3 propionates was observed. Our data supports the postulated scenario where the heme a3 propionates are involved in the proton pathway.

Immobilized cytochrome c bound to cardiolipin exhibits peculiar oxidation state-dependent axial heme ligation and catalytically reduces dioxygen.

Mitochondrial cytochrome c (cytc) plays an important role in programmed cell death upon binding to cardiolipin (CL), a negatively charged phospholipid of the inner mitochondrial membrane (IMM). Although this binding has been thoroughly investigated in solution, little is known on the nature and reactivity of the adduct (cytc-CL) immobilized at IMM. In this work, we have studied electrochemically cytc-CL immobilized on a hydrophobic self-assembled monolayer (SAM) of decane-1-thiol. This construct would reproduce the motional restriction and the nonpolar environment experienced by cytc-CL at IMM. Surface-enhanced resonance Raman (SERR) studies allowed the axial heme iron ligands to be identified, which were found to be oxidation state dependent and differ from those of cytc-CL in solution. In particular, immobilized cytc-CL experiences an equilibrium between a low-spin (LS) 6c His/His and a high-spin (HS) 5c His/- coordination states. The former prevails in the oxidized and the latter in the reduced form. Axial coordination of the ferric heme thus differs from the (LS) 6c His/Lys and (LS) 6c His/OH(-) states observed in solution. Moreover, a relevant finding is that the immobilized ferrous cytc-CL is able to catalytically reduce dioxygen, likely to superoxide ion. These findings indicate that restriction of motional freedom due to interaction with the membrane is an additional factor playing in the mechanism of cytc unfolding and cytc-mediated peroxidation functional to the apoptosis cascade.

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.

Potential‐Dependent Surface‐Enhanced Resonance Raman Spectroscopy at Nanostructured TiO2: A Case Study on Cytochrome b5

Nanostructured titanium dioxide (TiO2 ) electrodes, prepared by anodization of titanium, are employed to probe the electron-transfer process of cytochrome b5 (cyt b5 ) by surface-enhanced resonance Raman (SERR) spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2 , achieved by raising the anodization voltage from 10 to 20 V, the enhancement factor increases from 2.4 to 8.6, which is rationalized by calculations of the electric field enhancement. Cyt b5 is immobilized on TiO2 under preservation of its native structure but it displays a non-ideal redox behavior due to the limited conductivity of the electrode material. The electron-transfer efficiency which depends on the crystalline phase of TiO2 has to be improved by appropriate doping for applications in bioelectrochemistry.

Magnetic Silver Hybrid Nanoparticles for Surface-Enhanced Resonance Raman Spectroscopic Detection and Decontamination of Small Toxic Molecules

Magnetic hybrid assemblies of Ag and Fe3O4 nanoparticles with biocompatibly immobilized myoglobin (Mb) were designed to detect and capture toxic targets (NO2-, CN-, and H2O2). Mb was covalently attached to chitosan-coated magnetic silver hybrid nanoparticles (M-Ag-C) via glutaraldehyde that serves as a linker for the amine groups of Mb and chitosan. As verified by surface-enhanced resonance Raman (SERR) spectroscopy, this immobilization strategy preserves the native structure of the bound Mb as well as the binding affinity for small molecules. On the basis of characteristic spectral markers, binding of NO2-, CN-, and H2O2 could be monitored and quantified, demonstrating the high sensitivity of this approach with detection limits of 1 nM for nitrite, 0.2 μM for cyanide, and 10 nM for H2O2. Owing to the magnetic properties, these particles were collected by an external magnet to achieve an efficient decontamination of the solutions as demonstrated by SERR spectroscopy. Thus, the present approach combines the highly sensitive analytical potential of SERR spectroscopy with an easy approach for decontamination of aqueous solutions with potential applications in food and in environmental and medical safety control.

Analyzing the catalytic processes of immobilized redox enzymes by vibrational spectroscopies

Analyzing the structure and function of redox enzymes attached to electrodes is a central challenge in many fields of fundamental and applied life science. Electrochemical techniques such as cyclic voltammetry which are routinely used do not provide insight into the molecular structure and reaction mechanisms of the immobilized proteins. Surface-enhanced infrared absorption (SEIRA) and surface-enhanced resonance Raman (SERR) spectroscopy may fill this gap, if nanostructured Au or Ag are used as conductive support materials. In this account, we will first outline the principles of the methodology including a description of the most important strategies for biocompatible protein immobilization. Subsequently, we will critically review SERR and SEIRA spectroscopic approaches to characterize the protein and active site structure of the immobilized enzymes. Special emphasis is laid on the combination of surface-enhanced vibrational spectroscopies with electrochemical methods to analyze equilibria and dynamics of the interfacial redox processes. Finally, we will assess the potential of SERR and SEIRA spectroscopy for in situ investigations on the basis of the first promising studies on human sulfite oxidase and hydrogenases under turnover conditions.

Electron transfer dynamics of Rhodothermus marinus caa3 cytochrome c domains on biomimetic films

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.

Induced SER‐Activity in Nanostructured Ag–Silica–Au Supports via Long‐Range Plasmon Coupling

AbstractA novel Ag–silica–Au hybrid device is developed that displays a long‐range plasmon transfer of Ag to Au leading to enhanced Raman scattering of molecules largely separated from the optically excited Ag surface. A nanoscopically rough Ag surface is coated by a silica spacer of variable thickness from ∼1 to 21 nm and a thin Au film of ∼25 nm thickness. The outer Au surface is further functionalized by a self‐assembled monolayer (SAM) for electrostatic binding of the heme protein cytochrome c (Cyt c) that serves as a Raman probe and model enzyme. High‐quality surface‐enhanced resonance Raman (SERR) spectra are obtained with 413 nm excitation, demonstrating that the enhancement results exclusively from excitation of Ag surface plasmons. The enhancement factor is estimated to be 2 × 104–8 × 103 for a separation of Cyt c from the Ag surface by 28–47 nm, corresponding to an attenuation of the enhancement by a factor of only 2–6 compared to Cyt c adsorbed directly on a SAM‐coated Ag electrode. Upon immobilization of Cyt c on the functionalized Ag–silica–Au device, the native structure and redox properties are preserved as demonstrated by time‐ and potential‐dependent SERR spectroscopy.

Thermal Fluctuations Determine the Electron‐Transfer Rates of Cytochrome c in Electrostatic and Covalent Complexes

The heterogeneous electron-transfer (ET) reaction of cytochrome c (Cyt-c) electrostatically or covalently immobilized on electrodes coated with self-assembled monolayers (SAMs) of omega-functionalized alkanethiols is analyzed by surface-enhanced resonance Raman (SERR) spectroscopy and molecular dynamics (MD) simulations. Electrostatically bound Cyt-c on pure carboxyl-terminated and mixed carboxyl/hydroxyl-terminated SAMs reveals the same distance dependence of the rate constants, that is, electron tunneling at long distances and a regime controlled by the protein orientational distribution and dynamics that leads to a nearly distance-independent rate constant at short distances. Qualitatively, the same behavior is found for covalently bound Cyt-c, although the apparent ET rates in the plateau region are lower since protein mobility is restricted due to formation of amide bonds between the protein and the SAM. The experimental findings are consistent with the results of MD simulations indicating that thermal fluctuations of the protein and interfacial solvent molecules can effectively modulate the electron tunneling probability.

Selective Detection of HbA1c Using Surface Enhanced Resonance Raman Spectroscopy

In the current work, we report on selective detection of HbA1c, a marker for glycemic control in diabetic patients, using surface enhanced resonance raman spectroscopy (SERRS). We found a characteristic band around 770-830 cm(-1) in the SERRS spectrum of HbA1c which was not present in the SERRS spectrum of HbA. To examine the contribution of glucosyl moiety to the characteristic SERRS band of HbA1c, we investigated SERRS spectra for nonenzymatically glycosylated HbA. We found that the SERRS spectral features are essentially identical for both HbA1c and nonenzymatically glycosylated HbA. Furthermore, addition of HbA into colloidal solution of silver nanoparticles (Ag NPs) resulted in the formation of large aggregates of Ag NPs and subsequent sedimentation. On the other hand, aggregation of Ag NPs was considerably low in the case of HbA1c. The differential effect of HbA and HbA1c on colloidal solution of Ag NPs, probably due to their difference in hydrophilicity, enabled us to separate them in a mixture. The separation was characterized by electrophoresis and SERRS analysis. Thus, colloidal solution of Ag NPs and SERRS would be a promising tool for the selective detection of HbA1c.

Electrosynthesis of SER-Active Silver Nanopillar Electrode Arrays

Here we report a one step electrochemical method for the deposition of pillar-like silver nanostructures on graphite plate electrodes. The devices were tested as SER-active electrode arrays using covalently attached hemin. The nanopillar arrays provide reproducible surface enhanced resonance Raman (SERR) signals within 6% standard deviation and Raman enhancement factors of 1 × 105. They also show good electrochemical performance, as verified by potential-dependent SERR spectroscopy and cyclic voltammetry. Furthermore, the hemin-coated arrays display mediatorless electrocatalytic activity toward NO2−. Thus, the Ag nanopillar arrays present good prospects for their application in sensing devices and for SER/SERR based spectroelectrochemical applications in general.

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.

Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach

Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(-1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.