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Peter Hildebrandt – TU Berlin Electric field effects on the protein and electron transfer dynamics of immobilized heme proteins – vibrational spectroscopic approaches Acknowledgements People involved: Anja Kranich, TU Berlin Khoa H. Ly Murat Sezer Ingo Zebger Inez Weidinger Jiu-Ju Feng Diego Millo Gal Schkolnik Tillmann Utesch Maria Andrea Mroginski Nattawadee Wisitruangsakul, Bangkok Lisa Lorenz, Frankfurt Daniel H. Murgida, Buenos Aires Hainer Wackerbarth, Göttingen Financial support: Deutsche Forschungsgemeinschaft Sfb498, Cluster of Excellence „Unicat“ Collaborations Laszlo Zimanyi, Szeged Andreas Knorr, Berlin Content I. Why studying proteins at interfaces? II. Vibrational spectroscopies III. How to probe electric field effects? IV. What do we learn from SERR and SEIRA spectroscopy of biomimetic systems? V. Interfacial processes of cytochrome c 1. Dynamics of protein orientation and electron transfer 2. Dynamics of redox-linked protein structural changes 3. Electric field dependence of electron transfer mechanism and dynamics 4. Electric field dependence of electron tunneling 5. Electric field dependence of protein dynamics 6. Electric field dependence of the interfacial electron transfer process VI. Cytochrome c in apoptosis Why studying proteins at interfaces? Why studying proteins at interfaces? Most biological processes occur at interfaces: electron transfer sequence in the respiratory chain High electric fields (up to 109 V/m) at the reaction sites due to the ¾ transmembrane potential ¾ surface potential ¾ dipole potential Possible effects on the 9 Protein/cofactor structure and interactions 9 Alignment and induction of dipoles 9 Alteration of pKa´s 9 Activation energies 9 Energy levels Why studying proteins at interfaces? Electron transfer occurs within an electrostatic complex cytochrome c e- cytochrome c oxidase Vibrational spectroscopies Vibrational Spectroscopies Raman and IR spectroscopies probe molecular structures… ……..but are inherently insensitive! Requirements for appropriate methodology: ¾ High sensitivity ¾ High selectivity ¾ Adaptation to specific environments ¾ Adequate time resolution ¾ Molecular structure information Resonance Raman: probes exclusively the vibrational bands of the cofactor due to selective enhancement of upon resonant excitation IR difference: probes selectively the vibrational bands of those parts of the macromolecule that undergo reaction-induced changes Vibrational Spectroscopies Sensitivity and selectivity of RR and IR difference spectroscopy is not sufficient for probing proteins (sub-)monolayers ¾ Surface enhanced Raman spectroscopy: SERR ¾ Surface enhanced infrared absorption spectroscopy: SEIRA Dipole-dipole coupling of the radiation field with the surface plasmons: ¾ SEIRA: enhancement factor of ca. 102 ¾ SER: enhancement factor of ca. 105 Requirements: Nano-scale roughness Match of plasmon resonance and incident wavelength Restricted to Au and Ag Advantages: Vibrational spectroscopy of monolayers in proximity to metal surfaces Vibrational Spectroscopies Surface enhanced vibrational spectroscopy SERRS (= SER + RR): selectively probes the chromophores solely of the adsorbed molecules SEIRA (difference mode): Selectively probes the structural/orientational changes adsorbed molecules/molecular building blocks Electrodes as SER/SEIRA-active materials: probing potential-dependent processes Combination with the potential-jump technique: time-resolved SERR time-resolved SEIRA (rapid scan, step scan) Vibrational Spectroscopies Limitations of SERRS/SEIRA ...... Direct contact of proteins with metal surfaces may cause denaturation Vibrational Spectroscopies Limitations of SERRS/SEIRA and how to overcome them Direct contact of proteins with metal surfaces may cause denaturation Biocomaptible coating of metal surfaces by (e.g.) self-assembled monolayers of lipid analogues (functionalised mercaptanes) Vibrational Spectroscopies Limitations of SERRS ...... Wavelength dependence of the enhancement effect polydisperse nanostructures Ag Preferred spectral range for studying cofactor-protein complexes for combining RR and SER Au But: Au displays a much broader electrochemical potential range and is chemically more stable Vibrational Spectroscopies Limitations of SERRS and how to overcome them Layered hybrid devices Electrochemical roughening of Ag electrode Coating by a dielectric layer (SAM, SiO2) Deposition of a metal film or semiconductor film (Au, Pt, TiO2) 0.3 V -0.3 V 75 nm Functionalisation of the outer metal layer for protein binding C-O Metal film: ca. 20 nm dielectric layer: 2 – 30 nm Nanostructured Ag Ag Vibrational Spectroscopies Limitations of SERRS and how to overcome them Distance-dependence of the enhancement experimental theoretical CO Ag 25 20 Ag-spacer-Au Cyt 413 nm g0 15 10 Ag 5 0 0.6 0.8 1.0 r/R 1.2 1.4 How to probe electric field effects? How to probe electric field effects? Approach: Mimicking the protein-protein complex by model systems electron transfer e- eS S S S S S S S electrode SAM – membrane model electrostatic binding cytochrome c How to probe electric field effects? Quantification of the local electric field strength ω-carboxyl alkanethiol eCyt-c Parameters controlling the local electric field strength chain length (distance) - dc charge density at head groups σc electrode potential - E ionic strength - κ How to probe electric field effects? Determining the interfacial electric field strength by using the field-induced frequency shift of the CN stretching (vibrational Stark effect - VSE) Probing the VSE by SERS (Ag) and SEIRA (Au) as a function of E, dc, σc, and κ r r ⎞ 1 r ⎛ r r ~ Δ ν = ν ( E F ) − ν ( 0 ) = ⎜ Δμ ⋅ E F + E F ⋅ Δ α ⋅ E F ⎟ 2 ⎝ ⎠ How to probe electric field effects? Mapping the electric field in the SAM/protein interface & on the protein surface Cyt-c S S S S S S S S ¾ Introducing cysteine residues at different positions of the Cyt-c surface ¾ chemical modification by mercaptobenzonitrile SAM ¾ Measuring the VSE solution How to probe electric field effects? K39C r F = −1.5 ⋅ 108 V / m Δν = ν SEIRA − ν IR r r r r = Δμ ⋅ F ( x) = Δμ ⋅ F ( x) ⋅ cos(α ) experiment ⎛ Δν ( K 39C ) ⎞ ⎜⎜ ⎟⎟ = 0.4 ⎝ Δν ( K 8C ) ⎠ exp MD simulations ⎛ Δν ( K 39C ) ⎞ ⎜⎜ ⎟⎟ = 0.24 ⎝ Δν ( K 8C ) ⎠ calc r F = −6.2 ⋅108V / m K8C How to probe electric field effects? Determining the electric field from the VSE and by theoretical calculations Electrostatic calculations Molecular dynamics simulations and QM/MM hybrid methods SAM solution metaldependent Electric field strength increases with ¾ decreasing distance from the electrode ¾ with increasing potential difference with respect to the potential of zero-charge Epzc What do we learn from SERR and SEIRA spectroscopy of biomimetic systems? What do we learn from SERR and SEIRA spectroscopy? electrode SAM cytochrome c S S S S S S S S Electron transfer SERR: Excitation in resonance with the Soret transition of the heme: Probing oxidation marker bands What do we learn from SERR and SEIRA spectroscopy? electrode SAM Stationary SERR spectra cytochrome c S S S S S S S S Electron transfer equilibria Met Met Fe2+ Fe3+ His His What do we learn from SERR and SEIRA spectroscopy? electrode SAM cytochrome c S S S S S S S S Potential jump technique Electron transfer dynamics Met Met Fe2+ Fe3+ His His Time-resolved SERR spectra one-step relaxation process What do we learn from SERR and SEIRA spectroscopy? electrode SAM cytochrome c S S S S S S S S Redox-linked protein structural changes SEIRA: Difference spectra „oxidised“ minus „reduced“ Monitoring redox-sensitive amide bands Time-resolved SEIRA: (step & rapid scan) What do we learn from SERR and SEIRA spectroscopy? S S S S S S S S S S S S S S S S Protein re-orientation Differential enhancement of totally symmetric (A1g) and nontotally symmetric modes (B1g) depending on the heme orientation What do we learn from SERR and SEIRA spectroscopy? electrode SAM cytochrome c 1361 B1Red S S S S S S S S 1491 Met 1591 2+ His 1371 Met Electric-field induced structural changes B1Ox 1500 1581 3+ His 1369 3+ coordination and spin state changes of the heme (SERR) Met80 B2Ox HS 1577 1487 His 1374 His 3+ His18 B2Ox LS 1504 1586 His and protein structural changes (SEIRA) His26 1300 1350 1400 1450 1500 1550 1600 1650 His33 -1 ν / cm What do we learn from SERR and SEIRA spectroscopy? Stationary potential-dependent SERR and SEIRA spectroscopy: ¾ Heme structure ¾ Conformational and redox equilibria ¾ Orientational distribution Time-resolved potential-jump SERR and SEIRA spectroscopy: ¾ electron transfer ¾ protein and cofactor dynamics ¾ re-orientation Interfacial processes of cytochrome c 1. Dynamics of protein orientation and electron transfer Re-orientation Protein re-orientation: intensity ratio of modes of different symmetry Heme reduction: oxidation marker bands heme reduction S S S S S S S S Cyt c at C-15 SAM: Electron tunnelling is rate limiting 1. Dynamics of protein orientation and electron transfer Protein re-orientation: intensity ratio of modes of different symmetry Re-orientation Heme reduction: oxidation marker bands heme reduction S S S S S S S S Cyt c at C-5 SAM: Protein dynamics is rate limiting 2. Dynamics of redox-linked protein structural changes β-turn III aa 67-70 Protein structural changes monitored by SEIRA spectroscopy Lys 86,87 Lys 72,73 heme Amide I band changes of the β-turn III segment 67-70 occur simultaneously with electron transfer 3. Electric-field dependence of electron transfer mechanism and dynamics Re-orientation slows down with distance and increasing field strength: Electron tunneling k ET = A ⋅ exp(− dβ ) 3. Electric-field dependence of electron transfer mechanism and dynamics Change of ET mechanism with increasing the electric field strength Low electric field: High electric field: Electron tunnelling rate-limiting Gated electron transfer 4. Electric-field dependence of electron tunnelling Electron tunneling regime at C-15 SAMs: TR-SERR data Marcus-DOS ⎛ λ + eη erfc ⎜ ⎜ 4λ k T k ET (η ) B ⎝ = k ET (0) ⎛ λ erfc ⎜ ⎜ 4λ k T B ⎝ ⎞ ⎟ ⎟ ⎠ ⎞ ⎟ ⎟ ⎠ Reorganisation energy λ = 0.22 eV 4. Electric-field dependence of electron tunnelling Electron tunneling regime at C-15 SAMs on different electrodes SERR SERR SEIRA Metal-specific overpotential dependencies 4. Electric-field dependence of electron tunnelling Electric-field dependent retardation of electron tunnelling different for Au and Ag ) + e[a ( ⎛ λ + e E − E0 erfc ⎜ ⎜ k ET (E ) ⎝ = k ET (0) ⎛ λ + e a0 erfc ⎜ ⎜ ⎝ [ ] + a1 (E − E pzc ) ⎞ ⎟ ⎟ 4λ k B T ⎠ + a1 E 0 − E pzc ⎞ ⎟ ⎟ 4λ k B T ⎠ ( 0 )] 5. Electric-field dependence of protein dynamics Low electric field: High electric field: Electron tunnelling rate-limiting Gated electron transfer 5. Electric-field dependence of protein dynamics Molecular dynamics simulation and electron coupling analysis Low affinity binding site – strong electronic coupling – fast ET High affinity binding site – weak electronic coupling – slow ET 5. Electric-field dependence of protein dynamics General description: orientational distribution and rate distribution Preferred orientation Optimum ET 5. Electric-field dependence of protein dynamics Electric field effect on protein dynamics: viscosity and H/D dependence H2O viscosity 1.2 cp D2O viscositycorrected KIE of 2.0 5. Electric-field dependence of protein dynamics Gating regime: Increasing electric field slows down protein reorientation, rearrangement of hydrogen bond network, and electron tunnelling S S S S S S S S e- 5. Electric-field dependence of protein dynamics Gating via protein re-orientation: a general phenomenon 8 Observed for different 6 proteins but with different 4 ln k 0 app limiting rates even at the same electric field strength 2 - 1 - 2 Ag-S-(CH2)-CO2 Cyt c Au-S-(CH2)-CO2 Cyt c 0 3 Au-S-(CH2)-CH3 Azurin 4 Au-S-(CH2)-Py Cyt c + -2 Au-S-(CH2)-NH3 5 Cyt b562 6 Au-S-(CH2)-CH3 / -OH Cyt c6 7 -4 Au-S-(CH2)-CH3 / -OH CuA 0 2 4 6 8 10 12 14 16 Number of methylenes 18 20 22 6. The interfacial redox process of cytochrome c 6. The interfacial redox process of cytochrome c oxidation of cytochrome c 6. The interfacial redox process of cytochrome c oxidation at low electric fields Fast protein structural change Electron tunnelling is rate-limiting Fast re-orientation 6. The interfacial redox process of cytochrome c oxidation at high electric fields Retardation of electron tunneling & coupled protein structural changes Protein dynamics is rate-limiting slow re-orientation 6. The interfacial redox process of cytochrome c processes at very high electric fields Major protein structural change including the protein and the heme pocket Switch from the redox to the apoptotic function Cytochrome c in apoptosis Cytochrome c in apoptosis Observations: Cyt-c catalyses peroxidation of cardiolipin Increased permeability of the inner mitochondrial membrane Cyt-c release to the cytosol Involvement of Cyt-c in caspase-dependent apoptotic pathway What makes Cyt-c act as a peroxidase? Cytochrome c in apoptosis Cyt-c at very high electric fields (electrode, lipid versicles): Conformational changes involving the dissociation of the Met ligand from the heme – state B2 ¾ very low redox potential (cannot accept electrons from complex III) ¾ strongly increased peroxidase activity Met 3+ His 3+ His Met80 His 3+ His His18 His26 His33 Cytochrome c in apoptosis Transition from the native B1 state to the state B2 corresponds to a switch from the redox to the peroxidase function This switch is electric field dependent S S S S S S S S Conclusions 1. Proteins at biomimetic interfaces: providing novel insight into molecular processes under natural reaction conditions, such as electric field effects 2. SERR/SEIRA allow disentangling interfacial redox processes in terms of • protein (re-)orientation • electron tunneling • redox-linked structural changes 3. Coated electrodes allow probing the electric field dependent switch from the redox to the apoptotic function References Textbooks 1) Siebert, F.; Hildebrandt, P. „Vibrational Spectroscopy in Life Science“, Wiley-VCH, Weinheim, 2007. Review articles 1) 2) 3) 4) 5) 6) 7) Hildebrandt, P., Feng, J. J., Kranich, A., Ly, H. K., Martí, M.; Martín, D. F.; Murgida, D. H., Paggi, D. A.; Sezer, M., Wisitruangsakul, N., Weidinger, I., D. H., Zebger, I.,:”Electron transfer of proteins at membrane models”, in SERS – Analytical, Biophysical and Life Science Applications” (Schlücker, S., Ed.), WileyVCH, in press (2010). Feng, J.J., Wang, A. J., Hildebrandt, P.: “Electron transfer of proteins at nano-biointerfaces”, in “Biocompatible Nanomaterials: Synthesis, Characterization and Applications”, chapt. 9, (Kumar, A.; Wang, S.F., Eds.), Nova Publisher, New York, in press (2010). Murgida, D. 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A., Thomsen, C., Hildebrandt, P.: „Resonance Raman spectra of β-carotene in solution and in photosystems revisited: An experimental and theoretical study”, Phys.Chem.Chem.Phys.11, 11471-11478 (2009). De Biase, P. M., Paggi, D. A., Doctorovich, F., Hildebrandt,P., Estrin, D. A., Murgida, D. H., Marti, M. A.: “Molecular Basis for the Electric Field Modulation of Cytochrome c Structure and Function”, J. Am. Chem. Soc. 131, 16248-16256 (2009). Kranich, A., Naumann, H., Molina-Heredia, F. P., Moore, H. J., Lee, T.R., Lecomte, S., de la Rosa, M. A., Hildebrandt, P., Murgida, D. H.: “Gated Electron Transfer of Cytochrome c6 at Biomimetic Interfaces: A Time-Resolved SERR Study”, Phys. Chem. Chem. Phys.11, 7390-7397 (2009). Zuo, P., Albrecht, T. Barker, P. D. Murgida, D. H., Hildebrandt, P.: “Interfacial redox processes of cytochrome b562 “, Phys. Chem. Chem. Phys 11, 7430-7436, 2009 (hot article). Paggi, D. A.; Martín, D. F.; Kranich, A.; Hildebrandt, P.; Martí, M.; Murgida, D. H.:“Computer Simulation and SERR Detection of Cytochrome c Dynamics at SAM-Coated Electrodes”, Electrochim. Acta, 54, 4963-4970 (2009). Millo, D., Ranieri, A., Gross, P., Borsari, M., Ly, K. H., Hildebrandt, P., Wuite, G. J. L., Gooijer, C., van der Zwan, G.: “The electrochemical response of cytochrome c immobilized on smooth and roughened Ag and Au surfaces chemically modified with 11-mercaptounodecanoic acid”, J. Phys. Chem. C, 113, 28612866 (2009). Feng, J. J., Gernert, U., Sezer, M., Kuhlmann, U., Murgida, D. H., David, C., Richter, M., Knorr, A., Hildebrandt, P., Weidinger, I.:”A Novel Au-Ag hybrid device for surface enhanced (resonance) Raman spectroscopy”, Nano Lett. 9, 298-303 (2009). Todorovic, S., Verissimo, A., Wisitruangsakul, N., Zebger, I., Hildebrandt, P., Pereira, M. M., Teixeira, M., Murgida, D. H.: “SERR spectroelectrochemical study of a cbb3 oxygen reductase in a biomimetic construct”, J. Phys. Chem. B 112, 16952-16959 (2008). Feng, J. J., Murgida, D. 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