Article pubs.acs.org/JPCB
Surface Enhanced Resonance Raman Spectroscopy Reveals Potential Induced Redox and Conformational Changes of Cytochrome c Oxidase on Electrodes Murat Sezer,† Patrycja Kielb,† Uwe Kuhlmann,† Hendrik Mohrmann,‡ Claudia Schulz,† Dorothea Heinrich,‡ Ramona Schlesinger,‡ Joachim Heberle,‡ and Inez M. Weidinger*,† †
Institut für Chemie PC 14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany
‡
ABSTRACT: Immobilization of Cytochrome c oxidase (CcO) on electrodes makes voltage-driven reduction of oxygen to water possible. Efficient catalytic turnover in CcO/ electrode systems is, however, often observed at large overpotentials that cannot be rationalized by the redox properties of the enzyme itself. To understand the structural basis for this observation, CcO was electrostatically adsorbed on amino-functionalized Ag electrodes, and the redox transitions of heme a and a3 were monitored via surface enhanced resonance Raman spectroscopy (SERRS) as a function of applied potential. Under completely anaerobic conditions, the reduction of heme a3 could be seen at potentials close to those measured in solution indicating an intact catalytic center. However, in the immobilized state, a new non-native heme species was observed that exhibited a redox potential much more negative than measured for the native hemes. Analysis of the high and low frequency SERR spectra indicated that this new species is formed from heme a upon axial loss of one histidine ligand. It is concluded that the formation of the non-native heme a species alters the potential-dependent electron supply to the catalytic reaction and, thus, can have a impact on the applicability of this enzyme in biofuel cells.
■
INTRODUCTION Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water in the respiratory chain of living organisms. This reduction pathway requires four electrons and four protons that have to be supplied to the catalytic binuclear center (BNC) in time. In nature, four Cyt c molecules deliver one by one the electrons, while the protons are taken up from the inner side of the membrane.1,2 Understanding the mechanism of the proton and electron transfer induced catalytic reaction in CcO is important, particularly for the design of efficient oxygen reducing catalysts. It is, however, challenging to mimic the highly directed four electron reduction process that passes the four redox centers CuA−heme a−heme a3−CuB of CcO. Reduction by chemical reducing agents creates the fully reduced enzyme, but no directional electron transfer (ET) is present in this approach as the reducing agent interacts with all redox centers simultaneously. For directed ET and timeresolved analysis, either photosensitive electron donors have been attached to the enzyme3 or the fully reduced enzyme was first inhibited by CO and the oxygen splitting reaction was initiated by a laser flash.4,5 While for these approaches directional ET is given, only one-electron and two-electron partial reactions could be studied. Furthermore, the electrons are already within the enzyme when the reaction is triggered and are not sequentially delivered on a time scale that matches the one under physiological conditions. This is a very important point as it could be shown for biomimetic CcO model © 2015 American Chemical Society
compounds that the intermolecular electron transfer rate from the external redox partner to the catalytic center determines whether the reaction pathway proceeds via two-electron reduction to generate H2O2 or via four-electron reduction to yield water.6 One possibility to sequentially introduce four electrons one by one to CcO is to immobilize the enzyme on solid electrodes that act as source or sink for electrons. In this approach, not only directionality is given, but also the ET rate can be tuned upon variation of binding type and applied potential. Such solid-supported CcO/electrode systems are furthermore highly interesting with respect to their applicability as cathode material for biofuel cells. Regarding the latter, it is interesting that CcO, although it is known as one of the best oxygen reducing catalysts, has not been used in biofuel cells so far as high overpotentials are needed for effective oxygen reduction.7,8 Surface-enhanced resonance Raman spectroscopy (SERRS) provides unique insight into the structural changes of hemebased enzymes attached to electrodes.9,10 Although CcO has been extensively studied by resonance Raman spectroscopy in solution, only two studies have been published that investigated immobilized CcO via SERRS.11,12 In these works, it has been observed that heme a could be reduced by the electrode while Received: April 2, 2015 Revised: June 8, 2015 Published: July 2, 2015 9586
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
Article
The Journal of Physical Chemistry B
Figure 1. (A and B) UV−vis spectra of CcO in a spectro-electrochemical transmission cell recorded under different applied potentials in the absence (A) and presence (B) of oxygen. Spectra in (A) are shown for applied potentials +100, 0, −50, −75, −100, and −175 mV. Spectra in (B) are shown for applied potentials +300, +200, +100, 0, −100, and −200 mV. The absorbance was normalized to its maximum value at 444 nm. (C and D) Normalized absorbance of the 444 nm band as a function of potential in the absence (C) and presence (D) of oxygen. The continuous lines represent Boltzmann fits to the data.
heme a3 remained oxidized at potentials far below its redox potential in solution. The authors attributed this effect to a drastically slowed down intramolecular electron transfer between heme a and heme a3 under high electric fields, present close to the electrode surface, in combination with traces of remaining oxygen in the SERRS spectro-electrochemical cell. The latter led to constant turnover conditions, which interfered with the redox process. To eliminate the influence of catalytic oxygen reduction in the four electron transfer reaction of CcO, we have therefore developed a SERRS setup in an anaerobic box which allows us to study the structural changes of CcO as a function of potential under complete anaerobic conditions. Here, we present the first spectroscopic results of a complete electrochemical redox titration of CcO on electrodes and give insight into structural changes of the heme environment upon immobilization.
CcO with a C-terminal 6×His tag, was cultivated under aerobic conditions, and the enzyme was mainly purified as previously described.13 The main culture was grown on Sistrom’s succinate-based minimal medium,14 supplemented with streptomycin (50 μg/mL), spectomycin (50 μg/mL), and tetracycline (1 μg/mL) at 30 °C for 36 h with vigorous shaking at 120 rpm. The cells were harvested by centrifugation at 9000g for 30 min and resuspended to a concentration of 0.5 g cells/mL with 50 mM phosphate buffer, 50 mM MgCl2,, pH 8. For purification, the cells were disrupted by two passages through a HTU-DIGI-F Press (G. Heinemann, Ultraschall- and Labortechnik) at 20 000 psi pressure with DNase and 1 mM PMSF. The membranes with CcO were harvested at 244 000g for 2 h and resuspended in 2% n-dodecyl-β-D-maltoside, 50 mM phosphate buffer, 1 mM PMSF, pH 8 (1 g membrane pellet/5 mL buffer). To solubilize CcO, the solution was vigorously stirred overnight at 4 °C. To remove insoluble material, the extract was clarified by centrifugation at 244 000g for 2 h and the protein was subsequently purified via the Histag on a Ni-NTA affinity column (HisTrap FF crude, GE Healthcare).
■
EXPERIMENTAL SECTION Homologue Expression and Purification of CcO from Rhodobacter sphaeroides. The R. sphaeroides strain JS100, containing the genetic information on subunit I of the aa3-type 9587
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
Article
The Journal of Physical Chemistry B
Figure 2. (Left) Immobilization of CcO on NH2/OH terminated SAM-coated electrodes. (Right) Setup of the Raman experiments under anaerobic conditions. S, spectrometer; M, mirror; T, brass tube; Q, plane quartz window; BR, butyl rubber bridge; O, objective; E, spectro-electrochemical cell; W, working electrode; R, reference electrode; C, counter electrode; B, box; P, potentiostat. The laser beam is depicted in blue.
flexibility that was required for proper alignment. The objective was situated at the end of the brass tube in front of the spectroelectrochemical cell inside the box. The oxygen concentration was monitored constantly and was found to remain stable below 20 ppm. Data Evaluation. Intensity (I) vs potential (E) plots were fitted with the Boltzmann equation (I = I1 + (I1 − I2)/(1 + exp ((E − E0)/dE). Comparison with the Nernst equation shows that E0 can be set equal to the redox potential.
UV−Vis Spectroscopy. UV−vis spectro-electrochemical measurements in an anaerobic box were done using an OTTLE (optically transparent thin layer electrochemical) cell15,16 connected to an UV−vis spectrometer (Ocean Optics USB 2000) via fiber optics. In these experiments, a solution containing ca. 230 μM of CcO in the presence of mediators was used. UV−vis spectro-electrochemical measurements in the presence of oxygen were performed in a special quartz cuvette with 2 mm optical path length, under constant Ar flow. The CcO concentration was 20 μM in 10 mM PB at pH 8.0, containing 0.1% (w/v) β-DM, 0.1 M NaCl. In both setups, 20 μM of each of the following mediators were added: ferrocenylmethyl-trimethylammonium (+460 mV), ferrocene (+190 mV), N,N-dimethyl-p-phenylenediamin (+160 mV), 1,1-dimethylferrocene (+130 mV), N,N,N,N-tetramethyl-p-phenylenediamin (+60 mV), 2,6-dichloroindophenol (+10 mV), phenazinemethosulfate (−150 mV), 1,4-naphtochinone (−170 mV), methylene blue (0 mV), toluidene blue (−240 mV), indigocarmine (−330 mV), anthrachinone-2-sulfonate (−430 mV), phenosafranine (−460 mV), safranine T (−500 mV), deiquat (−350 mV). Spectra were recorded after ca. 2 h or 30 min equilibration time, for both cells, respectively, at each potential. A transparent gold mesh coated with a mixed 3mercaptopropionic acid/cysteamine (1:1 M/M) SAM, a platinum wire, and a Ag/AgCl (3 M KCl) was used as working, counter, and reference electrode, respectively. Prior to incubation into the SAM solution, the gold mesh was cleaned by repetitive CV in 0.1 M sulfuric acid in the potential window between +0.1 and +1.4 V. Raman Experiments. RR and SERRS measurements were performed with the 413 nm laser line of a Krypton ion laser (Coherent Innova 300 c) or the 442 nm line of a HeCd laser (VM-TIM, HCL-100). Spectra were recorded in backscattering mode using a confocal Raman spectrometer (LabRam HR-800, Jobin Yvon). The laser beam was focused onto the sample by a 20× objective, and the scattered light was collected by the same objective. The laser power on the sample was 200 μW in the SERR measurements to avoid photoreduction. Accumulation time was 30 s (20 cycles) for each spectrum. In the anaerobic SERRS measurements, the laser beam was aligned inside a brass tube that consists of two screw-mountable parts and extended into an anaerobic box through a circular hole at the wall of the box. The tube was attached to the box via a bridge made of butyl rubber. The gastight rubber material provides positional
■
RESULTS AND DISCUSSION First, a redox titration of CcO from R. sphaeroides in solution was performed using the UV−vis spectro-electrochemical transmission cell described in the Experimental Section. To exclude any influence of the oxygen reaction on the redox transition, the experiment was performed in an anaerobic box. The absorbance maximum at 444 nm in the UV−vis spectrum corresponds to the ferrous hemes a and a3, and its intensity was monitored to study the potential-dependent reduction of the heme cofactors (Figure 1C). Two redox potentials at −15 ± 12 and +205 ± 5 mV could be identified this way, which we assign to the redox transitions of heme a3 and heme a, respectively, according to literature.16 In contrast, similar measurements performed outside the anaerobic box revealed only one redox transition around −67 ± 2 mV (Figure 1D). In the next step, CcO was attached to solid electrodes and its redox behavior was analyzed via SERRS. A cylindrical Ag electrode was electrochemically roughened to create a SERS active support. Subsequently, the electrode was functionalized with a 3:1 mixture of 8-aminothiophenol (3 mM, C8-NH2) and 6-mercaptohexanol (1 mM, C6-OH) self-assembled monolayers to improve the biocompatibility of the electrode surface. Last, the electrode was incubated in a solution containing 0.2 μM CcO, 10 mM Na-PBS (pH 8.0), and 0.1% (w/w) β-DM. The amino termination of the SAM mimics the lysine rich binding site of CcOs natural redox partner cytochrome c (Cyt c) such that we expect CcO to bind electrostatically to the SAM in a similar manner as it does to to Cyt c, i.e., with the CuA containing site facing the electrode (Figure 2). The CcO functionalized electrode was inserted into a homemade spectro-electrochemical cell described previously.17 To investigate the redox properties of immobilized CcO without interference of the oxygen reduction reaction, a new 9588
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
Article
The Journal of Physical Chemistry B optical setup was developed that allows SERR spectroscopic measurements in an oxygen free environment. A detailed setup is depicted in Figure 2. SERR spectra of immobilized CcO were recorded under resonance conditions with 413 nm laser excitation. The potential-dependent spectra are depicted in Figure 3 and are compared to RR spectra in solution.
Figure 4. (Left and right) SERR spectra of CcO at 442 nm excitation for different applied potentials. (Middle) Intensity of the band at 1366 cm−1 (filled spheres) and 1663 cm−1 (filled squares) as a function of potential. The continuous lines represent Boltzmann fits to the data.
varied from experiment to experiment. While its contribution was only minor in the spectra shown in Figure 3, it was dominating at other times the spectral pattern in the ν3 region as shown in Figure 5 (left side, trace b). A potential-dependent Figure 3. From top to bottom: RR spectrum of CcO in solution (RR ox), (SE)RR spectra of CcO at different applied potentials on SAM coated Ag electrodes. RR spectrum of CcO in solution in the presence of an reducing agent (RR red). The SERR spectra were recorded inside (A) and outside (B) the anaerobic box.
Reduction of both hemes is monitored from the downshift of the frequency of the ν4 band from 1372 to 1356 cm−1, while the bands at 1663 and 1673 cm−1 belong to the vibration of the formyl group of reduced/oxidized heme a3, exclusively. It is evident that under strictly anaerobic conditions the SERR spectrum of CcO at −500 mV resembles the reduced RR spectrum. In particular, the band at 1673 cm−1 is entirely shifted to 1663 cm−1. Thus, we conclude that heme a3 is fully reduced by the electrode. This is different from SERRS measurements performed outside the box (Figure 3B). Here, almost no contribution of the 1663 cm−1 band could be seen in the probed potential range, leading to the conclusion that heme a3 remains almost completely oxidized even at very negative potentials if traces of oxygen are present in the experimental setup. SERRS measurements using 442 nm laser excitation selectively enhance the reduced CcO species and were used to monitor the potential-dependent heme reduction. Plotting the intensity of the 1663 cm−1 band versus applied potential yielded a sharp redox transition of heme a3 with a midpoint potential of E0 = +20 ± 10 mV (Figure 4). This midpoint potential is slightly more positive than the one measured in solution. This result can be explained by the positive charge of the SAM monolayer that increases the local potential at the heme site with respect to the applied potential.18 The ν4 band could not be satisfactorily fitted by two bands at 1354 and 1372 cm−1. Therefore, a third band with a maximum at 1366 cm−1 was included. This band showed an interesting redox behavior: One redox transition occurred similar to the 1663 cm−1 band with a redox potential at +20 mV. However, a second redox transition is evident at around −250 ± 30 mV that is not associated with one of the native hemes. Additionally a band at 1490 cm−1 could be detected exclusively in the SERR spectra. The intensity of this band
Figure 5. (a) RR spectrum of CcO in solution and (b) SERR spectrum of CcO on a SAM coated Ag electrode at open circuit potential. (Left) λexc = 413 nm. (Right) λexc = 442 nm.
redox titration showed a decrease of this band at more negative potentials (Figure 6). Therefore, it is assigned to an oxidized heme species with a redox potential of −306 ± 20 mV. This very negative redox potential is similar to the one that has been observed for the 1366 cm−1 band, and we conclude that both bands belong to a non-native heme species that is formed upon immobilization. The ν3 region is generally considered to be sensitive to the spin state of the heme. Upon comparison to Raman analysis of other heme complexes and proteins,19,20 it is therefore assumed that the band is indicative to the oxidized state of a high spin heme. If the 1490 cm−1 band would belong to a non-native heme a3 species, one should observe intensity changes of the formyl band at 1663 cm−1 around −300 mV. (Note that the position of this band is not altered by changes in axial heme ligation.21) However, no changes in intensity of the 1663 cm−1 band were observed at potentials below −100 mV. Thus, we exclude that the non-native species is related to heme a3. As a consequence of this conclusion, a fraction of heme a must have lost one or both of its axial Histidine ligands in the immobilized state. 9589
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
Article
The Journal of Physical Chemistry B
but also in this case, an increase of the 1356 cm−1 band is observed at potentials below −300 mV indicating the presence of a heme species with the same spectral pattern in the ν4 region as the native heme a, but a much more negative redox potential. We conclude from this observation that the contribution of the 5c heme a species is significant even in cases where the 1490 cm−1 band is hardly appearing in the spectrum. Loss of an axial ligand often occurs when the enzyme becomes denatured. However, the native redox potential of heme a3 point to an intact catalytic center and therefore makes a complete denaturation of the enzyme on the electrode not much likely. An electric field dependent change in coordination state has been observed for Cyt c that is accompanied by a strong negative shift of the redox potential.24 Most likely a similar transition is happening with heme a in CcO when exposed to high electric fields in the vicinity of an electrode surface. The very negative redox potential of this non-native heme a is a barrier for the electron transfer pathway to the BNC and further increases the intrinsically high overpotential needed for oxygen reduction in CcO based biofuel cells. In Cyt c, the change in heme coordination has a biological function as it changes its role from being an electron transfer protein to playing a role in cell apoptosis.25 Whether the change in coordination state of heme a of CcO has a biological function as well must be evaluated in further studies.
Figure 6. Intensity of the 1490 cm−1 band as a function of potential. (λexc = 413 nm). The continuous line represents a Boltzmann fit to the data.
To further verify this assumption, Raman experiments were performed in the low-frequency region where the Fe−His stretching vibration is expected to occur at around 214 cm−1 exclusively in reduced CcO complexes.22 In general, such Fe− His vibrations are only resonance-enhanced if the symmetry of the heme complex is lowered and, thus, can only be observed for a 5 coordinated (5c) Fe−histidine complex.23 Spectra were recorded by the following procedure: First RR and SERR measurements in the high-frequency region were performed on oxidized CcO using 413 nm laser excitation to monitor the presence or absence of the 1490 cm−1 band (Figure 5, left). Then, the enzyme was reduced by addition of sodium dithionite and Raman measurements in the low-frequency region were performed using 442 nm laser excitation (Figure 5, right). The low-frequency RR and SERR spectra showed an almost identical vibrational pattern. The only difference was the intensity of the Fe−His stretching vibration at 218 cm−1 which was more intense in the SERR spectrum. This observation can be explained assuming that only the Fe−His vibration of the 5c heme a3 complex is detected in the RR spectra while both hemes contribute to the signal in the SERR spectra. The increase in 218 cm−1 band intensity, therefore, supports our conclusion that an additional 5c species is formed in the immobilized state. Quantification of the 5c heme a contribution is difficult as the Raman cross section of this species for 413 nm excitation is not known. Comparison of the RR and SERR spectra in the region between 1500 and 1600 cm−1 (Figure 5) shows a decrease in intensity around 1550 cm−1, where preferentially vibrational bands of oxidized heme a occur (ν11 at 1543 cm−1 and ν38x at 1555 cm−1).21 While this confirms the decrease of an oxidized native heme a species, it does not provide strict evidence for the formation of a 5c species as the same effect would be expected upon heme a reduction. Unfortunately, the redox transition of the native heme a cannot be probed by SERRS as the Ag electrode starts to oxidize at potentials below +150 mV. Therefore, the native heme a will remain in its reduced state over the whole measured potential range. In the experiments recorded with excitation at 413 nm, the reduced species is hardly visible. Nevertheless, a shoulder of the ν4 band at 1355 cm−1 is detected (see Figure 3 at +100 mV) that we assign to the native reduced heme a species. The spectra in Figure 3 show only a very small contribution of the 5c species,
■
CONCLUSIONS CcO was electrostatically bound to amino functionalized electrodes, and the redox properties of heme a and heme a3 were investigated via SERRS. We have demonstrated that under rigorous anaerobic conditions the redox transition of heme a3 occurs in the same range as in solution indicating an intact catalytic center of CcO in the immobilized state. In contrast, we have strong indications that a significant portion of heme a undergoes a transformation into a non-native state exhibiting a redox potential that is 500 mV more negative than its value in the native state. We attribute the presence of this non-native species to a change in heme a coordination possibly induced by the high electric fields present at electrode surfaces. The structural changes of the heme a environment in the immobilized state might play a role for the very large overpotentials for oxygen reduction often observed in CcO/ electrode systems.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS Financial support from the DFG (SFB 1078 Projects A1, B4) is greatly acknowledged.
■
REFERENCES
(1) Michel, H. Cytochrome c Oxidase: Catalytic Cycle and Mechanisms of Proton Pumping-A Discussion. Biochemistry 1999, 38, 15129−15140. (2) Brzezinski, P.; Gennis, R. B. Cytochrome c Oxidase: Exciting Progress and Remaining Mysteries. J. Bioenerg. Biomembr. 2008, 40, 521−531.
9590
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
Article
The Journal of Physical Chemistry B
Proteins with Mesoporphyrin IX Analogues. J. Am. Chem. Soc. 1976, 98, 5482−5489. (20) Oellerich, S.; W, H.; Hildebrandt, P. Spectroscopic Characterization of Nonnative Conformational States of Cytochrome c. J. Phys. Chem. B 2002, 106, 6566−6580. (21) Kozuch, J.; von der Hocht, I.; Hilbers, F.; Michel, H.; Weidinger, I. M. Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome c Oxidase from Paracoccus Denitrificans. Biochemistry 2013, 52, 6197−6202. (22) Choi, S.; Lee, J. J.; Wei, Y. H.; Spiro, T. G. Resonance Raman and Electronic Spectra of heme a Complexes and Cytochrome Oxidase. J. Am. Chem. Soc. 1983, 105, 3692−3707. (23) Spiro, T. G. The resonance Raman spectroscopy of metalloporphyrins and heme proteins. In Iron Porphyrins; Lever, A. B. P., Gray, H. B., Eds.; Addison-Wesley: Reading, MA, 1983; Vol. 2, Chapter 3. (24) Murgida, D. H.; Hildebrandt, P. Redox and Redox-Coupled Processes of Heme Proteins and Enzymes at Electrochemical Interfaces. Phys. Chem. Chem. Phys. 2005, 7, 3773−3784. (25) McClelland, L. J.; Mou, T.-C.; Jeakins-Cooley, M. E.; Sprang, S. R.; Bowler, B. E. Structure of a Mitochondrial Cytochrome c Conformer Competent for Peroxidase Activity. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 6648−6653.
(3) Nilsson, T. Photoinduced Electron Transfer from tris(2,2′Bipyridyl)ruthenium to Cytochrome c Oxidase. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 6497−6501. (4) Varotsis, C.; Woodruff, W. H.; Babcock, G. T. Direct Detection of a Dioxygen Adduct of Cytochrome a3 in the Mixed Valence Cytochrome Oxidase/dioxygen Reaction. J. Biol. Chem. 1990, 265, 11131−11136. (5) Han, S.; Ching, Y. C.; Rousseau, D. L. Ferryl and Hydroxy Intermediates in the Reaction of Oxygen with Reduced Cytochrome c Oxidase. Nature 1990, 348, 89−90. (6) Collman, J. P.; Devaraj, N. K.; Decréau, R. A.; Yang, Y.; Yan, Y.L.; Ebina, W.; Eberspacher, T. A.; Chidsey, C. E. D. A Cytochrome C Oxidase Model Catalyzes Oxygen to Water Reduction under RateLimiting Electron Flux. Science 2007, 315, 1565−1568. (7) Su, L.; Hawkridge, F. M.; Rhoten, M. C. Electroreduction of Oxygen by Cytochrome c Oxidase Immobilized in ElectrodeSupported Lipid Bilayer Membranes. Chem. Biodiversity 2004, 1, 1281−1288. (8) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Enzymes as Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis. Chem. Rev. 2008, 108, 2439−2461. (9) Khoa Ly, H.; Sezer, M.; Wisitruangsakul, N.; Feng, J.-J.; Kranich, A.; Millo, D.; Weidinger, I. M.; Zebger, I.; Hildebrandt, P. SurfaceEnhanced Vibrational Spectroscopy for Probing Transient Interactions of Proteins with Biomimetic Interfaces: Electric Field Effects on Structure, Dynamics and Function of Cytochrome c. FEBS J. 2011, 278, 1382−1390. (10) Sezer, M.; Millo, D.; Weidinger, I. M.; Zebger, I.; Hildebrandt, P. Analyzing the Catalytic Processes of Immobilized Redox Enzymes by Vibrational Spectroscopies. IUBMB Life 2012, 64, 455−464. (11) Friedrich, M. G.; Giebeta, F.; Naumann, R.; Knoll, W.; Ataka, K.; Heberle, J.; Hrabakova, J.; Murgida, D. H.; Hildebrandt, P. Active Site Structure and Redox Processes of Cytochrome c Oxidase Immobilised in a Novel Biomimetic Lipid Membrane on an Electrode. Chem. Commun. (Cambridge, U. K.) 2004, 2376−2377. (12) Hrabakova, J.; Ataka, K.; Heberle, J.; Hildebrandt, P.; Murgida, D. H. Long Distance Electron Transfer in Cytochrome c Oxidase Immobilised on Electrodes. A Surface Enhanced Resonance Raman Spectroscopic Study. Phys. Chem. Chem. Phys. 2006, 8, 759−766. (13) Mitchell, D. M.; Gennis, R. B. Rapid Purification of Wild-Type and Mutant Cytochrome c Oxidase from Rhodobacter Sphaeroides by Ni2+-Affinity Chromatography. FEBS Lett. 1995, 368, 148−150. (14) Cohen-Bazire, G.; Sistrom, W. R.; Stanier, R. Y. Kinetic Studies of Pigment Synthesis by Non-Sulfur Purple Bacteria. J. Cell. Comp. Physiol. 1957, 49, 25−68. (15) Schlereth, D. D.; Mäntele, W. Redox-Induced Conformational Changes in Myoglobin and Hemoglobin: Electrochemistry and Ultraviolet-Visible and Fourier Transform Infrared Difference Spectroscopy at Surface-Modified Gold Electrodes in an Ultra-Thin-Layer Spectroelectrochemical Cell. Biochemistry 1992, 31, 7494−7502. (16) Hellwig, P.; Grzybek, S.; Behr, J.; Ludwig, B.; Michel, H.; Mantele, W. Electrochemical and Ultraviolet/visible/infrared Spectroscopic Analysis of heme a and a(3) Redox Reactions in the Cytochrome c Oxidase from Paracoccus Denitrificans: Separation of heme a and a(3) Contributions and Assignment of Vibrational Modes. Biochemistry 1999, 38, 1685−1694. (17) Wackerbarth, H.; Klar, U.; Gunther, W.; Hildebrandt, P. Novel Time-Resolved Surface-Enhanced (resonance) Raman Spectroscopic Technique for Studying the Dynamics of Interfacial Processes: Application to the Electron Transfer Reaction of Cytochrome c at a Silver Electrode. Appl. Spectrosc. 1999, 53, 283−291. (18) Sezer, M.; Spricigo, R.; Utesch, T.; Millo, D.; Leimkuehler, S.; Mroginski, A.; Wollenberger, U.; Hildebrandt, P.; Weidinger, I. M. Redox Properties and Catalytic Activity of Surface-Bound Human Sulfite Oxidase Studied by a Combined Surface Enhanced Resonance Raman Spectroscopic and Electrochemical Approach. Phys. Chem. Chem. Phys. 2010, 12, 7894−7903. (19) Spiro, T. G.; Burke, J. M. Protein Control of Porphyrin Conformation. Comparison of Resonance Raman Spectra of Heme 9591
DOI: 10.1021/acs.jpcb.5b03206 J. Phys. Chem. B 2015, 119, 9586−9591
