2492
J. Phys. Chem. B 2009, 113, 2492–2497
Electron Transfer Kinetics of Cytochrome c Probed by Time-Resolved Surface-Enhanced Resonance Raman Spectroscopy Marc Grosserueschkamp,†,| Marcel G. Friedrich,†,§,| Markus Plum,† Wolfgang Knoll,‡ and Renate L. C. Naumann*,† Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany, and Austrian Research Centers GmbH, Tech Gate Vienna, Donau-City Str. 1, 1220 Vienna, Austria ReceiVed: October 7, 2008; ReVised Manuscript ReceiVed: December 2, 2008
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. Timeresolved 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 kox ) 46 ( 7 s-1 and kred ) 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. Introduction Heterogeneous electron transfer of heme proteins adsorbed on metal surfaces can be investigated by spectro-electrochemistry.1-10 A combination of surface-enhanced infrared absorption spectroscopy (SEIRAS)11-14 and surface-enhanced Raman scattering (SERS) is particularly advantageous in this context. SEIRAS is designed to register changes of the protein backbone whereas SERS is sensitive to vibrations of the porphyrine ring, particularly in the resonance Raman (SERRS) mode.15,16 However, photodegradation of the protein by the incident laser beam is a serious problem in Raman scattering.17 In order to overcome this problem, a number of setups were designed to reduce the exposure time of the active area to the laser beam. This was achieved by using either a rotating Ag ring electrode,4 a linearly moving Ag disk electrode,5,6 or a rotating disk electrode (RDE).7,8 The latter solution seemed the most promising, particularly as far as the RDE was mounted upside down so that the laser beam could be focused onto the surface from above. A setup using this principle was described only once and not in great detail. Later on these authors returned to their earlier design of the rotating Ag ring electrode.9,10 The reason might be that some other details of the cell design were not solved properly, for example, the sealing of the fluid-containing cell body versus the rotating shaft of the electrode. Another problem is the electric contact to the rotating electrode. In the present paper, we present an improved setup including a cell design taking these problems into account. The power of the laser beam can thus be reduced to 100 µW or even lower compared to the rotating ring and linearly moving disk, which * To whom correspondence should be addressed. E-mail naumnnr@ mpip-mainz.mpg.de. Tel: +49 6131 379 157. Fax: +49 6131 379 100. † Max Planck Institute for Polymer Research. ‡ Austrian Research Centers GmbH. § Present address: Harvard University, Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, MA 02138. | These authors contributed equally to this work.
use 56 or 60 mW,4 respectively. The new setup was then used to perform time-resolved (tr) SERRS measurements on cytochrome c (cc). Cc is known to adsorb on self-assembled monolayers (SAMs) bearing different end groups in different orientations. The heme cleft is either oriented perpendicularly to the electrode or at a 90° angle in the case of the terminal carboxyl18,19 and pyridyl groups,20 respectively, thus facilitating electron transfer (eT). These orientations were investigated in detail by tr-SERRS. eT rates were found in the range of k0 ) 42-2400 s-1 depending on the tunnelling distance to the heme center given by the spacer length of the SAM.10 Cc adsorbed to OH-terminated layers, however, attracted less attention, as the heme cleft was directed away from the surface so as to hamper eT. Kinetic constants of cc adsorbed to mercaptoethanol (ME) layers obtained by electrochemistry were reported to be in the range of k0 ) 20 s-1.21 Recently, we used tr-SEIRAS to determine eT rates of cc on the same architecture, but on a surface roughened by the assembly of Au nanoparticles (NPs). Relatively high eT rates were found to be in the range of 1800 s-1.22 It was concluded from this study that the morphology of the surface might also play a role for the eT rate. SERRS also requires rough surfaces.23,24 So in the present study we used tr-SERRS to investigate eT rates of cc adsorbed on ME layers assembled on electrochemically roughened silver electrodes. Materials and Methods Potassium chloride, potassium dihydrogenphosphate, and cytochrome c from bovine heart was purchased from SigmaAldrich. 2-Mercaptoethanol 99% extra pure was supplied by Acros. Sodium perchlorate was purchased from Fluka Biochemika, Buchs, Switzerland. Preparation of the Roughened Silver Electrodes. The planes of silver rods were used as SERRS substrates and/or electrodes. They were polished prior to use with a polishing
10.1021/jp808865z CCC: $40.75 2009 American Chemical Society Published on Web 02/03/2009
Electron Transfer Kinetics of Cytochrome c by SERRS
J. Phys. Chem. B, Vol. 113, No. 8, 2009 2493
Figure 1. The SERRS setup is based on a confocal Raman microscope combined with a newly designed SERRS cell. Excitation is achieved by a Kr+ laser. An AOM allows blocking of the laser beam intermittently and therefore generating short laser pulses in the ms range.
machine (Buehler PHOENIX 4000) using a synthetic polishing cloth (ChemoMet, Buehler). The electrodes were successively polished with alumina (Al2O3) slurries with a grain size 1 (MicroPolish II (C) alpha, Buehler), 0.3 (MicroPolish II (A) alpha, Buehler), and 0.05 µm (MicroPolish II (B) gamma, Buehler), in that order. Each polishing step was performed for 3 min at 300 rpm. After each step, the electrodes were extensively rinsed with MilliQ-water and cleaned in an ultrasonic bath in order to prevent contamination with alumina particles. Thereafter the electrodes were electrochemically roughened by applying oxidation-reduction pulses provided by a potentiostat (Autolab, PGSTAT302, Eco Chemie, B.V. Utrecht, Netherlands). This treatment was performed in 0.1 M KCl solution changing the potential between +500 and -100 mV and holding it for 70, 20, 15, 10, and 5 s, respectively. The rms roughness of the electrochemically roughened silver electrodes was determined by atomic force microscopy using a Dimension3100 (Veeco Instruments Inc.). An area of 5 µm × 5 µm was scanned in the tapping mode, and the rms roughness was calculated from these data. The rms roughness finally obtained was 90 nm. Preparation of the Surface Layers. The roughened silver electrodes were immersed for 30 min into an aqueous solution of ME (1 mM). The excess of ME was removed by rinsing with water. Cc was adsorbed on the SAM of ME by immersing the functionalized silver electrode into PBS buffer (20 mM K2HPO4/100 mM NaClO4/pH ) 7) containing 50 µM cc for at least 30 min. Setup for tr-SERRS Measurements. The setup to perform SERRS measurements (Figure 1) was mounted onto a shockabsorbing optical table (Newport Corporation). Surfaceenhanced resonance Raman (SERR) spectra were acquired using a high resolution confocal Raman microscope (LabRam, HR800, HORIBA Jobin Yvon) equipped with a liquid nitrogen-cooled back-illuminated charge-coupled device (CCD) detector (Symphony, Jobin Yvon) optimized for near-ultraviolet light. The 413 nm emission line of a Kr+ laser (Innova 90C, Coherent) was used for excitation. The laser beam was first directed through a premonochromator (LaserspecIII, Spectrolab Research
Figure 2. Cross section of the novel SERRS cell design. High precision rotation of the electrode allows for minimal-invasive observation of the protein by low-power laser irradiation. Permanent electrical contact is achieved by a mercury contact connected by a conductive bolt to the Ag-RDE. The agile drive shaft is sealed against the cell body by an axial face sealing.
Laboratory, Newbury, England) in order to remove background radiation consisting of plasma lines produced by the laser medium. Time-resolved measurements were realized by periodic electrochemical excitation of the protein combined with synchronized illumination of the sample by short laser pulses in the ms range. For this purpose the laser beam was subsequently passing an acousto-optic modulator (AOM) (A.A. MT200/ A0,5@400nm01/24091, A.A. opto-e´le´ctronique,St. Remy, France). The AOM was mounted on a stage allowing free movement in x-, y-, z-directions and adjustment of the angular position in order to efficiently couple the laser beam onto the quartz crystal to achieve an optimum of light intensity. The AOM was controlled by a function generator (33250A 80 MHz, Agilent), which routed the exciting laser light onto the sample or to block the beam, respectively. The optical beam path was further guided by a series of adjustable mirrors and finally coupled into the
2494 J. Phys. Chem. B, Vol. 113, No. 8, 2009
Grosserueschkamp et al.
Figure 3. CV of cc adsorbed on a SAM of ME on the silver electrode shows cathodic and anodic peaks at 207 and 261 mV, respectively. Figure 5. Band assignment of the SERR spectra. The band ν4 resulting from a distortion mode of the pyrrole half-ring was used as a sensitive marker for the redox state of the protein.
Figure 4. Potentiostatic titration of cc by SERRS. Spectra of cc were acquired at different static potentials applied to the immobilized protein. (1) +20, (2) +130, (3) +170, (4) +190, (5) +210, and (6) +330 mV.
confocal microscope (BX41, Olympus). The laser beam was focused onto the protein sample, by a long-distance water immersion objective (Olympus LUMPLFL, 100 XW, WD ) 1.5, NA ) 1, BFOBJ) optimized for transmission of near UV light. The scattered light was then collected by the same objective and guided through a notch filter (413 nm) designed to filter out Rayleigh scattered light. The Raman scattered light was then focused onto the confocal pinhole of the microscope providing spatial resolution. Spectral resolution was achieved by an 1800 grooves/mm grating. The spectrum was imaged onto the CCD detector with an area of 2048 × 512 pixels. The focal length of the spectrograph was 800 mm. Thereby a spectral resolution of 3 cm-1 was achieved. The increment per data point was 0.4 cm-1. A software controlled (Gpes, Autolab) potentiostat (Autolab, PGSTAT302, Eco Chemie, B.V., Utrecht, Netherlands) was used for modulation of the potential applied to the protein. However, in time-resolved measurements a master signal provided by a function generator (20 MHz 8021, Tabor Electronics) was used to trigger the periodic step potential of
Figure 6. Deconvolution of a SERR spectrum recorded at +170 mV. Experimental data (black), overall fit (green), peaks from the reduced (blue), and oxidized (red) species of cc are displayed.
the potentiostat as well as the function generator controlling the AOM. The triggering process was screened by an oscilloscope (9354AM, LeCroy), and a synchronized electrochemical and optical excitation of the protein was achieved (Figure 8). SERRS Measuring Cell. A custom-made measuring cell was used designed for spectro-electrochemistry (Figure 2). It was based on a rotating disk electrode (RDE), turned upside down for illumination by means of a confocal microscope. To prevent photodegradation of the protein, the RDE was mounted on top of a rotating axis, which was belt-driven at a constant speed of 800 rpm by a DC-motor (FAULHABER 3863H012C 38A 1:5). In order for the surface of the electrode to stay exactly in the focal plane of the confocal microscope at all times, it had to be machined very precisely (deviation
