J. Phys. Chem. B 1999, 103, 10053-10064
10053
Dynamics of the Heterogeneous Electron-Transfer Reaction of Cytochrome c552 from Thermus thermophilus. A Time-Resolved Surface-Enhanced Resonance Raman Spectroscopic Study Sophie Lecomte,*,† Peter Hildebrandt,*,‡ and Tewfik Soulimane§ Laboratoire de Dynamique, Interactions et Re´ actiVite´ UPR-1580, CNRS-UniVersite´ Paris VI, 2 rue Henry Dunant, F-94320 Thiais, France, Max-Planck-Institut fu¨ r Strahlenchemie, Stiftstrasse34-36, D-45470 Mu¨ lheim, Germany, and Institut fu¨ r Biochemie, Klinikum Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany ReceiVed: June 3, 1999; In Final Form: August 18, 1999
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 Cytc552, which acts as an electron carrier in the respiratory chain of Thermus thermophilus, exists in a potentialdependent 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.
Introduction For a large number of redox proteins, electron transfer (ET) to their natural reaction partners occur over long distances of more than 10 Å.1 Most of these reactions take place at charged interfaces constituted by anionic and cationic binding domains of the partner proteins. Typical examples are the reduction processes of cytochrome c oxidase (CcO) and cytochrome c peroxidase by cytochrome c (Cyt-c).2 These long-range ET reactions require the involvement of amino acids to provide (specific) ET pathways. Furthermore, the electron has to be transferred across the electrical double layer of the proteinprotein interface. Most of the information about long-range ET reactions are obtained from photoinduced ET processes of metalloproteins in which additional photoactive redox sites are covalently attached to amino acids on the protein surface.3 However, in * To whom correspondence should be addressed. † CNRS-Universite ´ Paris VI. ‡ Max-Planck-Institut fu ¨ r Strahlenchemie. § Klinikum Aachem.
these reactions the electron is not transferred along the natural pathway that is used for the interprotein ET. Thus, the kinetic parameters derived from these studies are only of limited value for characterizing the biological process. This is particularly true for the reorganization energy which has to be corrected for the large contributions from the artificial solvent exposed redox site. Furthermore, this approach fails to consider the effect of the charged protein-protein interface on the ET process in an adequate manner.4 Such drawbacks can be overcome by studying ET processes of these redox proteins at electrodes.5 In the past years, considerable progress has been made in the development of electrochemical techniques as well as in the preparation of coated electrodes so that the determination of unimolecular heterogeneous rate constants of heme proteins is possible.6-10 Reorganization energies determined in electrochemical experiments exclusively refer to the natural redox site of the heme protein and the molecular environment and are in fact substantially smaller than those derived from photoinduced ET reactions of labeled proteins.3,8,9 Furthermore, appropriate choice of the electrochemical system generally allows the interaction of the
10.1021/jp991818d CCC: $18.00 © 1999 American Chemical Society Published on Web 10/27/1999
10054 J. Phys. Chem. B, Vol. 103, No. 45, 1999 redox protein with the electrode via the natural binding domain5 so that the ET is likely to follow the same pathway as that in the biological reaction. On the other hand, electrochemical techniques do not provide any structural information about the interfacial processes and, hence, are not appropriate to elucidate the mechanism of complex redox processes that may include additional reactions of the electroactive species coming before or after the ET. These are, for instance, conformational transitions of the redox protein that are induced by the electrostatic interactions in the electrical double layer of the electrode/electrolyte interface and that may also occur upon complex formation with the natural redox partners.11 This has indeed been shown for Cyt-c which undergoes the same structural changes upon binding to electrodes as well as to CcO.11-13 These conformational transitions may be of functional relevance for the biological ET providing a high efficiency and unidirectionality for the electron flow to the oxidase.13c Analogous processes may constitute the molecular basis also for conformationally gated ET of other redox couples as suggested from kinetic studies in solution.14 Consequently, a comprehensive analysis of the interfacial redox process requires the identification of intermediate species and the determination of their dynamics. Surface-enhanced Raman (SER) spectroscopy is a technique which can fulfill these requirements.15 Because of the selective enhancement of the Raman scattering upon adsorption on electrochemically roughened metal (Ag, Au, Cu) electrodes, it can be used to probe the molecular structure exclusive of the species in the electrical double layer. This technique has successfully been employed to study the potential-dependent equilibria of heme proteins where the SER effect and the molecular resonance Raman (RR) effect can be combined (surface-enhanced resonance Raman, SERR).12,16-19 Under these conditions, the sensitivity is further increased and the measured spectra exclusively display the vibrational bands of the heme group (i.e., the redox center) of the adsorbed protein. Furthermore, SERR spectroscopy has recently been extended to the time-resolved domain by combining the SERR spectroscopic measurements with the potential-jump technique. Thus, it is possible to probe the dynamics of the interfacial processes of heme proteins in the millisecond time scale.20,21 In this work we have carried out a systematic stationary and time-resolved SERR spectroscopic analysis of the interfacial redox process of cytochrome c552 (Cyt-c552) in continuation of our previous studies.19,20 Cyt-c552 is a mobile electron carrier in the respiratory chain of Thermus thermophilus where it transfers electrons to the terminal membrane-bound ba3oxidase.22 This heme protein exhibits some unique structural and functional properties which differ from those of other soluble c-type cytochromes.23 Cyt-c552 lacks the typical lysinerich cluster on the front surface that in Cyt-c serves as a binding domain for the reaction partner.2 Instead, the region around the exposed heme edge is shielded by an additional more unpolar peptide segment which is suggested to increase the thermostability of the protein.23 Hence, interactions with the ba3-oxidase may be qualitatively different compared to other eukaryotic and prokaryotic Cyt-c/CcO redox couples and may account for the high specificity of the Cyt-c552/ba3-oxidase reaction.22-25 Thus, a detailed analysis of the interfacial redox process of Cyt-c552, which is the goal of the present study, promises to elucidate the differences and similarities of the biological electron-transfer mechanism compared to Cyt-c.
Lecomte et al. Materials and Methods Cyt-c552 was isolated and purified as described previously.22,23 For RR and SERR spectroscopic experiments, the samples were dissolved in 10 mM Tris-HCl buffer (pH 7.0) with a final protein concentration of 15 and 0.1 µM, respectively. For the SERR experiments, the samples also include 50 mM KCl as a supporting electrolyte. RR and SERR spectra were measured with 413-nm cwexcitation of a Kr-ion laser with a power of ca. 50 mW at the sample. The spectra were recorded with a spectrograph (U1000, ISA) equipped with a liquid-nitrogen-cooled CCD camera. The spectral resolution was 2.5 cm-1 and the wavenumber increment per data point was 0.5 cm-1. The SERR spectra were measured with a rotating Ag cylinder electrode using a three-electrode cell controlled by a potentiostat. All potentials cited in this work refer to the saturated calomel electrode (SCE). The preparation of SER-active surfaces followed the protocol described previously.21 For time-resolved SERR experiments, a rapid potential jump was applied to the working electrode to perturb the equilibrium of the adsorbed species and the SERR signals were measured at a delay time δ subsequent to the potential jump. Following the measuring event, the potential was reset to the initial value so that the original equilibrium could be restored. An opto-mechanical device synchronized the sequences of potential jumps and measuring intervals ∆t which were repeated until a satisfactory signal-to-noise ratio was achieved. The experimental setup and the principles of the time-resolved experiments are described in detail elsewhere.21 Stationary and time-resolved SERR spectra were repeated several times under the same conditions to ensure reproducibility. The total (effective) accumulation time for each experiment was 20 and 3 s for the stationary and time-resolved experiments, respectively. To improve the signal-to-noise (S/ N) ratio, the individual spectra were combined only if their difference spectra yield straight lines. Prior to the spectra analysis, the structureless background was removed by polynomial subtraction. The spectra obtained in this way were analyzed by a band fitting or a component analysis.26 Results Conformational Equilibria of the Adsorbed Cyt-c552. The SERR spectra of Cyt-c552 that are obtained from a rotating and nonrotating electrode at -0.4 and 0.0 V are shown in Figure 1. SERR spectra of that quality could only be measured when KCl was used as a supporting electrolyte. In contrast to Cyt-c, much less intense signals were obtained upon replacement of KCl by Na2SO4.12,21 The spectra displayed in Figure 1 show the marker band region including the modes whose frequencies are characteristic for the oxidation, ligation, and spin state of the heme iron.27 At -0.4 V, the adsorbed Cyt-c552 is expected to be exclusively in the reduced form which is in fact confirmed by the position of the oxidation marker band ν4 at ca. 1360 cm-1 in the spectra obtained either with the rotating or the nonrotating electrode (Figure 1B,A). These two devices, however, yield substantially different spectra at 0.0 V (Figure 1C,D). When a rotating electrode is used, the resultant SERR spectrum includes an appreciable portion of the reduced form as indicated by the distinct shoulder at 1360 cm-1 adjacent to the prominent band at 1372 cm-1 (Figure 1C). Without the electrode being rotated, the oxidized species is clearly the dominant form (Figure 1D).19 Furthermore, the spectrum obtained with this device reveals significant changes also in the region above 1450 cm-1, which for example, includes new bands at 1491 and 1628 cm-1. These bands, which are characteristic of a five-coordinated (5c)
Reaction of Cytochrome c552 from Thermus thermophilus
J. Phys. Chem. B, Vol. 103, No. 45, 1999 10055 a nonrotating electrode reflect different equilibrium distributions between the various species. These changes of the equilibrium concentrations can be attributed to local heating of the adsorbed molecules in the laser focus which is significantly stronger for a nonrotating electrode than for a rotating electrode.21 Determination of the Component Spectra of the Adsorbed Cyt-c552. The conformational equilibria of the adsorbed Cytc552 depend on the potential so that relative contributions of the individual species to the spectra could easily be varied by a change in the potential. Thus, a global component analysis26 of the SERR spectra measured at various potentials allows one to determine the pure spectra of the individual species. In this analysis, i-measured spectra, Mi, are fitted by a set of complete component spectra, Nj, of the species j which are supposed to be involved according to the scheme in Figure 2. Thus, for all measured spectra, the relationship
Mi )
Figure 1. SERR spectra of Cyt-c552 measured with 413-nm excitation: (A) nonrotating electrode at -0.4 V; (B) rotating electrode at -0.2 V; (C) rotating electrode at 0.0 V; (D) nonrotating electrode at 0.0 V. Further experimental conditions are given in the text.
Figure 2. Scheme for the redox and conformational equilibria of the adsorbed Cyt-c552.
high-spin (HS) species,27 are much weaker in the spectrum obtained with the rotating electrode. Evidently, there are also new bands in the spectrum of Figure 1D which contribute to the broadening of the peaks at ca. 1550 and 1580 cm-1. Such a broadening, albeit not so pronounced, is also revealed by a careful inspection of the corresponding spectrum measured at -0.4 V (Figure 1A). All these spectral changes are fully reversible upon variation of the potential, implying that they do not reflect a denaturation of the adsorbed protein. These results are reminiscent of previous findings for the adsorbed mitochondrial Cyt-c which has been shown to exist in a conformational equilibrium between two states, i.e., state B1 which exhibits essentially the same spectrum as the dissolved Cyt-c and state B2 which comprises a five-coordinated highspin (5cHS) and a new six-coordinated low-spin (6cLS) form.12 Therefore, it is very likely that also the adsorbed Cyt-c552 exists in a similar conformational equilibrium (Figure 2).19,20 Then the differences between the spectra obtained with a rotating and
∑j gijNj
(1)
holds. The underlying assumption of this approach is that the component spectra Nj are potential-independent and only their relative amplitudes gij vary in the various measured spectra. Initial component spectra for the conformational state B2 required for the global fitting procedure were obtained by mutual subtraction of some of those measured spectra.19,26 Also, the previous results for Cyt-c served as a guideline for constructing the initial spectra.12 Only the component spectrum of the reduced state B1 (B1red) could be measured directly without interference from the contributions of other species using the rotating electrode at potentials -0.05 V, whereas at potentials -0.01 V, which can be ascribed to the effect of
J. Phys. Chem. B, Vol. 103, No. 45, 1999 10057 the charge redistribution in the electrical double layer upon approaching the oxidation potential of Ag/AgCl (see below). Attempts to apply a corresponding analysis to the redox equilibrium of state B2 failed because in the range of the expected redox potential (-0.3 V to -0.4 V) the equilibrium concentrations of B2 are very small because of the rapid conversion to B1red. For this reason, also, the time-resolved SERR studies had to be restricted to the redox process of the conformational state B1. Time-Resolved SERR Experiments of State B1. For the time-resolved SERR measurements, Cyt-c552 was adsorbed at -0.4 V to ensure that the protein was exclusively in state B1 as checked by a stationary SERR experiment. Subsequently, the potential was set to the initial potential Ei to start the timeresolved experiments. The initial dwell time at Ei (ca. 5 s) was sufficiently long to establish the redox equilibrium of B1. After each time-resolved experiment, a stationary SERR spectrum was measured at -0.4 V. Except for a slight lowering of the total intensity, these spectra reveal no differences compared to those measured prior to the time-resolved experiments. Thus, any denaturation processes of the adsorbed Cyt-c552 can be ruled out. The ranges of the potential jumps were chosen according to the following criteria: (i) large differences between the equilibrium concentrations of B1red/B1ox at the initial and the final potentials, Ei and Ef, which are essential for a reliable quantitative analysis of the time-resolved SERR spectra; (ii) large variations of the potential difference ∆E between Ef and E0 (i.e., the overpotential that determines the driving force for the ET), which are required for the determination of the reorganization energy. Because of the oxidation potential of Ag/AgCl and the concomitant loss of SERR activity at potentials close to E0(Ag/ AgCl), a value of 0.0 V constitutes the upper limit for the accessible potentials. For the heterogeneous oxidation this value corresponds to a maximum ∆E of only -0.044 V, which is not sufficient for determining the reorganization energy. Hence, the time-resolved SERR experiments were restricted to the reduction of the adsorbed Cyt-c552. Consequently, Ef was varied from Ef ) E0 (∆E ) 0 V) to negative values to increase the driving force for the heterogeneous reduction whereas Ei was confined to the range between 0.0 V and E0 (-0.044 V). Upon increase of the driving force, the processes to be monitored became faster so that a continuous improvement of the time resolution was required. With the present experimental setup, this could be achieved by increasing the rotational speed of the chopper wheel which in turn, however, reduced the recovery time for the initial equilibrium.21 Thus, eventually the dwell time at Ei became too small to guarantee the “freshsample” condition, leading to steady-state distributions between the reduced and the oxidized B1. It was found that this limit was reached at final potentials
