Orientation of Cytochrome c Adsorbed on a Citrate-Reduced Silver

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Orientation of Cytochrome c Adsorbed on a Citrate-Reduced Silver Colloid Surface I. D. G. Macdonald and W. E. Smith* Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, Scotland Received March 31, 1995. In Final Form: July 28, 1995X Eukaryotic membrane bound cytochrome c is believed to use areas of negative and positive charge to bind to the membrane and to interact in vivo with the corresponding oxidase and reductase. This specific charge distribution makes the protein ideal for the formation of self-assembled layers on charged surfaces. Citrate-coated, hexagonal silver colloidal particles were used to give a negatively charged surface for adsorption, and the effect of concentration and surface coverage was studied by surface enhanced resonance Raman scattering (SERRS). Excitation was with a preresonant frequency where the angular dependence of the scattering intensity is more pronounced than in resonance. The heme ring plane lies at a slight angle to the silver surface at low surface coverage. The adherence of the protein to the negatively charged surface is strong (indicated by no detectable SERRS from protein in supernatant), and as packing density is increased, a reorientation of about 5° to a more vertical orientation is observed. No alteration in frequency of the principal bands can be observed, and therefore, no denaturation occurs. A more subtle change in the relative intensities suggests that there are two forms of packing with the difference in packing density determining the protein/protein orientation. Increase in pH causes reorientation of the protein to a more inclined orientation. The extent of the change is dependent upon protein packing density. Greater reorientation occurs at low densities.

Introduction Eukaryotic mitochondrial cytochrome c is part of the energy transducing cycle on the outer surface of the inner mitochondrial membrane.1 It facilitates electron transfer between its embedded physiological oxidoreductases, cytochrome c oxidase and cytochrome c reductase. Important in its biological function is the exposure along one edge of the otherwise buried prosthetic heme. Alternative associations with its redox partners are mediated by favorable electrostatic interactions between the positively charged surface domain on cytochrome c and complementary negatively charged surface regions on the oxidoreductases.2 Moreover, the interaction domains on cytochrome c have been shown to be nearly identical for the reductase and oxidase.3 Inhibition of the reaction of mitochondrial cytochrome c with both redox partners by stoichiometric amounts of polylysine demonstrates the involvement of positively charged lysine residues.4 In addition, modeling of the protein surface charge localization indicates there is likely to be an optimum orientation on the membrane for electron transfer. Attainment of the functionally required relative orientation between cytochrome c and its partners is believed to proceed via a series of transient complexes dictated by relatively nonspecific hydrophobic and ionic interactions within a single encounter. A large number of casual precursor complexes may occur before the desired orientation is reached, and a complex capable of electron transfer is formed.5 It is difficult to study the factors controlling these orientational processes in situ. This study reports * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, December 1, 1995. (1) Dickerson, R. E.; Timkovich, R. In The Enzymes; Academic Press: New York, 1976; Vol. 11, p 397. (2) Salemme, F. R.; Kraut, J.; Kamen, M. D. J. Biol. Chem. 1973, 248, 7701. (3) Koppenol, W. H.; Ferguson-Miller, S.; Osheroff, N.; Speck, S. H.; Margoliash, E. In Oxidases and Related Redox Systems; Pergamon Press: Oxford, 1983; p 1037. (4) Salemme, F. R. Ann. Rev. Biochem. 1977, 46, 299. (5) Northrup, S. W.; Boles, J. O.; Reynolds, J. C. L. J. Phys. Chem. 1987, 91, 5991.

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the effect of concentration and pH on the orientation of adsorbed cytochrome c on a protected colloidal silver metal surface. The surface is chosen to enable surface enhanced resonance Raman scattering (SERRS) to be used to probe the orientation of cytochrome c at the surface and to monitor protein denaturation throughout. SERRS is sensitive and selective for the heme chromophore, and scattered intensity changes reflect alterations in the orientation of the heme ring and, therefore, cytochrome c relative to the surface. The technique requires that the protein be adsorbed on a roughened silver or related metal surface to obtain the enhancement. This may lead to conformational changes in the protein or to denaturation through possible reaction with silver ions. Previous studies using (unprotected) silver electrodes have elegantly demonstrated that conformational changes6 can be obtained in a controlled manner. Depending on conditions such as pH and ionic strength, both the native form of cytochrome c and a second conformation can be stabilized. To prevent denaturation or conformational change, the use of a biocompatible colloid was proposed.7,8 In this case, colloidal particles formed by careful reduction of silver with citrate are coated with a layer of citrate, and consequently the protein is adsorbed onto a negatively charged layer. The presence of a surface citrate species can be verified by surface enhanced Raman scattering from aggregated colloid in the absence of protein. Long term stability of the colloid is dependent on the correct formulation of the surface layer. The retention of a mixed spin state in cytochrome P-450 on this colloid and not on other colloids demonstrates the protection offered by this biocompatible surface. A recent evaluation of this method has been carried out.9 Individual colloidal particles, within this almost monodispersed suspension, have well-defined, hexagonal shapes. Therefore, the protein adsorption process from solution, under controlled conditions, occurs (6) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710. (7) Rospendowski, B. N.; Schegel, V. L.; Holt, R. E.; Cotton, T. M. Biosystems-2; Plenum Publishing Corp.: New York, 1989. (8) Rospendowski, B. N.; Kelly, K.; Wolf, C. R.; Smith, W. E. J. Am. Chem. Soc. 1991, 113, 1217. (9) Munro, C. H.; Smith, W. E.; Garner, M.; Clarkson, J.; White, P. C. Langmuir 1995, 11, 3712.

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Orientation of Cytochrome c

onto well-characterized silver surfaces, which give relatively flat templates for adsorption. Previous papers have reported SERRS of cytochrome c from a colloid,10,11 but the nature of the surface is not clearly defined. Experimental Section Colloidal silver was prepared according to a modified Lee and Meisel12 method. Approximately 470 mL of distilled water was stirred mechanically [Citenco motor driven glass rod] and heated rapidly in a 1 L round-bottomed flask. At 50-60 °C 90 mg of silver nitrate (Aldrich, >99%, ACS reagent), dissolved in 30 mL of distilled water, was added to the stirring solution. Continuous rapid heating and stirring was maintained until boiling, whereupon 10 mL of 1% w/v trisodium citrate (Fisons, Analar Grade) was immediately added. Vigorous stirring was continued under boiling action for a further 75 min. Colloidal suspensions were cooled and corrected for volume loss with distilled water, thereby maintaining the suspension at 500 mL. Colloidal suspensions, with acceptable characteristics, exhibited a UV/vis absorption maximum between 408 and 415 nm with a typical half-height width of 50-60 nm. Colloids generated by the above method were stable over a considerable time period and were regarded as usable for up to 6-8 weeks. Cytochrome c (horse heart, 95% purity, Sigma) was dissolved in 100 mM phosphate buffer (13.62 mg/25 mL of 0.1 M phosphate buffer Analar Grade; pH ) 7.35) giving a concentration of 4.4 × 10-5 M. From this stock solution, cytochrome c solutions of various concentrations between 22.2 and 2.2 × 10-8 M were prepared. Two hundred µL of each solution was added in turn to 2 mL of colloid and mixed thoroughly in a 1 cm fluorimeter cell, yielding a 9.9 mM buffered solution at an effective pH of 7.6. For all concentrations studied aggregation occurred immediately upon addition of cytochrome c solution to the colloidal suspension. This aggregation gave rise to clearly visible but transient turbidity changes in the colloid. Aggregation was induced by the presence of protein in the buffered solution. Each sample was stoppered and left standing to equilibrate thermally for 20 min at 18 °C. SERR spectra were recorded 30 min after aggregation. To quantify any effect of heterogeneity of the protein further purification of one sample of cytochrome c was carried out. A small protein sample (20 mg) was dissolved in 1 mL of 50 mM Tris/HCl (pH ) 7.5) and applied to a Sephacryl S-200 HR (Pharmacia) column (1 m × 2 cm). A peristaltic pump (LKB 2232 Microperspex S) provided a throughput of 10 mL/h. A Pharmacia Redifrac collector was used for obtaining 3.5 mL fractions. The peak fraction was centrifuged briefly. A UV/vis spectrophotometer (Shimatzu UV2101) recorded absorption spectra in the 250-600 nm range. Comparison of the absorbance ratio of the peaks at 410 and 280 nm implied further hemoprotein purification, and this sample was estimated to be greater than 99% pure. The same procedure of sample preparation and spectroscopic analysis was carried out for this purified protein. Cytochrome c stock solutions were prepared separately in 100 mM sodium dihydrogen phosphate (May and Baker, Analar Grade) and 100 mM disodium hydrogen orthophosphate (Fisons, Analar Grade). Dilution produced two separate concentrations in the aforementioned buffers. Differing pH values were obtained by altering the ratio of 100 mM monosodium to disodium phosphate buffer. pH measurements were obtained with a Mettler 320 pH meter. Two hundred µL of the mixed cytochrome c solutions ((23.43 and 4.73) × 10-7 M) were added to 2 mL of colloid in a 1 cm fluorimeter cell, yielding respective final protein concentrations of 21.3 × 10-8 and 4.3 × 10-8 M. After being allowed to stand for 20 min to achieve thermal equilibrium, the aggregated solutions were transferred to the Raman spectrometer. Spectra-Physics 2020/2045 Argon ion lasers provided 514.5 nm of exciting radiation. All samples were placed in a quadrant shutter cell holder with sensitive adjustment, thereby enabling accurate and precise sample cell positioning. Unless otherwise indicated, spectra were obtained using 100 mW irradiation, 3.6 (10) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986, 90, 6017. (11) Cotton, T. M.; Tirkovich, R.; Cork, M. S. FEBS Lett. 1981, 39. (12) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391.

Langmuir, Vol. 12, No. 3, 1996 707 cm-1 slit width, and a scanning rate of 1 cm-1 s-1, corresponding to a resolution of 2 cm-1. Scattered radiation was collected using conventional 90° geometry and analyzed by an Anaspec-modified Cary 81 scanning monochromator. A Peltier cooled Thorn-EMI 9658R photomultiplier tube was used with photon counting electronics (Anaspec Series 2000 quantum photometer) for data acquisition.

Results and Discussion Acid-induced aggregation of the colloidal suspensions in the absence of protein leads to the appearance of citrate signals from the surface. Signals are sharp and well defined and indicate that one form of citrate predominates on the surface.9 The surface is negatively charged due to uncomplexed carboxylate groups from the citrate. Positively charged molecules adhere strongly to this surface, exhibiting SERRS activity.13 Aggregation is a prerequisite for efficient scattering. Cytochrome c is believed to adsorb specifically on negative areas on the inner mitochondrial membrane surface.14 The negatively charged citrate coated silver particles form a suitable homogeneous surface for cytochrome c adsorption by the same groups involved in membrane binding. The frequencies of the bands due to SERRS from cytochrome c adsorbed on the surface correlate with cytochrome c solution resonance Raman studies15-17 of a single species, indicating that only one form of the protein is present as the predominant species. Other experiments have shown that at significantly less than monolayer coverage, denaturation or conformational changes occur,10 characterized by frequency and intensity shifts in SERRS. A similar effect can be induced by incorrect addition of the protein to the surface, pH,18 ionic strength factors,1 or perhaps most importantly, the nature of the surface. However, as demonstrated by the similarity of SERRS and resonance under controlled conditions, a monolayer of protein in its native state can be maintained at the surface, and considerable care was taken to ensure that this was the case in this study. The colloid used in this experiment was carefully monitored. The peak position and half width of the unaggregated colloid was regularly checked and corresponds to nearly monodispersed colloid, in which the particles are known to be relatively well-formed hexagons with flat surfaces. Thus, protein adsorption occurs onto well-defined flat surfaces. To calculate the approximate concentration at which monolayer coverage was expected, the hexagons were approximated to a sphere of radius 18 nm and the protein to a sphere of radius 1.7 nm estimated from the crystal structure.15 This very approximate calculation predicts that complete monolayer coverage would occur at 3 × 10-7 M. To determine whether surface desorption of protein occurred, aggregated colloid was centrifuged and the supernatant collected. No SERRS signals could be obtained when this supernatant was added to fresh colloid, indicating strong adherence between cytochrome c and the colloidal surface. The major changes in the relative intensities of the bands discussed later correspond approximately to concentrations of protein roughly equivalent to that required for the monolayer (13) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1984, 88, 5935. (14) Heinburg, T.; Hildebrandt, P.; Marsh, D. Biochemistry 1991, 30, 9084. (15) Cartling, B. Biological Applications of Raman Spectroscopy; Wiley-Interscience: New York, 1988; Vol. 3, p 217. (16) Kitagawa, T.; Ozaki, Y. Struct. Bonding 1987, 64, 71. (17) Spiro, T. G.; Czernuszewicz, R. S.; Li, Y. Y. Coord. Chem. Rev. 1990, 100, 541. (18) Theorell, H.; A° kesson, A° . J. Am. Chem. Soc. 1941, 63, 1804.

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Figure 1. Cytochrome c surface adsorption performed as described in the Experimental Section. SERR spectra of ferricytochrome c (1.5 × 10-7 M) recorded immediately after protein-induced colloidal aggregation (A), 10 min after aggregation (B), after 30 min (C), and after 105 min (D). Exciting line: 514.5 nm,100 mW. Figure 3. Protein adsorption (see Experimental Section). SERR spectra of chromatographically purified ferricytochrome c at sample concentrations of 4 × 10-8 M (A), 7.5 × 10-8 M (B), and 1.5 × 10-7 M (C). Spectra recorded 30 min after aggregation. Exciting line: 514.5 nm,100 mW. Table 1. Comparison of the Full Width Half Heights (FWHH) for Vibrational Bands ν4, ν3, and ν10 for “Impure” Ferricytochrome c (Supplied by Sigma) and Chromatographically Purified Hemoprotein full width half height/cm-1 (error is 3 cm-1) impure cytochrome c

Figure 2. Protein adsorption performed as described in the Experimental Section. SERR spectra of ferricytochrome c (1 × 10-8 M) recorded 1 min after aggregation (A), after 60 min (B), and after 80 min (C). Exciting line: 514.5 nm,100 mW.

coverage as calculated above. Thus, changes observed are attributed to the buildup of the first surface layer. Given the spacing of the chromophore from the surface, it is not surprising that the second layer effect is small and not detected in these experiments. Assignment of Bands SERRS is likely to be a single process, but is perhaps most easily understood, and is usually described, in terms of two separate components, namely the surface enhanced

pure cytochrome c

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Raman scattering (SERS) component and the resonance scattering component. SERS scattering arises from an interaction between the surface plasmon on a roughened surface and the adsorbed molecule.19-23 Silver has favorable dielectric properties at optical frequencies enabling large enhancement of this scattering phenomenon.24-26 (19) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163. (20) Moskovits, M.; Didella, D. P. In Surface Enhanced Raman Scattering; Plenum Publishing: New York, 1982; p 243. (21) Lund, P. A.; Smardzewski, R. R.; Tevault, D. E. Chem. Phys. Lett. 1982, 89, 508. (22) Liao, P. F.; Stern, B. Opt. Lett. 1982, 7, 483. (23) Krasser, W.; Renouprez, A. J. Solid State Commun. 1982, 41, 231. (24) Creighton, J. A. Spectroscopy of Surfaces; Wiley: New York, 1988; Vol. 16, p 37. (25) Weitz, D. A.; Moskovits, M.; Creighton, J. A. In Chemistry and Structural Interfaces, New Laser and Optical Techniques; VCH: Deerfield Beach, FL, 1986; p 197. (26) Gersten, J. I.; Nitzan, A. J. J. Chem. Phys. 1980, 73, 3023.

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Figure 4. Protein adsorption (see Experimental Section). Recorded SERR of ferricytochrome c at final sample concentrations of 4 × 10-8 M (A), 7.5 × 10-8 M (B), and 15 × 10-8 M (C) together with a graph of the intensity ratio of ν10 to ν4 with increasing surface coverage. Error bars are calculated to account for noise and base line slope. Spectra obtained 30 min after aggregation. Exciting line: 514.5 nm,100 mW.

The SERS effect is greatly amplified by aggregation of the colloid, shifting the surface plasmon resonance to the red where large metallic dielectric values occur.24 Furthermore, intense induced electric fields are created in the interstesis upon aggregation, leading to scattering enhancement. Under the controlled conditions applied, the maximum in the SERS effect is expected between 600 and 700 nm, coincident with the surface plasmon resonance of the aggregate.25,27 There are two well established components which are believed to be responsible for the SERS phenomenon: an electromagnetic component that is effective up to 50 Å28,29 from the SERS-active surface and a chemical component, the occurrence of which requires the presence of chemically “bonded” molecules at the surface.30-32 The use of a citrate spacer and the additional distance between the SERRS active chromphore and the surface precludes any strong chemical contribution, allowing electromagnetic SERS to dominate in this case. Furthermore, the application of surface selection rules for electromagnetic SERS can yield, via relative intensities of the bands, orientational information on the adsorbed surface species. For a porphyrin with an idealized D4h symmetry, Raman-active A1g, A2g, B1g, and B2g modes show surface enhancement when each mode involves a polar(27) Wang, D. S.; Kerker, M. Phys. Rev. 1981, B24, 1777. (28) Murray, C. A.; Allara, D. L.; Rhinewine, M. Phys. Rev. Lett. 1981, 46, 57. (29) Murray, C. A.; Allara, D. L. J. Chem. Phys. 1982, 76, 1290. (30) Otto, A. Light Scattering in Solids; Springer-Verlag: Berlin, 1984; Vol. 4. (31) Avouris, Ph.; Demuth, J. E. J. Chem. Phys. 1981, 75, 4783. (32) Billman, J.; Otto, A. Solid State Commun. 1982, 44, 105.

ization component perpendicular to the surface.24,33,34 Inplane modes such as ν3, ν4, and ν10, irrespective of symmetry labels, will exhibit Raman scattering when the porphyrin plane is perpendicular to the silver surface. Conversely, only A1g vibrations (ν3,ν4) would be expected to have appreciable intensity with the ring plane aligned parallel to the silver surface.24 Thus, changes in orientation of the heme plane relative to the surface should be observable using SERS. Assuming no specific destructive interference or selfabsorption, the maximum resonance intensity would be expected when the laser excitation frequency coincides with the electronic absorption maximum of the chromophore. However, resonance excitation involves a significant depolarization of the scattered radiation,35 and consequently, surface orientation is best achieved at an off-resonance position, where preresonance may still enhance the SERS effect, thus retaining the specificity of the heme chromophore while increasing the likely contribution to the signal from SERS. For this reason the excitation wavelength used in this investigation was 514.5 nm (preresonant with Q band) rather than 413 nm irradiation (resonant with Soret band) utilized by others in previous studies.6,10 Excitation at 413 nm induces a strong resonance from the chromophore. This strong contribution concomitant with a weaker surface plasmon enhancement in the blue region tends the SERRS effect toward resonance, reducing the applicability of surface selection rules which depend on the SERS contribution. (33) Moskovits, M. J. Chem. Phys. 1982, 77(9), 4408. (34) Moskovits, M.; Suh, J. S. J. Phys. Chem. 1984, 88(23), 5526. (35) Zgierski, M. Z.; Pawlikowski, M. Chem. Phys. 1982, 65, 335.

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Figure 5. Protein adsorption (see Experimental Section). Recorded SERR spectra of ferricytochrome c at final solution concentrations of 15 × 10-8 M (A), 18.2 × 10-8 M (B), and 20 × 10-8 M (C). Spectra obtained 30 min after aggregation. Exciting line: 514.5 nm, 100 mW.

Interpretation is greatly aided by the many previous studies of the cytochrome c porphyrin system by resonance Raman scattering. Extensive normal coordinate analyses have been carried out on this system.36,37 From these studies clear assignments of the main bands (>1350 cm-1) have been formulated. Of particular relevance to this study is the positioning of the heme sensitive oxidation (ν4) and spin state (ν3, ν10) Raman active bands for cytochrome c in the frequency range 1300-1700 cm-1. Under the experimental conditions applied, a sixcoordinate low spin (6cls) heme is observed and not the mixed five-coordinate high spin (5chs) species identified by other authors. The presence of the 6cls form was established by ν3, ν10, and ν4, at 1506, 1640, and 1375 cm-1, respectively (Figures 1 and 2). Indeed, SERRS obtained at 514.5 nm (291 K) from this laboratory are in reasonable agreement with previously recorded spectra at -196 °C.10 Moreover, there were no discernable frequency shifts between the resonance Raman spectrum of cytochrome c and the SERR spectra. This suggests that cytochrome c conformation is unaltered by adsorption onto the colloidal surface. At no stage was any 6cls-5chs conversion observed on the experimental time scales used. In a previous study, the presence of a substantial temperature-induced high spin component for cytochrome c adsorbed on silver was observable in the growth of ν19 [spin state marker band (A2g)] at 1568 cm-1. The intensity of the high spin ν19 band was monitored over an extended period relative to the ν19 low spin band at 1587 cm-1 (Figure 1). The relatively stable intensity relationship between (36) Parthasarathi, N.; Hansen, C.; Yamaguichi, S.; Spiro, T. G. J. Am. Chem. Soc. 1987, 109, 3865. (37) Hu, S.; Morris, I. K.; Singh, J. P.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1993, 115, 12446.

Macdonald and Smith

Figure 6. Protein adsorption (see Experimental Section). SERR spectra of ferricytochrome c at final solution concentrations of 2 × 10-8 M (A), 4 × 10-8 M (B), and 6 × 10-8 M (C). Spectra recorded 30 min after aggregation. Exciting line: 514.5 nm,100 mW.

these two bands suggests minimal or slow 5chs formation on the citrate-reduced silver colloid. This conversion was not appreciably accelerated even at well below (a tenth) monolayer coverage (Figure 2). Moreover, observation (457.9 nm irradiation) of ν3 at 1506 cm-1 confirmed the absence of a 5chs form, and only with large variations in pH coupled with very dilute (4 × 10-8 M) concentrations of protein were significant frequency shifts, characteristic of denaturation or perturbation of the heme environment of cytochrome c, observed. However, some band broadening over this extended period (see Figure 2) could be discerned, suggesting some less extensive change in the form of the protein. The above evidence strongly supports the presence of a stable, essentially six-coordinate low spin single heme form of cytochrome c adsorbed onto the citrate surface. The relative intensities of the vibrations ν10 (1640 cm-1) and ν4 (1375 cm-1) change, reflecting orientational changes of the heme. These intensity changes are discussed later. The possibility of interference from dimers or oligomers present in the protein was eliminated by repeating a scattering (SERR) experiment using different concentrations of chromatographically purified cytochrome c. Results are presented in Table 1 with accompanying illustrative spectra in Figure 3. Comparison of the peak positions and full width half height (FWHH) of three sensitive vibrational bands (ν4 ,ν3 and ν10) for cytochrome c (used as supplied by Sigma) and purified protein indicates no detectable differences in band frequencies and only small changes within experimental error in FWHH. Significant FWHH differences would be expected if a

Orientation of Cytochrome c

Figure 7. Protein adsorption (see Experimental Section). SERR spectra of ferricytochrome c at 6 × 10-8 M (final sample concentration) recorded immediately after aggregation (A), after 15 min (B), and 30 min (C) after aggregation. Exciting line: 514.5 nm,100 mW.

different species resided on the surface. This is indicative of a single and identical 6cls species being present on the colloidal surface for both protein purities. The actual frequencies given in this study vary slightly from those in some other studies. The available data from a number of laboratories has been assessed, and the small differences in frequency quoted appear to be relatively consistent for each study. The relative differences in frequency between modes appear to be very consistent. The differences in absolute frequency appear to reflect differing instrumentation systems rather than alterations in protein adsorption. Spectra obtained by this laboratory are usually within one to two wave numbers of at least one other group.38,39 For this reason, the matter of absolute frequency reported in any of the papers must be treated with some caution. Cytochrome c Surface Coverage and Surface Orientation Modeling of cytochrome c (pI ) 9.8) by ourselves has suggested that there are two areas of positive charge which can most effectively bind to a negatively charged surface at neutral pH. One region is a lysine rich surface (Lys54, Lys-55, Lys-72, Lys-73, and His-39), while the other has surface residues of histidine and lysine (His-33, His36, Lys-22, and Lys-27). Both regions could compete for surface adsorption sites, and binding to the coated surface by either region would position the heme plane at a shallow incline relative to the surface. This incline is similar but may not be identical upon binding by either region. (38) Cotton, T. M.; Schlegel, V.; Holt, R. E.; Swanson, B.; Ortiz de Montellano, P. Proc. SPIE-Int. Soc. Opt. Eng. 1989, 1055, 263. (39) Cotton, T. M.; Schultz, S.; Van Dyne, R. P. J. Am. Chem. Soc. 1980, 102, 7960.

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Moreover, these orientations would allow favorable electrostatic contacts between two protein surfaces by coupling areas of opposite charge. Favorable contacts between positive residues (Lys-4, Lys-5, Lys-86, Lys-87, Lys-89, Arg-13, and Arg-91) and negatively charged residues (Glu60, Glu-61, Glu-66, Glu-88, Asp-90, and Asp-93) would seem most likely, positioning the heme moeity at a shallow incline relative to the silver surface. Other interactions with polar residues and close van der Waals contacts between hydrophobic residues will stabilize these orientations. At this stage the effect of these forces has not been quantified. A similar orientation was deduced from a study of cytochrome c adsorbed on a negatively charged multilayer surface using optical linear dichroism.40 A calculation based upon monodispersed spherical particles and treating cytochrome c molecules as spheres enable an estimate of monolayer coverage to be achieved. A concentration of 3 × 10-7 M completely adsorbed on the surface is equivalent to monolayer coverage in this idealized situation. However, the presence of buffered hydration spheres enveloping the protein, adsorbed on the flat silver surfaces of the hexagonal particles, may create some discrepancies. Nevertheless, the experimental data indicate monolayer coverage is achieved between 5 × 10-8 and 20 × 10-8 M, not dissimilar from the calculated value. Throughout this concentration study no frequency changes were noted, but marked differences in relative band intensities were observed. The protein matrix of cytochrome c spaces the heme chromophore from the surface and distances second protein layer hemes from the aggregated colloid surface. Classical electromagnetic theory for colloidal particles makes it likely that SERRS intensities will be dominated by contributions from the adsorbed first monolayer. The intensity of the oxidation and spin state marker bands ν4 (A1g) and ν10 (B1g) indicates enhancement of ν10 intensity relative to ν4 upon increasing protein concentration from below to above monolayer surface coverage (Figure 4). As discussed earlier, ν10 should increase in relative intensity as the plane of the heme ring reorientates to an increased angular position relative to the metal surface plane. Confirmation of a more vertically inclining prosthetic group is indicated by the reduced intensity of ν29 (B2g; 1408 cm-1) relative to ν10, upon increasing protein coverage. Therefore, during monolayer formation and conversion to multilayer coverage, protein self-organization on the surface alters to give a more inclined orientation of the heme ring (Figure 4). An extended surface adsorption isotherm was precluded by the occurrence of denaturation at lower concentration (