THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biologq, Inc.

Vol. 263, No. 9, Issue of March 25, pp. 4374-4380,IW Printed in U.S.A.

Surface-enhanced Resonance Raman Scattering Spectroscopy of Bacterial Photosynthetic Membranes THE CAROTENO~D OF R ~ O ~ O S P I R ~ L ~ ~ ~ R~~~~~

(Received for publication, July 13, 1987)

Rafael Picorel, Randall E. Halt$, Therese M. Cotton$, and Michael SeibertBI From the Photoconversion Branch, IISolur Energy Research Institute, Golden, Colorado 80401, the $Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, and the $Department of Biobgieat Sciences, University of Denver, Denver, Colorado 80208

Resonance Raman scattering by the carotenoid, spi- rilloxanthin (Spx), in a suspension of chromatophores (cytoplasmic side out) isolated from the photosynthetic bacterium, Rhodo8pir i~~m rubrum, is greatly en- hanced when the membranes are adsorbed onto the surface of an anodized Ag electrode. The phenomenon is the basis for surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The Spx SERRS peaks observed were at 1505-1510,1150-1155, and 1000-1005 cm” with laser excitation wavelengths ranging between 457.9 and 568.2 nm. Similar peaks were not observed with spheroplasts (periplasmic side out) isolated from the same species. The difference in signal detected in chromatophores and spheroplasts is not due to differences in membrane surface charge, presence of residual cell wall on the spheroplast sur- face, lack of adhesion of spheroplasts to metals, or large differences in pigment content per unit mem- brane area. Instead, the results indicate an asymmetric distribution of Spx in vivo across the membrane (i.e. it is located on the cytoplasmic side of the membrane). The results also demonstrate that the SERRS effect is extremely distance sensitive, and the thickness of a single bacterial membrane (separating the Ag electrode from the carotenoid) is sufficient to prevent detection of Spx spectra. Studies of chromatophores from the F24 strain (a reaction centerless mutant) have pin- pointed B880 antenna complex as the source of the Spx SERRS spectra, and a schematic model of the minimal structural unit of B8SO is presented, This work dem- onstrates the potential of the SERRS technique as a probe for surface topology of pigmented membranes.

The i m ~ r t a n c e of biological membranes for cellular func- tion is unquestioned. A large body of literature has examined the constituents and structure of a wide variety of membranes from many different organisms (1). Membranes are composed of lipid bilayer sheets which provide the structural framework for anchoring proteins and fiposoluble cofactors. The mem- branes themselves provide for compartmentation and spatial biochemical separation of different parts of the cell, and

* This work was supported by Chemistry of Life Processes Grant CHE-8509594 from the National Science Foundation to (T. M. C. and M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

W To whom correspondence should be addressed. I Operated by the Midwest Research Institute for the United States

Department of Energy under contract DE-AC-02-83CH10093.

bound protein complexes account for membrane function, including electron transport, ion transport, and phosphoryl- ation (2).

In photosynthesis pigment-protein antenna complexes ab- sorb light and transfer the energy to a specialized pigment- protein complex called a reaction center (3). The reaction center is the site of primary photochemistry associated with photos~thet ic electron transport (4), and in fact the reaction center isolated from the photosynthetic bacterium, Rhodo- pseudomonas viridis, was the first integral m$mbrane protein to be crystallized (5). An x-ray structure at 3 A resolution was reported recently (6). Several antenna complexes have been purified and characterized from purple photosynthetic bacte- ria (see Ref. 7 for a review). All contain two small hydrophobic polypeptides (a- and &-subunits) that bind bacteriochloro- phyll (BChl)’ and carotenoid with a precise stoichiomet~. Bacterial reaction centers are thought to span the membrane in the intact system (8,9), and so the antenna complexes (10) and chromophores associated with these complexes may be located close to the surface of the membrane. Furthermore, due to the asymmetric nature of the photosynthetic apparatus (111, some chromophores may be found preferentially on one side of the membrane. To test this possibility, we have devel- oped methods to adapt a new Raman scattering technique, called surface-enhanced resonance Raman scattering (SERRS) spectroscopy, for the study of bacterial membrane surface properties.

The unique feature of SERRS spectroscopy is the fact that resonance Raman scattering at the interface between an anodized Ag electrode under potentiostated conditions and a sample adsorbed onto the surface of the electrode is greatly enhanced over resonance Raman scattering of the sample in suspension (12). Furthermore, the effect is extremely distance sensitive (13). These properties provide a powerfu1 means for probing membrane surfaces for highly scattering molecules such as the pigments involved in light absorption and energy transfer processes of photosynthesis. Although structural and functional aspects of biological macromolecules have been investigated over the past 6 years using SERRS spectroscopy (14), the technique has been applied only recently to problems involving biological membranes (15, 16).

This paper reports on the use of SERRS to probe for the presence of the carotenoid, spirilloxanthin (Spx), on the ex- posed membrane surface of chromatophore (cytoplasmic side out) and spheroplast (periplasmic side out) vesicles isolated

The abbreviations used are: BChl, bacteriochlorophyl~ HEPES, N-2-hy~oxyethylpiperazine-N-2-ethanesu~onic acid; SCE, standard calomel electrode; SERRS, surface-enhanced resonance Raman scat- tering; Spx, spirilloxanthin.

4374

SERRS of Bacterial Photosynthetic Membranes 4375

from the photosynthetic bacterium, Rhodospirillum rubrum. The results provide evidence for the location of Spx on the cytoplasmic side of the membrane. The potential importance of this work lies in the apparent ability of this spectroscopic technique to provide structural information about intact in vivo membrane systems on a nondestructive basis using very small amounts of sample material.

MATERIALS AND METHODS

Growth of Mkroorganisms-". rubrum S1 wild type, reaction cen- terless mutant F24 (17), and spirilloxanthinless mutant G9 were grown aerobically in the dark at 30 "C on the medium of Lascelles (18) supplemented with 2 &/liter of yeast extract (Difco). Cells were cultured in 1-liter flasks filled to 80% capacity on a rotary shaker (Lab-Line Orbit Environ-Shaker, Lab-Line Instruments, Melrose Park, IL) a t 200 rpm, Cells to be used for chromatophore preparation were harvested from stationary phase cultures. Those used to prepare spheroplasts were harvested in early stationary phase when the culture reached an equivalent optical density of 1.2 (1-cm pathlength) at 660 nm, corresponding to a BChl concentration of about 1.5-2.5 pg/ml. The cells were washed once in 200 mM Tris-HC1, pH 8.0, and kept in the dark at 4 "C until use.

Prepara t~n of C h ~ ~ ~ o p ~ ~ s - T h e cells in 50 rnM Tris-HCl, pH 8.0, were broken by two passages through a precooled French pressure cell a t 20,000 pounds per square inch. The homogenate was centri- fuged at 20,000 X g for 20 min to remove unbroken cells and large debris, and the resultant supernatant was pelleted at 120,000 X g for 90 min. The chromatophore pellet was suspended in 10 mM Tris- HC1, pH 8.0, and further purified in a 30-55% (w/v) linear sucrose density gradient made up in the same buffer. The material was centrifuged in a Beckman SW 41 rotor a t 150,000 X g for 12 h. The main pigment band at about 40% sucrose was recovered, diluted 3- fold with 10 mM Tris-HC1, pH 8.0, and centrifuged a t 120,000 X g for 90 min. The pellet was resuspended in 50 mM Tris-HC1, pH 8.0, and constituted the purified chromatophore preparation. All procedures were performed at 4 "C, and the samples were kept in the dark at 4 "C until use.

Preparation of Spheropbts-To a F24 cell suspension of final optical density 10 at 660 nm, the following reagents were added at the indicated final concentrations: 200 mlvl Tris-HC1, pH 8.0, 10% (w/v) sucrose, 2 mg/ml egg lysozyme (Sigma, EC 3.2.1.17, Grade I), and 6 mM EDTA previously neutralized at pH 7.3 with NaOH. At the end of a 2-h incubation at 35 "C, almost a11 of the spiral-shaped cells were converted to nonmotile spherical forms as observed under phase contrast light microscopy. Magnesium sulfate crystals up to a final concentration of 50 mM were added to Spheroplast suspension and incubated for 15 min at room temperature with gentle stirring. The suspension then was centrifuged at low speed (7000 x g) for 20 min at 4 "C. The spheroplasts, recovered as a pellet, were carefully resuspended in 200 mM Tris-HC1, pH 8.0, 10% (w/v) sucrose, and 50 mM MgSOc by using a small soft paint brush. After washing three times in the same buffer, the spheroplasts were kept in the dark at 4 "C in the same buffer until use.

Electron Microscopy-Samples were prepared for transmission electron microscopy by the method of Snyder et al. (19). They were first pelleted and resuspended in 3% glutaraldehyde containing 100 mM cacodylate buffer and 5 mM sucrose for 30 min at room temper- ature. Next, they were washed three times for 5 min each in cacodylate buffer. The spheroplasts then were imbedded in 5% agar. The agar was cut into 1-mm cubes, fixed in 1% osmium tetraoxide in distilled water for 1 h, and then washed three times in distilled water. Spher- oplasts were stained in 1% aqueous uranyl acetate for 1 h and washed three times in distilled water. They were then dehydrated in a series of graded ethanol solutions. The cubes were in 15% for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min, 95% twice for 10 min each, and 100% three times for 10 min each. Agar cubes were then infiltrated in 50% ethanol and 50% Epon 812 (E. G. Fullam Go., New York) for 2 h. Each agar block was transferred to 100% Epon 812 for 30 min and transferred to fresh 100% Epon 812 for 1 h. The cubes were placed in a 60 "C oven for 18 h, sectioned (70-80 nm thickness), and poststained first with 2% methanotic uranyl acetate for 10 min then with Reynolds lead citrate for 2 min. The sections were observed using a Hitachi HU-11C electron microscope.

D e t e r m i ~ t ~ n of the ~ C h l / ~ n i ~ of M e ~ r a ~ Surface Area of Spheroplast and Chromatophore Suspensions-The size distribution of F24 spheroplasts was measured directly from photographs taken

W -Working

4-4 Dye iaser 1 Argori C -Counter ton laser R -- Reference

E - Electrode S - Scattered hqht

I

1 hlonochroniator speclroqraph

I I Muillchannel

~ Interface

L""""l

FIG. 1. A schematic diagram of the instrumentation used for resonance Raman and surface-enhanced resonance Raman scattering experiments. Light from an argon ion laser is focused onto a SERRS active Ag electrode located in the sample cuvette or elect,rochemical cell. The Ag working electrode (WE), a Ag/AgCl reference electrode (RE), and a platinum counter electrode (CE) are all connected to a potentiostat. The cuvette contained 15 ml of SERRS buffer (100 mM Na2S04, 300 mM sucrose, and 20 mM HEPES, pH 7.5, unless stated otherwise) and the sample membranes. Light scattered from the interface between the surface of the Ag electrode and the adsorbed membranes is collected by a lens ( S ) and focused onto the input slits of a triplet monochromator. The output is detected by an optical multichannel analyzer.

with a Nikon Optiphot microscope. From the average diameter of a spheroplast, we could calculate the average surface area of a typical spheroplast. The number of spheroplasts per milliliter of suspension was determined using a hemacytometer (VWR Scientific). Knowing the concentration of spheroplasts in the suspension, the surface area of an average spheroplast, and the BChl content of the suspension, one can easily calculate the concentration of BChl per unit area of membrane surface. The BChl concentration per unit of membrane surface area for chromatophores was calculated using the 80 nm diameter and 2.5 X lo"* fig of total BChl content of an average chromatophore (20). These literature values can be used because they do not vary under different cell growth conditions in R. rubrum. BChl concentration was determined from acetone/methanol (72) extracts using a millimolar extinction coefficient of 70 at 770 nm, which is the average of the six most commonly used values for this solvent com- bination (21-23).

Spec~rosco~-Abso~tion spectra were obtained with a Gary 17D spectrophotometer at room temperature using 1-cm pathlength cu- vettes. Resonance Raman and SERRS spectra were recorded using the 457.9, 488.0, or 514.5 nm line (10 mW) of an argon ion laser (INNOVA 90-5, Coherent Inc., Palo Alto, CA) or the 530.9 or 568.2 nm line (10 mW unless otherwise specified) of a krypton ion laser (Innova 100) as an excitation source. The spectrometer geometry is shown in Fig. 1. man-scattered light from the sample cuvette was collected in the backscattering mode and focused through a Canon 55 mm, f/1.2 camera lens onto the slits of the monochromator. A Spex 1877 Triplemate monoc~romator/spect~graph, equipped with a 1200 grooves/mm grating (D-' = 1.4 nm/mm) with a slit width of 0.200 mm in the spectrograph stage, was used to disperse the light across the detector. The detector, an intensified silicon photodiode array (PAR 14201, was maintained at 0 'C. Spectra were acquired and manipulated using OMA I1 (PAR 1215 console and 1218 con- troller) software. Single scans were collected using 60 delays (approx- imately 1-s integration time), and 16 scans were accumulated.

The electrochemical cell (sample cuvette in Fig. 1) used in these experiments was the same a8 described previously (15, 24): Electro- Iyte, consisting of 20 mM HEPES, pH 7.5, 100 mM Na2S0,, and 300

* M. Seibrt, T. M. Cotton, and J. G. Metz, submitted for publica- tion.

4376 SEXRS of Bacterial Pho tos~n~he t~c Membranes mM sucrose in deionized/distilled water, was degassed by N, purging prior to addition of the biological samples and subsequent anodization of a polished Ag electrode. The Ag electrode was prepared from flattened polycrystalline Ag wire (Goldsmith, Evanston, IL) and sealed into a glass holder using Torr Seal (Varian Assw., Palo Alto, CA). Anodization of the electrode was accomplished in the presence of the sample but in the absence of light. The anodization procedure itself consisted of stepping the electrode from -0.6 V uersm SCE (all potentials will be referenced to SCE hereafter) to +0.45 V and allowing 25 mC/cm2 charge to pass, followed by stepping the electrode back to -0.6 V, This procedure allowed us to observe enhancement of resonance Raman signals. Similar enhancement of spectra were also observed when sample membranes were added after anodization of the Ag electrode (data not shown). This demonstrates that SERRS spectra reported here are due to chemical species in their natural environment and not to pigment molecules extracted from the mem- brane during the anodization step. During all experiments reported below, the electrode potential was maintained at -0.2 V. Samples were not exposed to light between monitoring scans since extended exposure (several minutes) to laser light decreased the amount of observable Spx SERRS signals (data not shown).

RESULTS

Fig. 2 shows the absorption spectra of chromatophores isolated from R. rubrum S1, F24, and G9. All spectra display a main absorption band in the near infrared region due to the B880 antenna complex (B870 in G9), the only antenna com- plex present in R. rubrum (25, 26). The minor bands at 800 and 755 nm are the result of absorption by the reaction center, which is absent in the F24 strain. The three main absorption bands in the visible region (450-570 nm) correspond to the carotenoid, spirilloxanthin, which represents Z99% of the colored carotenoid present in R. rubrum (27). This carotenoid is completely absent in the G9 strain as seen from its spec- trum. Fig. 2 also shows the chemical structure of the Spx, a symmetric isoprenoid chain with conjugated double bonds.

Resonance Raman spectra of S1 and F24 chromatophores have peaks at 1508 (ul), 1150 (u2), and 1003 ( u s ) cm", but no equivalent peaks are observed in G9 chromatophores (Fig. 3). The frequency of the above-mentioned peaks and the fact that they are absent in G9 demonstrate that 488.0 nm of light excites Spx resonance Raman spectra. Furthermore, the ob- servation of a Spx resonance Raman spectrum in chromato- phores of the reaction-centerless mutant F24 demonstrates that the antenna complex is the source of the Spx spectra. The peak at 1508 is assigned to the C=C stretching mode of the isoprenoid chain of the carotenoid (28, 29). The band at 1150 is generally attributed to C-C bond stretching of the isoprenoid chain, but important contributions from other modes are very probable. The 1003 cm" peak is assigned

$31

~ ' 0 8 a 1150

1003

F24

G9

Raman Shffi (em-')

FIG. 3. Resonance Raman spectra of chromatophores iso- lated from the S1, F24, and G 9 strains of R. rubrum. The frequencies of the peaks are reported in em", and in the case of S1 and F24, the spectra correspond to scattering by the carotenoid, spirilloxanthin. X,, = 488.0 nm, BChl = 0.9 mg/ml.

1508

A 1151

s1 /i % 1000

I

F24 1001

I Ramen Shlft (em-')

FIG. 4. SERRS spectra of chromatophores isolated from the S1, F24, and GB strains of R. rubrum. The frequencies of the peaks are reported in cm", and the accompanying trace in each case is a spectrum of the sample on the polished electrode prior to anodization. In the case of the S1 and F24 strains, the labeled peaks are those of spirilloxanthin. = 488.0 nm; BChl = 0.33 pg/ml.

450 550 650 750 850 Wavelength (nm)

FIG. 2. Absorption spectra of R. rubrum chromatophores isolated from the S1 (-), F24 (-----), and G9 (---) strains. The vesicles were suspended in 10 mM Tris-HC1, pH 8.0. The struc- ture shown is that of s~irilloxant~ln.

mainly to the stretching of C-C bonds between the methyl groups and the main chain. No BChl spectral peaks at 1640, 1609, 1529, 1502, 1344, 1286, 1153, and 1060 cm" (28) are observed in any strain. This is predictable because BChl is a weaker resonance Raman scatterer at these excitation wave- lengths than carotenoids (Le. BChl does not have a major electronic absorption band in this region of the spectrum).

Fig. 4 presents SERRS spectra (Aex = 488.0 nm) of chro- matophores from the three different R. rubrum strains. Again Spx peaks at 1508, 1151-1152, and 1000-1001 cm" are ob- served with S1 and F24 while no carotenoid peaks are ob- served with G9. It should be emphasized that there is a large difference in the BChl concentration of the samples used in the experiments of Figs. 3 and 4. This illustrates the extreme sensitivity associated with the SERRS technique. Spirilloxan- thin resonance Raman spectra at the BChl concentration

SERRS of Bacterial Photosynthetic Membranes 4377

used to obtain SERRS spectra are barely detectable (see Fig. 4).

To characterize better resonance Raman and SERRS spec- tra from Spx, F24 chromatophores were excited at different Wavelengths. Fig. 5 shows resonance Raman spectra excited with 457.9, 488.0, 5145,530.9, or 568.2 nm of laser light. All spectra show similar shape, but the intensity ratio between the 1508 and 1150 peaks varies depending on the excitation wavelength. The ratio changes are due to resonance enhance- ment arising from the individual vibronic bands comprising the lowest energy electronic transition. Fig. 6 presents SERRS spectra excited at the same wavelengths. As can be seen, the SERRS spectra are all of similar shape but again display different peak intensity ratios. This similarity suggests that a large resonance contribution to the enhancement mechanism is operative. The intensity ratios of the major resonance Raman and SERRS peaks are comparable at each excitation wavelength, as seen in Table I.

Fig. 7 shows coverage-dependence curves (areas under the SERRS peaks corrected for the amount of bulk resonance

1507 n 1150

4 5 7 * g J u . . . .

Raman Shift (cm-')

FIG. 5. Resonance Raman spectra of chromatophores iso- lated from the F24 strain of R. rubrum excited at 467.9, 488.0, 614.6, 630.9, and 668.2 nm. The laser excitation power was 25 mW at 568.2 mm and 10 mW at all other wavelengths. BChl = 50 pg/ml.

Raman Shift (cm-I)

FIG. 6. SERRS spectra of chromatophores isolated from the F24 strain of R. rubrum excited at 467.9, 488.0, 514.6, 530.9, and 668.2 nm. Laser power was the same as in Fig. 5. BChl = 0.33 rg/ml.

TABLE I Intensity ratio of the 1508 c m - ~ / l l 5 0 cm" resoname Raman and

SERRS peaks at different ~ a u e k ~ t ~

Type of Raman Excitation wavelength spectroscopy 457.9 488.0 514.5 530.9 568.2

nm Resonance Raman 1.1 1.2 1.8 1.1 0.9 SERRS 1.0 1.0 1.6 1.0 0.9

1 I

BChl @a) FIG. 7. SERRS signals obtained at different sample BChl

concentrations in the sample cuvette. Open symbols (0, 0, A) designate areas under the 1150 cm" peak of Spx, and closed symbols (0, H, A) denote the 1508 cm" peak. Circks indicate chromatophores from the F24 strain of R. rubrum suspended in the normal buffer while squares represent F24 chromatophores suspended in buffer with 50 mM MgSOO, substituted for 100 mM Na2S0,. The triangles repre- sent F24 spheroplasts suspended in ~ g S O ~ - s u ~ t i t u t e d SERRS buffer. Samples with indicated BChl values in m i c ~ ~ ~ s were suspended in 15 rnl of SERRS buffer. See tex t for an explanation of the numbers in parentheses. = 488.0 nm.

Raman scattering present in the sample) for the 1508 and 1150 cm-l SERRS peaks of F24 chromatophores where the BChl content in the sample cuvette is varied. The rise in signal is due to increasing coverage of the Ag electrode with membrane. Saturation is reached at a BChl concentration of about 0.7 pg/ml. The decrease in signal at higher BChl con- centration is due to reabsorption of SERRS light by the increasing concentration of chromatophore membranes used to generate this part of the saturation curve. Also shown on the same plot are the results when 50 mM MgSO, is substi- tuted for 100 mM Na,SU, normally present in the SERRS buffer. The substitution of MgSO, decreases the apparent SERRS signal by about 20%, and data for chromatophores under these conditions are given for comparison because spheroplasts (to be examined next} require the presence of MgSO, for stability and maintenance in suspension.

In an attempt to determine on which side of the membrane Spx i s located, SERRS spectra of F24 chromatophores, and spheroplasts are compared in Fig. 8. As can be seen, there is little evidence for SERRS peaks attributable to Spx in the spheroplasts while there are large signals associated with chromatophores. This suggests that Spx is located on, or near to, that surface of the chromatophore membrane which cor- responds to the cytoplasmic side of the whole cell membrane. However, several possible artifacts might account for the lack of SERRS signal in spheroplasts. These include the presence of cell wall around the spheroplast membrane, nondeinvagin- ation of the intracytoplasmic membranes during spheroplast preparation, low BChl content/unit membrane surface area in spheroplasts, lack of spheroplast absorption onto the Ag electrode, and different surface charges on the periplasmic and cytopiasmic side of the membrane.

Fig. 9 shows a transmission electron micrograph of an ultrathin section of F24 cells and spheroplasts. In whole cells,

4378 SERRS of Bacterial Photosynthetic Membranes

1506 A 1151

J Raman Shift (cm-l)

FIG. 8. SERRS spectra obtained f rom chromatophores (Chr) and spheroplasts (Sp) of R. rubrum, F24 st ra in. Note that little evidence for the presence of spirilloxanthin signal is seen in the spheroplast preparation. X,, = 488.0 nm; RChl = 0.33 pg/ml (chromatophores) or 0.66 pg/ml (spheroplasts).

the cell wall is easily distinguishable from the cytoplasmic membrane. The intracytoplasmic membranes constitute the chromatophore fraction which contains most of the photosyn- thetic pigments. In spheroplasts, the original intracytoplasmic membranes are deinvaginated, and the remaining cell wall covers less than 20% of all spheroplast membrane. Therefore the spheroplast membrane used in the present work has direct contact with the Ag electrode surface, a prerequisite for de- tection of SERRS spectra.

Another possible explanation for the lack of SERRS signal in the spheroplast preparation might be a vastly lower pig- ment content per unit surface area of membrane in the case of the spheroplast preparation compared to the chromato- phores. This might be due to preferential location of pigments on the intracytoplasmic membranes. If the ratio of cyto- plasmic to intracytoplasmic membranes were very large, little Spx might be detectable in the case of the spheroplasts which are composed of both types of membrane. (Since the sphero- plasts are highly devoid of cell wall, the original intracyto- plasmic membranes are deinvaginated and also are available for direct contact with the Ag SERRS electrode). As can be seen in Table 11, the pigment content on a BChl per unit of membrane surface area basis for the two different prepara- tions is found to be within a factor of 1.7. This information and the SERRS saturation curve data of Fig. 7 can resolve this issue. Compare point (I) in Fig. 7 for F24 chromatophores, which is the BChl concentration a t which we can just detect a SERRS signal, with point (2), which is about the BChl concentration one would expect to see maximum SERRS Spx signals in spheroplasts if they displayed the same saturation properties as chromatophores. As can be seen, spheroplasts,

\

ICM

FIG. 9. Transmission electron micrographs of whole cells ( A ) and spheroplasts ( B ) f rom R. rubrum, F24 strain. The spherical invaginations located on the periphery of the whole cell cytoplasm are the intracytoplasmic membranes ( I C M ) isolated as chromatophores in this study. The spheroplast preparation shows little residual cell wall ( C W ) material. Such material, if present, might separate the Ag electrode from the surface of the spheroplast membrane by a sufficient distance to prevent our detectingcarotenoid SERRS peaks. Since little cell wall is left, SERRS signals should be observable if carotenoids are present on the periplasmic side of the spheroplast membrane. Cytoplasmic membrane ( C M ) .

at 210 times the BChl concentration required for observation of Spx signals in chromatophores, do not display significant Spx signals. Thus, a factor of 1.7 difference in pigment content

SERRS of Bacterial Photosynthetic Membranes 4379

TABLE I1 Properties of s p h e r o ~ ~ ~ and c h ~ m a t o p ~ ~ s used

in the present work Vesicle

area Sample Diameter surface BChl/vesicle surface area BChljunit

p m pm2 d m a

S p h e ~ p l ~ t 3.06 29.45 2.19 X lo-' 0.74 X 10"' Chromatophore 0.08" 0.02 2.50 X 10-'*" 1.25 X 10"' ' From Ref. 20.

-0.8 -0.6 -0.4 -0.2 0 Potential (V vs. SCE)

FIG. 10. Potential profile of Spx SERRS signals. The 1508- cm" SERRS peak of chromatophores (m) and spheroplasts (A) of R. rubrum S1 is plotted as a function of potential (V uersus SCE) applied to the Ag working electrode in the sample cuvette. X.. = 488.0 nm; BChl = 0.67 pg/ml.

per unit of membrane surface area cannot account for the lack of Spx SERRS peaks in the spheroplast preparation.

In order to determine if the apparent lack of signal in the spheroplasts is due to differences in surface charges on the periplasmic and cytoplasmic sides of the membrane, we in- vestigated the amount of SERRS signal observed as a function of the potential imposed on the Ag electrode. Fig. 10 shows that chromatophore SERRS signals are seen between poten- tials of -0.8 V (where H, is evolved) and "0.1 V (where the Ag on the surface of the electrode starts to oxidize). No signal is observed over the same potential range for spheroplasts. The lack of signal in the case of spheroplasts is not due to the lack of spheroplast absorption onto the SERRS electrode since spheroplasts are known to absorb onto metal surfaces (30), and since we have observed SERRS signals associated with FAD in spheroplasts?

DISCUSSION

This paper represents an extensive application of SERRS spectroscopy to the investigation of a biological membrane topology problem. The work clearly demonstrates our ability to use the technique to detect the presence of chromophores on the surface of chromatophore membranes isolated from photosynthetic bacteria. The frequencies of the major reso- nance Raman and SERRS peaks (1505-1510,1150-1155, and 1000-1005 cm") in chromatophores isolated from the S1 strain of R. rubrum, and the fact that the bands are absent in chromatophores isolated from the G9 strain (a spirilloxan- thinless mutant) allow us to conclude that the spectra arise from spirilloxanthin, the major colored carotenoid present in this organism (27). Spirilloxanthin is a chemical constitutent of both the bacterial reaction center and the B880 antenna complex of R. rubrum (21, 25, 26) but is present in different structural conformations in the two complexes. Lutz et al. (31) have shown that Spx is "cis-trans" in the reaction center and "all-trans'' in the B880 antenna complex. Since the same

R. Picorel, T. Lu, R. E. Holt, T. M. Cotton, and M. Seibert, manuscript in preparation.

Spx spectra are seen in the reaction centerless F24 mutant of R. rubrum as in the $31 strain (Figs. 3 and 41, we conclude that the B880 complex is the primary source of the resonance Raman and SERRS spectra.

SERRS spectroscopy is a distance-sensitiv? technique ( i e . extremely dependent on the distance [on an A scale] between the surface of the Ag electrode and the location of the chro- mophore of interest), Thus, the fact that we observe Spx spectra with chromatophores (cytoplasmic side out) but not with spheroplasts (periplasmic side out) in Fig. 8 indicates that the carotenoid is preferentially located in close proximity to, or on, the cytoplasmic side of the bacterial photosynthetic membrane in this species. Appyently, the thickness of a single bacterial membrane (-50 A from the outside point of the lipid polar head groups across the bilayer) is sufficient separation from the Ag electrode to eliminate detectable Spx SERRS spectra. We also have demonstrated previously that a single 33-kDa extrinsic protein can mask a surface-enhanced Raman signal, which is indirectly related to Mn, located on the surface of isolated photosystem I1 membrane fragments from spinach (E).'

The results of the current study are consistent with a preliminary SERRS study showing that carotenoids are pref- erentially detected in chromatophores uersw spheroplast- derived vesicles (24). Small amounts of carotenoid spectra seen in the latter material were related to chromatophore contamination of the spheroplast-derived vesicle preparation. The results of the present work indicate that there is no significant chromatophore con~mination in our spheroplast preparation. Furthermore, the structural intactness of our spheroplast preparations was over 92% as judged by control (results not shown) malate dehydrogenase activity measure- ments (32). Thus, in the case of R. rubrum, spheroplast preparations are better material than spheroplast-derived ves- icles for membrane surface topology studies because they are a more homogeneous preparation than spheroplast-derived vesicles. This is reasonable considering that less physical manipulation is needed to prepare spheroplasts.

Since the intensity ratios of the two major resonance Ra- man and SERRS peaks are comparable at each excitation wavelength (Table I), we cannot determine what end of the Spx molecule is closer to the surface of the membrane from our data. This is because Spx is symmetric on both ends, and the observable SERRS peaks are not characteristic of any specific parts of the molecule. However, from the above con- clusions and the results of several other laboratories, we can make some statements about the organization of the B880 antenna complex. Linear dichroism experiments, for example, show that the isoprenoid chain of carotenoids in chromato- phores of several photosynthetic bacteria is oriented on the average at about 45" to the plane of the membrane (33), but there is no specific information on B880 complex of R. rub- rum. In addition, in R. rubrum chromatophores: (a) the ele- mentary structural unit of the B880 antenna complex prob- ably consists of one a-apoprotein, one ~-apoprotei~, two BChl, and one Spx (34); ( b ) BChl associated with the B880 complex is supposed to be located near the periplasmic side of the membrane based on protein sequencing data (35); ( c ) Spx has to be close to BChl due to the high efficiency of energy transfer (36); ( d ) the length of the Spx molecule in the all-trans c o n ~ ~ r a t i o n is between 35 and 40 A, depending on the exact conformation of the two ends of the molecule4; ( e ) the protein moiety of the B880 complex contains a high proportion of a-

The length of the Spx molecule was calculated using the XIC- AMM" (Xiris Corporation, Monmouth, NJ) molecular modeling sys- tem.

4380 SERRS of Bacterial Photosynthetic Membranes

FIG. 11. A proposed model for the elementary structural unit of the BSSO antenna complex of R. rubrum. This ~ i n i ~ elementary unit in vivo would be aggregated to other equivalent units making a large complex that surrounds the reaction center in the membrane. The a- and @-subunits span the membrane with their C- terminal and N-terminal regions exposed to the periplasm and cyto- plasm, respectively. The orientation and the structure of the protein subunits determine the specific positions of the pigments (2 BChl and 1 Spx) bound to the complex. The two interacting BChls (the ring orientation is indicated by the planes denoting the Qx and Q, transitions) are coordinated to the histidines of the hydrophobic (Y-

helix domains of the apoproteins. The Spx in the all-trans configu- ration is located close to the BChl, but at the same time one end of the molecule is exposed to the cytoplasm. In this model the structure of the polar domains of the subunits is schematic.

helix (37, 38); ( f ) the a-helices are oriented at an angle of about 70" to the plane of the membrane (37); (g) the plane of the BChl rings are tilted out of the membrane plane (3!); and (hf the distance between the BChl planes is about 10 A (39). Fig. 11 is a schematic model of the elementary structural unit of the B880 complex taking the above information into ac- count. Our data suggests that one end of the Spx molecule must be very close to the cytoplasmic surface in order to be detectable by SERRS in chromatophores but undetectable in spheroplasts.

Finally, our conclusion regarding the asymmetric location of Spx on the cytoplasmic side of the bacterial membrane supports previous predictions from antibody (40) and protein- ase treatment (40,41) studies. However, the most far-reaching effect of the present study is the demonstration of the poten- tial role for SERRS spectroscopy in probing the surface topology of biological membranes on a nondest~ctive basis.

A c & n o w ~ d g ~ n ~ - W e would like to thank Dr. J. Snyder for doing the electron microscopy, Dr. J. S. Connolly for calculating the length

of the Spx molecule, and Drs. P. F. Weaver, T. H. Lu, and P. Callahan for helpful discussions.

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