Plrororhrmisrry and Phofobiology. 1975. Vol. 22. pp. 3 7 - 4 6
Pergamon Press. Printed in Great Britain
ISOLATION AND PARTIAL CHARACTERIZATION OF THE PHOTOCHEMICAL REACTION CENTER OF CHROMATIUM VINOSUM (STRAIN D) LILYLINand J. PHILIPTHORNBER Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024, U.S.A.
(Received I 1 November 1974; accepted 7 March 1975) Abstract-A preparation of the photochemical reaction center of Chromatiurn has been obtained by chromatography of lauryldimethylamineoxide-solubilized chromatophores on hydroxylapatite and Sepharose columns. The procedure has yielded a reaction center preparation from both carotenoidcontaining and carotenoid-deficient Chromatiurn cells. Preliminary analysis of the isolated component indicates that the photochemical reaction center of the Thiorhodaceae is homologous to that of the Athiorhodaceae. In particular, the near infrared absorption spectrum of the Chromatiurn reaction center preparation shows the same triple-peaked spectrum observed for reaction center preparations from the Athiorhodaceae. The Chromatiurn preparation undergoes a rapid light-induced oxidation and dark reduction of the reaction center. The ratio of the reaction center to the two membrane-bound cytochromes (cytochrome c s 5 2 and c 5 s 5 )is greatly increased over the ratio observed in chromatophores or in other previously isolated, reaction center-enriched subchromatophore fractions of this organism.
INTRODUCTION
Photochemical reaction centers of photosynthetic bacteria have been successfully isolated essentially devoid of light-harvesting chlorophylls from several members of the Athiorhodaceae, predominantly from Rhodopseudomorias spheroidrs (Reed and Clayton, 1968; Jolchine et al.. 1969; Feher, 1971; Segen and Gibson, 1971; Slooten, 1972) and Rhodospirillum rubrum (Gingras and Jolchine, 1969; Niiel et al., 1972; Smith et al.. 1972; Wang and Clayton, 1973). While chromatophores of both wild type and carotenoidless mutant strains have yielded a reaction center preparation after treatment with a variety of detergents, such as the anionic sodium dodecyl sulfate (SDS), the cationic cetyl trimethyl ammonium bromide (CIAB), and the zwitterionic lauryl dimethylamine oxide (LDAO), only carotenoidless mutant strains have yielded to solubilization with the nonionic detergent Triton X-100. In the family of the 7hiorhodaceae, specifically Chrornatiurn Strain D, complete dissociation of the reaction center from light-harvesting bacteriochlorophyll has not been achieved previously by treatment with Triton X-100(Garcia et al., 1966; Ke et al., 1968; Ke and Chaney, 1971) nor with SDS (Thornber, 1970). The best enrichment of the reaction center of Chronlatiurn is Fraction A of Thornber (1970). Fraction A was obtained by hydroxylapatite chromatography of SDS-solubilized chromatophores. The mol wt was estimated to be over 6 x 10'. The molar ratio of reaction center bacteriochlorophyll (P883) to the light-harvesting bacteriochlorophyll (B890) in Fraction A was 1/40. The fraction also contained both the high potential c-type cytochrome, C555, and the low potential, C5.52. in the ratio of
P883:C555:C552 = 1:2:7: carotenoids were also present. The following year Ke and Chaney (1971) attempted to isolate the reaction center from carotenoid-deficient cells of Chromatiurn using Triton X100. The active fraction, termed the Heavy Band (Hband), was enriched in the reaction center relative to the whole cells. However, both preparations (Fraction A and the H-band) still contained substantial amounts of light-harvesting bacteriochlorophyll and were spectrally similar. The zwitterionic detergent, LDAO, has been used successfully to isolate the reaction center from carotenoidless mutants of Rhodopseudomonas spheroides (Feher, 1971) and Rhodospirillum rubrum (Wang and Clayton, 1973) as well as from wild type cells of Rsp. rubrurn (Noel et al., 1972). We have found that Chromatiurn chromatophores will also yield to LDAO solubilization. It is the purpose of this communication to present some preliminary characteristics of the reaction center isolated from wild type and carotenoid-deficient cells of this organism. MATERIALS AND METHODS
An inoculum of Chromatiurn was kindly provided by Dr. R. G . Bartsch (University of California, San Diego). The cells were grown anaerobically in completely filled 1 screw-cap bottles in a modified Bartsch medium- (Bose, 1963). Continuous illumination was provided by 25 W tungsten bulbs giving an intensity of 1080 21601ux/m2 falling on the glass surface. Temperature remained between 28-30°C. Cells were harvested by centrifugation, 4000 g for 5 min, during the stationary phase (7-day-old cultures). Packed cells were wet frozen (- 18°C) if not used immediately. Chromatiurn cells were cultured both in the presence (Wassink and Kronenberg, 1962) and absence of diphenylamine (DPA). Cells growing in the presence of DPA appear green due to inhibition of carotenoid synthesis,
-
37
LILYLIN and J PHILIP THORNRER
38
whercas cells grown in the absence of DPA appear red due to the presence of the normal full complement of carotenoids. Hereafter, cells grown in the presence of DPA will 08be termed green cells. and those grown in its absence will be termed red cells. Packed cells were resuspended in an equal vol of 50 mM 06Tris-HCl, pH 8.0, passed twice through the French Pres- 0 sure Cell at 1 x 10' N/m2 (15,000 p.s.i.), and centrifuged at 20,OOOg for 15 min. The supernate contained the pig- d 0.4mented membranes, and its absorbance at 590 nm was a used to estimate the bacteriochlorophyll concentration assuming cS9"= 20mM-' cm-' (Thornber, 1970). A 1% 0.2(v/v) solution of LDAO in the ratio of detergent/bacteriochlorophyll = 40/1 (g/mmol) was added slowly to the 001 I I I I I I I I supernate and stirred for 3 h at room temp. Detergent300 400 500 600 solubilized material was loaded onto a hydroxylapatite Wavelength, nm column (Siegelman ef al., 1965). For the column. a 50cm3 Luer-Lok glass syringe was packed to give a vol of 30 m f I n. hydroxylapatite. and equilibrated with 5 0 d of 0.01 M sodium phosphate 0.2M NaCl, pH 7.0. This size column easily accommodated 25 pmol bacteriochlorophyll in the detergent extract derived on the average from 5 g of packed cells. The light-harvesting bacteriochlorophyll complexes are adsorbed onto the stationary phase allowing m 0 6 the reaction centercontaining fraction to move down the c column. The column was washed with 50 mC of the equilig 0.4brating buffer. The reactive material was precipitated from the eluate by adding solid (NH4)S04 to a final con- a centration of 25% (w/v). and centrifuged at 20,OOOg for 15 min. Precipitate adhering to the walls of the tubes and 0.2 as a floating pellet was removed and dissolved in 50mM Tris-HC1, pH 8.0. For further purification, the ammonium 0.0 600 700 sulfate precipitate was redissolved in a minimal volume BOO 900 (2 m/) of 50mM Tris-HC1, 0 2 M NaC1. 0.057" LDAO, Wavelength, nm pH 8.0 and chromatographed on a 3 x 40 cm Sepharose 6B (Pharmacia, Piscataway, New Jersey) column previously Figure 1. Absorption spectra of the reaction center prepequilibrated with the same buffer. The flow rate was kept aration; (a) in the visible region and (b) in the near IR at 0.2 m/ per min; 4 mf fractions were collected and assayed region. The solid trace was recorded by a weak beam of spectrophotometrically. This procedure removed a fraction monochromatic light, whereas the dotted trace was containing oxidized bacteriochlorophyll and bacteriopheo- recorded while a strong white light illuminated the sample. phytin from the photochemically active material. The absorption spectra were measured on an Aminco-Chance DW-2 Dual Wavelength spectrophotometer. Photochemical activity was measured by examining light-induced ab- creased in absorbance with a concomitant loss of sorbance changes of the reaction center in the near infrared absorbance at 800 and 875nm. The 589nm absor(8W900nm). The actinic light source was obtained from bance is attributed to the Q,-band of the bacterioa 30 W. GE 1493 tungsten lamp of a microscope slide ilchlorophyll a molecules in the preparation. The three luminator (American Optical Company). Actinic light was filtered through a Corning glass filter number 5433. or peaks at 530, 503 and 480nm are observed in the alternatively a I M copper sulfate solution I cm thick was reaction center preparation from the red cells, but used. A Corning red cut-off glass filter number 2403 was only the 530nm absorbance is present in the caroused to prevent stray exciting light from falling on the tenoid-deficient green cell preparation. It thus is very photomultiplier.
g g -
+
g
RESULTS
The absorption spectrum of the reaction center preparation from Chrornatiurn is shown in Fig. 1. The most probable assignment of the absorption bands to components in the preparation is as follows: The absorbance at 875 nm is due to the primary electron donor molecule, P870 and the 800 nm absorbance is due to the closely associated pigment, P800. The 756 nm-peak is attributed to bacteriopheophytin a while the 689nm-peak is most likely a degradation product, oxidized bacteriochlorophyll (Reiss-Husson and Jolchine, 1972). If a LDAO/BChl ratio greater than 50/1 is used to dissociate the pigmented membranes in the isolation of the photochemical reaction center, or on prolonged exposure of the preparation to light and air, the 756 and 689nm-peaks are in-
probable that the 480 and 503nm absorbances are due to carotenoids, as is also the absorbance at 445 nm. Absorbance in the region of 530 nm has been seen in all reaction center preparations obtained so far from purple photosynthetic bacteria regardless of whether wild type cells or carotenoidless mutants were used as the starting material, and regardless of the nature of the detergent used to dissociate such membranes. Various authors (Jolchine et al.. 1969; Reed, 1969; Reed and Peters, 1972; Slooten, 1972) have ascribed this absorbance to bacteriopheophytin a. In the Soret region, three peaks are observed at 366 nm, 386 nm. and 404 nm. Hemoprotein absorption may account for the 404 nm-component (Smith rt al., 1972) whereas the 366nm and 386nm-peaks are almost certainly due to the Soret peaks of bacteriochlorophyll and bacteriopheophytin respectively. In general, the reaction center of Chrounariutir bears a
39
The photochemical reaction center of chromatiurn
I W t
t
I
AA-O.OO5
t No additions
Ascorbate
Dithionite
sodium dithionite difference spectrum (Fig. 3a) shows decreases at 552 and 422 nm which are almost certainly due to absorbance changes in the a- and ybands of the low potential c-type cytochrome, C552, of Chromatiurn chromatophores. In Fig. 3b, the oxidized (K3Fe(CN),)-minus-reduced (sodium ascorbate) spectrum reveals the presence of the high potential c-type cytochrome, C555. Reduction of the isolated material by sodium ascorbate (Fig. 3b) shows that most of the C555 is in the reduced state on isolation,
Figure 2. Light-induced absorption changes at 875 nm of the reaction center preparation, recorded as described in the Results section. The arrows indicate when the actinic light was turned on and off. Sodium ascorbate (100 mM) and sodium dithionite (10 mM) were added to the cuvette as indicated.
0 02
g
great spectral similarity to the reaction center preparations from other bacteriochlorophyll a-containing organisms. Upon exposure of the preparation to strong actinic light, or on chemical oxidation, the preparation exhibits a behavior typical of other light-oxidized reaction ccnter preparations. There is a disappearance of the peak at 875 nm and a hypsochromic shift of P800 from 799 nm to 796 nm. Figure 2 illustrates the kinetics of light-induced changes in the near infrared. On illumination, there is a reversible bleaching with a rapid absorbance decrease a t 875 nm and an almost equally rapid dark recovery. Interestingly, the kinetics of the light-induced absorption changes in this preparation are much more rapid than those in fractions previously obtained for Cltrormtiurti (cf. Garcia rt al., 1966; Ke, ut a/., 1968; Thornber. 1970; Ke and Chaney, 1971). Photo-oxidation and dark reduction occur with no additions of exogenous electron donors or acceptors. On the addition of sodium ascorbate the apparent light-induced absorbance change is lessened while the even stronger reducing agent, sodium dithionite, completely obliterates the change (Fig. 2). This complete loss of change in absorbance is almost certainly due to the chemical reduction of the primary acceptor, such that no photochemistry can occur. The precise location of this long wavelength maximum in the LDAO-prepared material varies from 877 nm to 869 nm with increasing detergent concentration. The stability of the reaction center preparation decreases in relation to the position of the wavelength maximum: the shorter the wavelength, the less stable is the preparation; that is, within a few hours after isolation a preparation with a maximum a t 869 nm loses most of its ability to undergo reversible photobleaching. The chemically oxidized (K,Fe(CN),)-minusreduced (Na2S204)difference spectrum -(Fig. 3c) in the near infrared also shows a maximum absorbance decrease at 875 nm together with an equally large decrease a t 808nm. The absorbance difference at 600 nm is very probably due to the loss of absorbance of the Q,-band of the P870 chlorophyll molecules. In the visible region, the sodium ascorbate-minus-
-
001
c $
0
::-001 n 4
-
-0.02
-003-
-
-0.04-
442 I
I
-005
I
I
I
I
Wavelength
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I
004-
I
-
\
406
0.021-
-
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-
5 600
700 1
-00311 -004
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300
0
0
400
500
Wavelength
0.03
0.02
\I \
-0.02 -0.03
"""
900
I -
1000
Wavelength
Figure 3. Oxidized-minus-reduced difference spectrum of the reaction center preparation (a) in the visible region of sodium ascorbate minus sodium dithionite, (b) -untreated-minus-sodium ascorbate; - - - potassium ferricyanide-minus-sodium ascorbate; ' . . potassium ferricyanide-minus-sodiumdithionite; (c)in the near IR region of oxidized (potassium ferricyanide)-minus-reduced (sodium dithionite).
LILYLINand J. PHILIP THORNBER
40
and furthermore, that some of the low potential cytochrome, C552 is reduced by ascorbate. It is perhaps noteworthy that the maximum absorbance decrease in this case occurs at 553 and not 552 nm. The ratio of cytochrome/reaction center is greatly decreased in this reaction center preparation from that observed in chromatophores or subchromatophore fractions of this organism. The light-induced absorbance change is almost negligible (one twentieth) compared to the change at 875 nm. Nevertheless, the cytochromes are the same two present in chromatophores (Parson, 1969), subchromatophore fractions (Case et al., 1970; Thornber, 1970), and in a cholate-solubilized cytochrome preparation (Kennel and Kamen, 1971). These two cytochromes are both associated with the reaction center bacteriochlorophyll P870 (For a review of this topic, see Parson, 1974). The number of polypeptides present in the isolated material has been briefly examined. The preparation was brought to 100°C for 1 min in 1% SDS. and the resulting dissociated material was electrophoresed on 8% SDS-polyacrylamide gels in 50 mM Tris-HC1, 0.1% SDS, pH 8.0 (Weber and Osborn, 1969). After staining with Amido Black, 5 protein zones were observed, 3 of which were particularly prominent and m a y possibly correlate with the 3 polypeptides observed in other reaction center preparations (Feher. 1971; Okamura er al.. 1974). There was little difference between the preparation from the green and red cells apart from the expected decreased carotenoid content in the fraction from the green cells.
bacteriochlorophyll by further fractionation of Fraction A were not successful. It was better to start with whole chromatophores and to use a different detergent (LDAO) than that which yielded Fraction A. Chromatography of LDAO-treated pigment membranes provided a reaction center preparation from both red and green cells. The preparation is essentially devoid of light-harvesting bacteriochlorophyll (B800,B850, B890); studies are in progress to try to eliminate the small amount of contaminating 689 nm absorbance from the fraction. It is noteworthy that after removal of bulk bacteriochlorophyll and most of the carotenoids from the photochemical reaction center, the maximum absorbance change due to P870 is shifted to wavelengths shorter than the 883 nm observed for Fraction A. The characteristics of the Chror~atiurii reaction center preparation determined so far indicate that the reaction center preparations of purple photosynthetic bacteria are spectrally quite similar. Thus, although Chromatium is classified in a taxonomic family (Thiorhodaceae) different from that of Rhodopseudomonas and Rkodospirilluni (Athiorhodacrar) on the basis of metabolism, the basic mechanism for survival. the photochemical reaction center, maintains spectral homology among these different families of bacteria. The premise that the reaction center is homologous between the two families will be tested by further characterization of the biophysical and biochemical properties of the isolated reaction center preparation.
DISCUSSION
Ackriowlrdgmrnts-Research was supported by NSF GB 31207 to J.P.T. and by NSF Traineeship to L.L. LDAO was kindly donated by the ONYX Chemical Company (Los Angeles office) of Jersey City, New Jersey.
Preliminary experimentsin which we tried to obtain a reaction center preparation free from light-harvesting
REFERENCES
Bose. S. K. (1963) In Bacterial Photosyrithrsis (Edited by H. Gest, A. San Pietro, and L. P. Vernon), pp. 501-510. Antioch, Yellow Springs, Ohio. Case, G. D., W. W. Parson and J. P. Thornber (1970) Biochim. Biophys. Acta 223. 122-128. Feher. G. (1971) Photochem. Photobiol. 14. 373-387. Garcia, A,, L. P. Vernon and H. Mollenhauer (1966) Biochem. 5. 2399-2407. Gingras, G., and G. Jolchine (1969) In Progress in Photosynthesis Research (Edited by H. Metzner), Vol. I, pp. 209-216, Tubingen, Germany. Jolchine. G., F. Reiss-Husson and M. D. Kamen (1969) Proc. Natl. Acad. Sci. U S 64, 65s653. Ke, B., and T. H. Chaney (1971) Biochim. Biophys. Acta 226. 341-353. Ke, B.. L. P. Vernon, A. Garcia and E. Ngo (1968) Biochem. 7. 311-318. Kennel. S. J., and M. D. Kamen (1971) Biochim.Biophys. Acta 253. 153-166. Noel, H., M. Van Der Rest and G. Gingras (1972) Biochim. Biophys. Acta 275. 219-230. Okamura. M. Y..L. A. Steiner and G. Feher (1974) Biochem. 13, 13941403. Parson, W. W. (1969) Biochim. Biophys. Acta 189, 397-403. Parson, W. W. (1974) A 7 r 1 . Rev. Microhiol. 28. 41-59. Reed, D. W. (1969) J. Biol. Chem. 244, 49364941. Reed, D. W., and R. K. Clayton (1968) Biochem. Res. Cornmuti. 30, 471-475. Reed, D. W.. and G. A. Peters (1972) J. Biol. Chrm. 247, 7148-7152. Reiss-Husson, F., and G. Jolchine (1972) Biochim. Biophys. Acra 256. 440-451. Segen, B. J., and K. D. Gibson (1971) J . Bacteriol. 105, 701-709. Siegelman, H. W., G. A. Wieczorek and B. C. Turner (1965) Anal. Biochem. 13, 402-404. Slooten, L. (1972) Biochim. Biophys. Acra 2J6, 452-466. Smith, W. R. Jr., C. Sybesma and K. Dus (1972) Biochim. Biophys. Acta 267, 609-615. Thornber, J. P. (1970) Biochem. 9, 2688-2698. Wang, R. T.. and R. K. Clayton (1973) Photochem. Photohiol. 17, 57-61. Wassink, E. C., and G. H. M. Kronenberg (1962) Nature lW,553-554. Weber. K., and M. Osborn (1969) J. Biol. Chem. 244, 44OW12.
Pergamon Press. Printed in Great Britain
ISOLATION AND PARTIAL CHARACTERIZATION OF THE PHOTOCHEMICAL REACTION CENTER OF CHROMATIUM VINOSUM (STRAIN D) LILYLINand J. PHILIPTHORNBER Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024, U.S.A.
(Received I 1 November 1974; accepted 7 March 1975) Abstract-A preparation of the photochemical reaction center of Chromatiurn has been obtained by chromatography of lauryldimethylamineoxide-solubilized chromatophores on hydroxylapatite and Sepharose columns. The procedure has yielded a reaction center preparation from both carotenoidcontaining and carotenoid-deficient Chromatiurn cells. Preliminary analysis of the isolated component indicates that the photochemical reaction center of the Thiorhodaceae is homologous to that of the Athiorhodaceae. In particular, the near infrared absorption spectrum of the Chromatiurn reaction center preparation shows the same triple-peaked spectrum observed for reaction center preparations from the Athiorhodaceae. The Chromatiurn preparation undergoes a rapid light-induced oxidation and dark reduction of the reaction center. The ratio of the reaction center to the two membrane-bound cytochromes (cytochrome c s 5 2 and c 5 s 5 )is greatly increased over the ratio observed in chromatophores or in other previously isolated, reaction center-enriched subchromatophore fractions of this organism.
INTRODUCTION
Photochemical reaction centers of photosynthetic bacteria have been successfully isolated essentially devoid of light-harvesting chlorophylls from several members of the Athiorhodaceae, predominantly from Rhodopseudomorias spheroidrs (Reed and Clayton, 1968; Jolchine et al.. 1969; Feher, 1971; Segen and Gibson, 1971; Slooten, 1972) and Rhodospirillum rubrum (Gingras and Jolchine, 1969; Niiel et al., 1972; Smith et al.. 1972; Wang and Clayton, 1973). While chromatophores of both wild type and carotenoidless mutant strains have yielded a reaction center preparation after treatment with a variety of detergents, such as the anionic sodium dodecyl sulfate (SDS), the cationic cetyl trimethyl ammonium bromide (CIAB), and the zwitterionic lauryl dimethylamine oxide (LDAO), only carotenoidless mutant strains have yielded to solubilization with the nonionic detergent Triton X-100. In the family of the 7hiorhodaceae, specifically Chrornatiurn Strain D, complete dissociation of the reaction center from light-harvesting bacteriochlorophyll has not been achieved previously by treatment with Triton X-100(Garcia et al., 1966; Ke et al., 1968; Ke and Chaney, 1971) nor with SDS (Thornber, 1970). The best enrichment of the reaction center of Chronlatiurn is Fraction A of Thornber (1970). Fraction A was obtained by hydroxylapatite chromatography of SDS-solubilized chromatophores. The mol wt was estimated to be over 6 x 10'. The molar ratio of reaction center bacteriochlorophyll (P883) to the light-harvesting bacteriochlorophyll (B890) in Fraction A was 1/40. The fraction also contained both the high potential c-type cytochrome, C555, and the low potential, C5.52. in the ratio of
P883:C555:C552 = 1:2:7: carotenoids were also present. The following year Ke and Chaney (1971) attempted to isolate the reaction center from carotenoid-deficient cells of Chromatiurn using Triton X100. The active fraction, termed the Heavy Band (Hband), was enriched in the reaction center relative to the whole cells. However, both preparations (Fraction A and the H-band) still contained substantial amounts of light-harvesting bacteriochlorophyll and were spectrally similar. The zwitterionic detergent, LDAO, has been used successfully to isolate the reaction center from carotenoidless mutants of Rhodopseudomonas spheroides (Feher, 1971) and Rhodospirillum rubrum (Wang and Clayton, 1973) as well as from wild type cells of Rsp. rubrurn (Noel et al., 1972). We have found that Chromatiurn chromatophores will also yield to LDAO solubilization. It is the purpose of this communication to present some preliminary characteristics of the reaction center isolated from wild type and carotenoid-deficient cells of this organism. MATERIALS AND METHODS
An inoculum of Chromatiurn was kindly provided by Dr. R. G . Bartsch (University of California, San Diego). The cells were grown anaerobically in completely filled 1 screw-cap bottles in a modified Bartsch medium- (Bose, 1963). Continuous illumination was provided by 25 W tungsten bulbs giving an intensity of 1080 21601ux/m2 falling on the glass surface. Temperature remained between 28-30°C. Cells were harvested by centrifugation, 4000 g for 5 min, during the stationary phase (7-day-old cultures). Packed cells were wet frozen (- 18°C) if not used immediately. Chromatiurn cells were cultured both in the presence (Wassink and Kronenberg, 1962) and absence of diphenylamine (DPA). Cells growing in the presence of DPA appear green due to inhibition of carotenoid synthesis,
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37
LILYLIN and J PHILIP THORNRER
38
whercas cells grown in the absence of DPA appear red due to the presence of the normal full complement of carotenoids. Hereafter, cells grown in the presence of DPA will 08be termed green cells. and those grown in its absence will be termed red cells. Packed cells were resuspended in an equal vol of 50 mM 06Tris-HCl, pH 8.0, passed twice through the French Pres- 0 sure Cell at 1 x 10' N/m2 (15,000 p.s.i.), and centrifuged at 20,OOOg for 15 min. The supernate contained the pig- d 0.4mented membranes, and its absorbance at 590 nm was a used to estimate the bacteriochlorophyll concentration assuming cS9"= 20mM-' cm-' (Thornber, 1970). A 1% 0.2(v/v) solution of LDAO in the ratio of detergent/bacteriochlorophyll = 40/1 (g/mmol) was added slowly to the 001 I I I I I I I I supernate and stirred for 3 h at room temp. Detergent300 400 500 600 solubilized material was loaded onto a hydroxylapatite Wavelength, nm column (Siegelman ef al., 1965). For the column. a 50cm3 Luer-Lok glass syringe was packed to give a vol of 30 m f I n. hydroxylapatite. and equilibrated with 5 0 d of 0.01 M sodium phosphate 0.2M NaCl, pH 7.0. This size column easily accommodated 25 pmol bacteriochlorophyll in the detergent extract derived on the average from 5 g of packed cells. The light-harvesting bacteriochlorophyll complexes are adsorbed onto the stationary phase allowing m 0 6 the reaction centercontaining fraction to move down the c column. The column was washed with 50 mC of the equilig 0.4brating buffer. The reactive material was precipitated from the eluate by adding solid (NH4)S04 to a final con- a centration of 25% (w/v). and centrifuged at 20,OOOg for 15 min. Precipitate adhering to the walls of the tubes and 0.2 as a floating pellet was removed and dissolved in 50mM Tris-HC1, pH 8.0. For further purification, the ammonium 0.0 600 700 sulfate precipitate was redissolved in a minimal volume BOO 900 (2 m/) of 50mM Tris-HC1, 0 2 M NaC1. 0.057" LDAO, Wavelength, nm pH 8.0 and chromatographed on a 3 x 40 cm Sepharose 6B (Pharmacia, Piscataway, New Jersey) column previously Figure 1. Absorption spectra of the reaction center prepequilibrated with the same buffer. The flow rate was kept aration; (a) in the visible region and (b) in the near IR at 0.2 m/ per min; 4 mf fractions were collected and assayed region. The solid trace was recorded by a weak beam of spectrophotometrically. This procedure removed a fraction monochromatic light, whereas the dotted trace was containing oxidized bacteriochlorophyll and bacteriopheo- recorded while a strong white light illuminated the sample. phytin from the photochemically active material. The absorption spectra were measured on an Aminco-Chance DW-2 Dual Wavelength spectrophotometer. Photochemical activity was measured by examining light-induced ab- creased in absorbance with a concomitant loss of sorbance changes of the reaction center in the near infrared absorbance at 800 and 875nm. The 589nm absor(8W900nm). The actinic light source was obtained from bance is attributed to the Q,-band of the bacterioa 30 W. GE 1493 tungsten lamp of a microscope slide ilchlorophyll a molecules in the preparation. The three luminator (American Optical Company). Actinic light was filtered through a Corning glass filter number 5433. or peaks at 530, 503 and 480nm are observed in the alternatively a I M copper sulfate solution I cm thick was reaction center preparation from the red cells, but used. A Corning red cut-off glass filter number 2403 was only the 530nm absorbance is present in the caroused to prevent stray exciting light from falling on the tenoid-deficient green cell preparation. It thus is very photomultiplier.
g g -
+
g
RESULTS
The absorption spectrum of the reaction center preparation from Chrornatiurn is shown in Fig. 1. The most probable assignment of the absorption bands to components in the preparation is as follows: The absorbance at 875 nm is due to the primary electron donor molecule, P870 and the 800 nm absorbance is due to the closely associated pigment, P800. The 756 nm-peak is attributed to bacteriopheophytin a while the 689nm-peak is most likely a degradation product, oxidized bacteriochlorophyll (Reiss-Husson and Jolchine, 1972). If a LDAO/BChl ratio greater than 50/1 is used to dissociate the pigmented membranes in the isolation of the photochemical reaction center, or on prolonged exposure of the preparation to light and air, the 756 and 689nm-peaks are in-
probable that the 480 and 503nm absorbances are due to carotenoids, as is also the absorbance at 445 nm. Absorbance in the region of 530 nm has been seen in all reaction center preparations obtained so far from purple photosynthetic bacteria regardless of whether wild type cells or carotenoidless mutants were used as the starting material, and regardless of the nature of the detergent used to dissociate such membranes. Various authors (Jolchine et al.. 1969; Reed, 1969; Reed and Peters, 1972; Slooten, 1972) have ascribed this absorbance to bacteriopheophytin a. In the Soret region, three peaks are observed at 366 nm, 386 nm. and 404 nm. Hemoprotein absorption may account for the 404 nm-component (Smith rt al., 1972) whereas the 366nm and 386nm-peaks are almost certainly due to the Soret peaks of bacteriochlorophyll and bacteriopheophytin respectively. In general, the reaction center of Chrounariutir bears a
39
The photochemical reaction center of chromatiurn
I W t
t
I
AA-O.OO5
t No additions
Ascorbate
Dithionite
sodium dithionite difference spectrum (Fig. 3a) shows decreases at 552 and 422 nm which are almost certainly due to absorbance changes in the a- and ybands of the low potential c-type cytochrome, C552, of Chromatiurn chromatophores. In Fig. 3b, the oxidized (K3Fe(CN),)-minus-reduced (sodium ascorbate) spectrum reveals the presence of the high potential c-type cytochrome, C555. Reduction of the isolated material by sodium ascorbate (Fig. 3b) shows that most of the C555 is in the reduced state on isolation,
Figure 2. Light-induced absorption changes at 875 nm of the reaction center preparation, recorded as described in the Results section. The arrows indicate when the actinic light was turned on and off. Sodium ascorbate (100 mM) and sodium dithionite (10 mM) were added to the cuvette as indicated.
0 02
g
great spectral similarity to the reaction center preparations from other bacteriochlorophyll a-containing organisms. Upon exposure of the preparation to strong actinic light, or on chemical oxidation, the preparation exhibits a behavior typical of other light-oxidized reaction ccnter preparations. There is a disappearance of the peak at 875 nm and a hypsochromic shift of P800 from 799 nm to 796 nm. Figure 2 illustrates the kinetics of light-induced changes in the near infrared. On illumination, there is a reversible bleaching with a rapid absorbance decrease a t 875 nm and an almost equally rapid dark recovery. Interestingly, the kinetics of the light-induced absorption changes in this preparation are much more rapid than those in fractions previously obtained for Cltrormtiurti (cf. Garcia rt al., 1966; Ke, ut a/., 1968; Thornber. 1970; Ke and Chaney, 1971). Photo-oxidation and dark reduction occur with no additions of exogenous electron donors or acceptors. On the addition of sodium ascorbate the apparent light-induced absorbance change is lessened while the even stronger reducing agent, sodium dithionite, completely obliterates the change (Fig. 2). This complete loss of change in absorbance is almost certainly due to the chemical reduction of the primary acceptor, such that no photochemistry can occur. The precise location of this long wavelength maximum in the LDAO-prepared material varies from 877 nm to 869 nm with increasing detergent concentration. The stability of the reaction center preparation decreases in relation to the position of the wavelength maximum: the shorter the wavelength, the less stable is the preparation; that is, within a few hours after isolation a preparation with a maximum a t 869 nm loses most of its ability to undergo reversible photobleaching. The chemically oxidized (K,Fe(CN),)-minusreduced (Na2S204)difference spectrum -(Fig. 3c) in the near infrared also shows a maximum absorbance decrease at 875 nm together with an equally large decrease a t 808nm. The absorbance difference at 600 nm is very probably due to the loss of absorbance of the Q,-band of the P870 chlorophyll molecules. In the visible region, the sodium ascorbate-minus-
-
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-
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004-
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-
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5 600
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Figure 3. Oxidized-minus-reduced difference spectrum of the reaction center preparation (a) in the visible region of sodium ascorbate minus sodium dithionite, (b) -untreated-minus-sodium ascorbate; - - - potassium ferricyanide-minus-sodium ascorbate; ' . . potassium ferricyanide-minus-sodiumdithionite; (c)in the near IR region of oxidized (potassium ferricyanide)-minus-reduced (sodium dithionite).
LILYLINand J. PHILIP THORNBER
40
and furthermore, that some of the low potential cytochrome, C552 is reduced by ascorbate. It is perhaps noteworthy that the maximum absorbance decrease in this case occurs at 553 and not 552 nm. The ratio of cytochrome/reaction center is greatly decreased in this reaction center preparation from that observed in chromatophores or subchromatophore fractions of this organism. The light-induced absorbance change is almost negligible (one twentieth) compared to the change at 875 nm. Nevertheless, the cytochromes are the same two present in chromatophores (Parson, 1969), subchromatophore fractions (Case et al., 1970; Thornber, 1970), and in a cholate-solubilized cytochrome preparation (Kennel and Kamen, 1971). These two cytochromes are both associated with the reaction center bacteriochlorophyll P870 (For a review of this topic, see Parson, 1974). The number of polypeptides present in the isolated material has been briefly examined. The preparation was brought to 100°C for 1 min in 1% SDS. and the resulting dissociated material was electrophoresed on 8% SDS-polyacrylamide gels in 50 mM Tris-HC1, 0.1% SDS, pH 8.0 (Weber and Osborn, 1969). After staining with Amido Black, 5 protein zones were observed, 3 of which were particularly prominent and m a y possibly correlate with the 3 polypeptides observed in other reaction center preparations (Feher. 1971; Okamura er al.. 1974). There was little difference between the preparation from the green and red cells apart from the expected decreased carotenoid content in the fraction from the green cells.
bacteriochlorophyll by further fractionation of Fraction A were not successful. It was better to start with whole chromatophores and to use a different detergent (LDAO) than that which yielded Fraction A. Chromatography of LDAO-treated pigment membranes provided a reaction center preparation from both red and green cells. The preparation is essentially devoid of light-harvesting bacteriochlorophyll (B800,B850, B890); studies are in progress to try to eliminate the small amount of contaminating 689 nm absorbance from the fraction. It is noteworthy that after removal of bulk bacteriochlorophyll and most of the carotenoids from the photochemical reaction center, the maximum absorbance change due to P870 is shifted to wavelengths shorter than the 883 nm observed for Fraction A. The characteristics of the Chror~atiurii reaction center preparation determined so far indicate that the reaction center preparations of purple photosynthetic bacteria are spectrally quite similar. Thus, although Chromatium is classified in a taxonomic family (Thiorhodaceae) different from that of Rhodopseudomonas and Rkodospirilluni (Athiorhodacrar) on the basis of metabolism, the basic mechanism for survival. the photochemical reaction center, maintains spectral homology among these different families of bacteria. The premise that the reaction center is homologous between the two families will be tested by further characterization of the biophysical and biochemical properties of the isolated reaction center preparation.
DISCUSSION
Ackriowlrdgmrnts-Research was supported by NSF GB 31207 to J.P.T. and by NSF Traineeship to L.L. LDAO was kindly donated by the ONYX Chemical Company (Los Angeles office) of Jersey City, New Jersey.
Preliminary experimentsin which we tried to obtain a reaction center preparation free from light-harvesting
REFERENCES
Bose. S. K. (1963) In Bacterial Photosyrithrsis (Edited by H. Gest, A. San Pietro, and L. P. Vernon), pp. 501-510. Antioch, Yellow Springs, Ohio. Case, G. D., W. W. Parson and J. P. Thornber (1970) Biochim. Biophys. Acta 223. 122-128. Feher. G. (1971) Photochem. Photobiol. 14. 373-387. Garcia, A,, L. P. Vernon and H. Mollenhauer (1966) Biochem. 5. 2399-2407. Gingras, G., and G. Jolchine (1969) In Progress in Photosynthesis Research (Edited by H. Metzner), Vol. I, pp. 209-216, Tubingen, Germany. Jolchine. G., F. Reiss-Husson and M. D. Kamen (1969) Proc. Natl. Acad. Sci. U S 64, 65s653. Ke, B., and T. H. Chaney (1971) Biochim. Biophys. Acta 226. 341-353. Ke, B.. L. P. Vernon, A. Garcia and E. Ngo (1968) Biochem. 7. 311-318. Kennel. S. J., and M. D. Kamen (1971) Biochim.Biophys. Acta 253. 153-166. Noel, H., M. Van Der Rest and G. Gingras (1972) Biochim. Biophys. Acta 275. 219-230. Okamura. M. Y..L. A. Steiner and G. Feher (1974) Biochem. 13, 13941403. Parson, W. W. (1969) Biochim. Biophys. Acta 189, 397-403. Parson, W. W. (1974) A 7 r 1 . Rev. Microhiol. 28. 41-59. Reed, D. W. (1969) J. Biol. Chem. 244, 49364941. Reed, D. W., and R. K. Clayton (1968) Biochem. Res. Cornmuti. 30, 471-475. Reed, D. W.. and G. A. Peters (1972) J. Biol. Chrm. 247, 7148-7152. Reiss-Husson, F., and G. Jolchine (1972) Biochim. Biophys. Acra 256. 440-451. Segen, B. J., and K. D. Gibson (1971) J . Bacteriol. 105, 701-709. Siegelman, H. W., G. A. Wieczorek and B. C. Turner (1965) Anal. Biochem. 13, 402-404. Slooten, L. (1972) Biochim. Biophys. Acra 2J6, 452-466. Smith, W. R. Jr., C. Sybesma and K. Dus (1972) Biochim. Biophys. Acta 267, 609-615. Thornber, J. P. (1970) Biochem. 9, 2688-2698. Wang, R. T.. and R. K. Clayton (1973) Photochem. Photohiol. 17, 57-61. Wassink, E. C., and G. H. M. Kronenberg (1962) Nature lW,553-554. Weber. K., and M. Osborn (1969) J. Biol. Chem. 244, 44OW12.
