ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 192, No. 1, January, pp. 158-163, 1979
A Partial DAVID
*Department
Purification B. KNAFF,*
of Membrane-Bound b and c Cytochromes from Chromatium vinosum THOMAS M. WORTHINGTON,* AND RICHARD MALKIN’F
CRAIG
C. WHITE,t
qf Chemistry, Texas Tech University, Lubbock, Texas 79409, and TDepartment Physiology, University of California-Berkeley, Berkeley, California 947’20 Received July 10, 1978; revised September
of Cell
11, 1978
Three membrane-bound cytochromes from the photosynthetic purple sulfur bacterium Chromatium vinosum have been partially separated from other membrane components by treatment with deoxycholate followed by ammonium sulfate fractionation and chromatography on Rio-Gel A1.5. Cytochrome c 55j, present in relatively small amounts in the deoxycholate extract, has an a-band that splits into two bands at 548 and 552.5 nm at liquid nitrogen temperature. The major components of the deoxycholate extract are cytochromes csjJ and b,,,, which are present in essentially equimolar amounts through all the stages of the purification procedure.
It has been known for some time that the photosynthetic electron transport chains of all oxygen-evolving organisms contain membrane-bound cytochromes of both the b and c type (1, 2). Until recently it was thought that a different situation existed insofar as photosynthetic bacteria were concerned. Only photosynthetic membranes from the purple nonsulfur bacteria were thought to contain both types of cytochromes, while the membranes of purple sulfur and green sulfur bacteria were considered to possess only c-type cytochromes (see references cited in 3). Recent experiments have provided evidence that both the green sulfur bacterium Chlorobium limicola f. thiosulfalophilum and two purple sulfur bacteria (Chromatium vinosum and Ectothiorhodospira mobilis) contained, in addition to c-type cytochromes, membranebound b-type cytochromes (3). Evidence suggested that these b cytochromes functioned in light-driven electron transfer reactions (3). The presence of a b type cytochrome in green sulfur bacteria was also noted in other laboratories (4, 5) but the report of a b-type cytochrome in purple sulfur bacteria remained unconfirmed. In order to document further and better char0003-9861/79/010158-06$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
acterize the b cytochrome of the photosynthetic membranes of purple sulfur bacteria, we have partially purified the b-type cytochrome from C. vinosum chromatophores and determined its spectral properties. MATERIALS
AND METHODS
Normal cells of Chromatium vinosum were grown on the malate-containing medium described previously (3). Green cells, grown under conditions that inhibit carotenoid biosynthesis, were obtained by growth of C. vinosum in the normal medium to which 30 @M diphenylamine had been added (6). Chromatophores were prepared by sonication as described previously (3). Cytochrome c,~, content was estimated assuming a reduced minus oxidized extinction coefficient of 13.1 mM-’ cm-’ at 553 nm (8). Nonheme iron was determined as described by Miller and Massey (9). Absorption spectra were obtained using an Aminco DW-2a spectrophotometer. Bacteriochlorophyll was estimated after extraction into acetone:methanol as described by Clayton (7). Sodium deoxycholate and sodium cholate were obtained from Sigma Chemical Company. Bio-Gel Al.5 was obtained from Bio-Rad Laboratories. RESULTS
In our initial studies of the membranebound b cytochrome in C. vinosum chro158
Chromatium
vinosum
CYTOCHROMES
b AND c
159
vinosum contains a membrane-bound b-type
FIG. 1. Soret band reduced minus oxidized difference spectrum of C. vinosum low-potential cytochromes. Chromatophores prepared from normal C. vinosum cells at a bacteriochlorophyll concentration of 30 pM in the presence of 1 mM hydroquinone were placed in the sample and reference cuvettes of the spectrophotometer. Sodium dithionite crystals were added to the sample cuvette until the maximum absorbance was reached.
matophores, we were able to measure its absorbance spectrum only in the a-band region. Figure 1 shows a dithionite-reduced minus hydroquinone-reduced difference spectrum of C. vinosum chromatophores in the Soret region. This spectrum shows only the low-potential (E, < +200 mV) cytochromes. Chromatophores reduced with hydroquinone or ascorbate showed only the single peak at 422 nm characteristic of reduced cytochrome css5(E, = +340 mV, Refs. (10-12)). As can be seen in Fig. 1, in addition to the peak at 424 nm characteristic of reduced cytochrome es58(10, ll), there is a shoulder at 430 nm in the region characteristic of reduced b-type cytochromes. The Soret region dithionite minus hydroquinone difference spectra of chromatophores that had been washed twice to remove soluble cytochromes was identical to that shown in Fig. 1. These results are in agreement with our earlier report (3) that C.
cytochrome present at a level of approximately one cytochrome b per reaction center bacteriochlorophyll. It was found that membrane-bound cytochromes in C. vinosum chromatophores could be partially separated from other membrane components by the following procedure: Chromatophores were adjusted to 1 InM bacteriochlorophyll concentration in 50 mM Tris buffer (pH 7.8) containing 150 mM KCl. Sodium deoxycholate (DOC)’ was added to a final concentration of 0.5% and the mixture was incubated for 45 min at 2°C. The reaction mixture was centrifuged for 2 h at 3OO,OOOg, the pellet was discarded, and the supernatant was dialyzed against 50 mM Tris buffer (pH 7.8) containing 0.1% sodium cholate. Ammonium sulfate was added to the dialyzed supernatant and the fraction precipitating between 20 and 40% saturation was collected. After dialysis against Tris buffer containing 0.1% sodium cholate, chromatography on a Bio-Gel Al.5 column (3 x 70 cm), preequilibrated with the same buffer, was carried out and the fractions with the highest cytochrome content were pooled. Only one cytochromecontaining peak was detected. As can be seen from the dithionite-reduced minus hydroquinone-reduced difference spectrum shown in Fig. 2, the preparation clearly contains both cytochrome b,,, and cytochrome css3.The two cytochromes appear to be present in approximately equimolar amounts. The results from a typical preparation using normal C. vinosum cells are summarized in Table I. Cytochromes b,,, and css3 were present in approximately equimolar amounts at all stages of the purification. Although a considerable separation of cytochrome from pigments were obtained, the cytochrome-containing fractions still contain significant amounts of bacteriochlorophyll and carotenoids. We have as yet been unable to achieve a further separation of the cytochromes from these pigments. It should be pointed out that no cytochrome was found in the 0 to 20% ammonium ’ Abbreviation
used: DOC, deoxycholate.
160
KNAFF
ET AL.
Generally, results similar to those shown in Table I were obtained using chromatophores from green C. vinosum (grown in the presence of diphenylamine) except that the cytochrome-containing fraction was essentially free of carotenoids. One major difference of considerable interest between the two types of chromatophores was that while essentially no cytochrome cSs5was found in the DOC-supernatant from normal chromatophores, the DOC-supernatant from green chromatophores contained considerable amounts of cytochrome cSsS. The cytochrome-containing fraction from green chromatophores (after DOC extraction and Bio-Gel Al.5 chromatography) typically had a cytochrome c,,,:cytochrome cSSsratio near 5. The cytochrome cj,,:cytochrome czs:,ratio in the preparations from green chromatophores was generally somewhat less than 1. The chromatophore memFIG. 2. a-band reduced wcilzus oxidized difference branes from the green and normal cells spectrum of partially purified low-potential C. vi~osum apparently differ enough so that deoxychocytochromes. The cytochrome-containing fraction late releases cytochrome csSjfrom the former after Bio-Gel Al.5 chromatography of a preparation from normal chromatophores in the presence of 1 mM but not from the latter. Difference spectra hydroquinone was treated as described in Fig. 1. of the 300,OOOgpellet obtained after DOC treatment confirm that the majority (almost 100% in the case of chromatophores from sulfate fraction, but considerable cyto- normal C. vinosum cells) of the cytochrome chrome remained in the 40% ammonium cSSzis not released by this detergent treatsulfate supernatant. The cytochrome recov- ment. Neither DOC preparations from normal ery at this step could be increased by increasing the ammonium sulfate concentra- nor green chromatophores contained any tion but only at the cost of a higher bacterio- detectable cytochrome c’ (13). The nonheme chlorophyll:cytochrome ratio. The recovery iron content (9) of the preparations was of cytochromes csz3and b,,, was 1.2% and variable. Electron paramagnetic resonance an eightfold purification (based on bacterio- spectroscopy at liquid helium temperatures of dithionite-reduced preparations did not chlorophyll) was obtained. TABLE PARTIAL PURIFICATION OFC.
Preparation Chromatophores DOC-supernatant ZO-40% ammonium sulfate precipitate Bio-Gel Al.5 chromatography
WNOSUM
I MEMBRANE-BOUND
Cytochrome cgjZh (nmol) 202 (100%) 26.2 (13%) 2.88 (1.4%) 2.46 (1.2%)
CYTOCHROMES~
Bacteriochlorophyll
kmol) 30.9 (100%) 1.3 (4.2%) 0.097 (0.30%) 0.046 (0.14%)
BacteriochlorophylV cytochrome cSS8 153 50 34 19
n Chromatophores from normal C. vinosum cells were treated as described in the text. p Cytochrome bjsa was present in amounts essentially equimolar to cytochrome cSag, as judged by equal a-band absorbances in the DOC supernatant and at all subsequent stages of the procedure.
Chromatium
vinosum
CYTOCHROMES
reveal the presence of any iron-sulfur centers in preparations from normal chromatophores. As spectroscopy at cryogenic temperatures is often a useful tool for resolving cytochromes that are difficult to distinguish at room temperature (1,14), we have determined the absorbance characteristics at 77°K of the C. vinosum cytochromes obtained after Bio-Gel chromatography of the material solubilized from green chromatophores by deoxycholate. The ascorbate reduced minus oxidized difference spectrum of this preparation at 77”K, shown in Fig. 3, exhibits a peak at 552.5 nm and a distinct shoulder at 548 nm. Since spectra at 300°K indicate that only cytochrome csjsis reduced under these conditions, it can be concluded that the 555nm a-band absorbance of this cytochrome shifts to shorter wavelength and splits at liquid nitrogen temperatures.
b AND c
161
Such splitting has previously been observed for otherc-type cytochromes (1,15). Figure 4 shows the dithionite-reduced minus ascorbate-reduced difference spectrum for this preparation at 77°K. Such a spectrum would be expected to show the a-bands of cytochrome es53and cytochrome b,,, and two bands can be clearly seen in the difference spectrum. The band at 549-550 nm can be assigned to cytochrome es53while that at 556-557 nm can be assigned to cytochrome b,,,. Similar blue shifts in the a-band maxima of b-type cytochromes at liquid nitrogen temperatures have been observed in chloroplasts (1, 15) and mitochondria (14). We have also characterized the Soret region bands of these three C. vinosum cytochromes at 77°K. Figure 5 shows that that reduced cytochrome cs3j (ascorbate-reducible) has a Soret band at 420 nm at 77°K (shifted from 422 nm at 300°K). The two dithionitereducible cytochromes also exhibit spectral shifts at 77”K, with the Soret band of cytochrome cj53 shifted to 421 nm (compared to 424 nm at 300°K) and that of cytochrome
552 5
FIG. 3. n-band reduced minus oxidized difference spectrum of cytochrome cjss at 77°K. To the cytochrome-containing fraction after Bio-Gel Al.5 chromatography of a preparation from green chromatophores, glycerol was added to give a final glycerol concentration of 60%. The material was placed in the low-temperature cuvette of the Aminco DW-2a spectrophotometer and sodium ascorbate (final concentration, 5 mM) was added to the sample side. The spectrum was obtained at liquid nitrogen temperature.
FIG. 4. a-band reduced minus oxidized difference spectrum of low-potential C. vinosum cytochromes at 77°K. Conditions as in Fig. 3 except that both sample and reference cuvettes contained 5 mM sodium ascorbate and the sample cuvette contained, in addition, sodium dithionite.
KNAFF
ET AL.
contains a membrane-bound b-type cytochrome. The spectral characteristics of this cytochrome, as well as those of the two membrane-bound c-type cytochromes found in C. vinosum, have been determined at both physiological and cryogenic temperatures. Some progress has been made in purifying these cytochromes but the complete separation of the cytochromes from bacteriochlorophyll and carotenoids has not yet been achieved. It is of considerable interest that deoxycholate treatment preferentially releases cytochromes b,,, and es53from C. vinosum chromatophores and releases little cyto. chrome css5from the membrane. The results reported above are quite different from those reported by Kennel and Kamen (8), 440 430 410 410 who found that sodium cholate solubilized Wavelength (“ml, a complex containing cytochromes cjjB and css5in a 2:l ratio but containing no cytoFIG. 5. Soret band reduced minus oxidized difchrome b. Presumably, these differences ference spectra of C. vinosum cytochromes at 77°K. arise from the fact that Kennel and Kamen Conditions as in Figs. 3 and 4. extracted all the pigments with acetone b,,, shifted to 427 nm (compared to 430 nm prior to detergent treatment and used conat 300°K). The spectral characteristics of siderably higher concentrations of detergent. The fact that cytochrome b,,, and cytothe three cytochromes are summarized in chrome cjjs are found in all fractions from Table II. normal C. vinosum cells in essentially equimolar ratios raises the possibility that a DISCUSSION complex of the two cytochromes exists. The data presented above clearly confirm Further purification studies are required the earlier report, based on protoheme to determine whether a true cytochrome analyses and in situ a-band spectra, that c. cytochrome b complex occurs in C. vinothe purple sulfur bacterium C. vinosum sum but the existence of such complexes in higher plants (16) and respiratory electron transfer chains (1’7, 18) suggests the TABLE II possibility of such a complex in photosynSPECTRALPROPERTIESOFC.VINOSUM CYTOCHROMES" thetic bacteria. -.
a-band maximum
Sore&band maximum
300°K
77°K
300°K
‘77°K
552.5 (548) 549.5 556.5
422
420
424 430
421 427
Cytochrome
cjs5
555
Cytochrome Cytochrome
css3 b,,,
553 560
a The wavelength maxima (nm) were measured on the deoxycholate-solubilized material from green chromatophores after ammonium sulfate fractionation and Bio-Gel Al.5 chromatography. The 300°K values are identical to those in situ. The 77°K values were measured in a 60% glycerol:40% water glass.
ACKNOWLEDGMENTS The authors would like to thank Mr. Don Carlson and Mr. Bud Faris for growing the C. vinosum cells and Professor Graham Palmer for the use of his epr facilities. This investigation was supported in part by National Science Foundation PCM76-24131 and Robert A. Welch Foundation (D-710) grants to David B. Knaff and a National Institutes of Health grant (GM-207511 to Richard Malkin. REFERENCES 1. BOARDMAN,N. K. (1968)Adv. Enzymol. 30,149. 2. KNAFF, D. B. (1978) Coord. Chem. Rev. 26, 47-70.
Chromatium
vinosum
CYTOCHROMES
3. KNAFF, D. B., AND BUCHANAN, B. B. (1975) Biochim. Biophys. Acta 376, 549-560. 4. FOWLER, C. (1974) Biochim. Biophys. Acta 357, 327-331. 5. OLSON, J. M., GIDDINGS. T. H., AND SHAW, E. K. (1976) Biochim. Biophys. Acta 449, 197-208. 6. CASE, G. D., ANDPARSON, W. W. (1973)Biochim. Biophys. Acta 325, 441-453. 7. CLAYTON, R. K. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A., and Vernon, L. P., eds.), p. 498, Antioch Press, Yellow Springs, Ohio. 8. KENNEL, S. J., AND KAMEN, M. D. (1971) Biochim. Biophys. Acta 253, 153-166. 9. MILLER, R. W., AND MASSEY, V. (1965) J. Biol. Chem. 240, 1453-1465. 10. PARSON, W. W. (1969) Biochim. Biophys. Acta 189, 397-403.
b AND c
163
11. CUSANOVICH, M. A., AND KAMEN, M. D. (1968) Biochim. Biophys. Acta 153, 376-396. 12. CASE, G. D., AND PARSON, W. W. (1971) Biochim. Biophys. Acta 253, 187-202. 13. BARTSCH, R. G., AND KAMEN, M. D. (1960) J. Biol. Chem. 235, 825-831. 14. SATO, N., WILSON, D. F., AND CHANCE, B. (1971) Biochim. Biophys. Acta 253, 88-97. 15. BOARDMAN, N. K., AND ANDERSON, J. M. (1967) Biochim. Biophys. Acta 143, 187-203. 16. NELSON, N., AND NEUMANN, J. (1972) J. Bid. Chem. 247, 1817-1824. 17. HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E. (1962) J. Biol. Chem. 237, 1681-1685. 18. ZAUGG, W. S., AND RIESKE, J. S. (1962) Biochem. Biophys. Res. Commun. 9, 213-217.
A Partial DAVID
*Department
Purification B. KNAFF,*
of Membrane-Bound b and c Cytochromes from Chromatium vinosum THOMAS M. WORTHINGTON,* AND RICHARD MALKIN’F
CRAIG
C. WHITE,t
qf Chemistry, Texas Tech University, Lubbock, Texas 79409, and TDepartment Physiology, University of California-Berkeley, Berkeley, California 947’20 Received July 10, 1978; revised September
of Cell
11, 1978
Three membrane-bound cytochromes from the photosynthetic purple sulfur bacterium Chromatium vinosum have been partially separated from other membrane components by treatment with deoxycholate followed by ammonium sulfate fractionation and chromatography on Rio-Gel A1.5. Cytochrome c 55j, present in relatively small amounts in the deoxycholate extract, has an a-band that splits into two bands at 548 and 552.5 nm at liquid nitrogen temperature. The major components of the deoxycholate extract are cytochromes csjJ and b,,,, which are present in essentially equimolar amounts through all the stages of the purification procedure.
It has been known for some time that the photosynthetic electron transport chains of all oxygen-evolving organisms contain membrane-bound cytochromes of both the b and c type (1, 2). Until recently it was thought that a different situation existed insofar as photosynthetic bacteria were concerned. Only photosynthetic membranes from the purple nonsulfur bacteria were thought to contain both types of cytochromes, while the membranes of purple sulfur and green sulfur bacteria were considered to possess only c-type cytochromes (see references cited in 3). Recent experiments have provided evidence that both the green sulfur bacterium Chlorobium limicola f. thiosulfalophilum and two purple sulfur bacteria (Chromatium vinosum and Ectothiorhodospira mobilis) contained, in addition to c-type cytochromes, membranebound b-type cytochromes (3). Evidence suggested that these b cytochromes functioned in light-driven electron transfer reactions (3). The presence of a b type cytochrome in green sulfur bacteria was also noted in other laboratories (4, 5) but the report of a b-type cytochrome in purple sulfur bacteria remained unconfirmed. In order to document further and better char0003-9861/79/010158-06$02.00/O Copyright Q 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
acterize the b cytochrome of the photosynthetic membranes of purple sulfur bacteria, we have partially purified the b-type cytochrome from C. vinosum chromatophores and determined its spectral properties. MATERIALS
AND METHODS
Normal cells of Chromatium vinosum were grown on the malate-containing medium described previously (3). Green cells, grown under conditions that inhibit carotenoid biosynthesis, were obtained by growth of C. vinosum in the normal medium to which 30 @M diphenylamine had been added (6). Chromatophores were prepared by sonication as described previously (3). Cytochrome c,~, content was estimated assuming a reduced minus oxidized extinction coefficient of 13.1 mM-’ cm-’ at 553 nm (8). Nonheme iron was determined as described by Miller and Massey (9). Absorption spectra were obtained using an Aminco DW-2a spectrophotometer. Bacteriochlorophyll was estimated after extraction into acetone:methanol as described by Clayton (7). Sodium deoxycholate and sodium cholate were obtained from Sigma Chemical Company. Bio-Gel Al.5 was obtained from Bio-Rad Laboratories. RESULTS
In our initial studies of the membranebound b cytochrome in C. vinosum chro158
Chromatium
vinosum
CYTOCHROMES
b AND c
159
vinosum contains a membrane-bound b-type
FIG. 1. Soret band reduced minus oxidized difference spectrum of C. vinosum low-potential cytochromes. Chromatophores prepared from normal C. vinosum cells at a bacteriochlorophyll concentration of 30 pM in the presence of 1 mM hydroquinone were placed in the sample and reference cuvettes of the spectrophotometer. Sodium dithionite crystals were added to the sample cuvette until the maximum absorbance was reached.
matophores, we were able to measure its absorbance spectrum only in the a-band region. Figure 1 shows a dithionite-reduced minus hydroquinone-reduced difference spectrum of C. vinosum chromatophores in the Soret region. This spectrum shows only the low-potential (E, < +200 mV) cytochromes. Chromatophores reduced with hydroquinone or ascorbate showed only the single peak at 422 nm characteristic of reduced cytochrome css5(E, = +340 mV, Refs. (10-12)). As can be seen in Fig. 1, in addition to the peak at 424 nm characteristic of reduced cytochrome es58(10, ll), there is a shoulder at 430 nm in the region characteristic of reduced b-type cytochromes. The Soret region dithionite minus hydroquinone difference spectra of chromatophores that had been washed twice to remove soluble cytochromes was identical to that shown in Fig. 1. These results are in agreement with our earlier report (3) that C.
cytochrome present at a level of approximately one cytochrome b per reaction center bacteriochlorophyll. It was found that membrane-bound cytochromes in C. vinosum chromatophores could be partially separated from other membrane components by the following procedure: Chromatophores were adjusted to 1 InM bacteriochlorophyll concentration in 50 mM Tris buffer (pH 7.8) containing 150 mM KCl. Sodium deoxycholate (DOC)’ was added to a final concentration of 0.5% and the mixture was incubated for 45 min at 2°C. The reaction mixture was centrifuged for 2 h at 3OO,OOOg, the pellet was discarded, and the supernatant was dialyzed against 50 mM Tris buffer (pH 7.8) containing 0.1% sodium cholate. Ammonium sulfate was added to the dialyzed supernatant and the fraction precipitating between 20 and 40% saturation was collected. After dialysis against Tris buffer containing 0.1% sodium cholate, chromatography on a Bio-Gel Al.5 column (3 x 70 cm), preequilibrated with the same buffer, was carried out and the fractions with the highest cytochrome content were pooled. Only one cytochromecontaining peak was detected. As can be seen from the dithionite-reduced minus hydroquinone-reduced difference spectrum shown in Fig. 2, the preparation clearly contains both cytochrome b,,, and cytochrome css3.The two cytochromes appear to be present in approximately equimolar amounts. The results from a typical preparation using normal C. vinosum cells are summarized in Table I. Cytochromes b,,, and css3 were present in approximately equimolar amounts at all stages of the purification. Although a considerable separation of cytochrome from pigments were obtained, the cytochrome-containing fractions still contain significant amounts of bacteriochlorophyll and carotenoids. We have as yet been unable to achieve a further separation of the cytochromes from these pigments. It should be pointed out that no cytochrome was found in the 0 to 20% ammonium ’ Abbreviation
used: DOC, deoxycholate.
160
KNAFF
ET AL.
Generally, results similar to those shown in Table I were obtained using chromatophores from green C. vinosum (grown in the presence of diphenylamine) except that the cytochrome-containing fraction was essentially free of carotenoids. One major difference of considerable interest between the two types of chromatophores was that while essentially no cytochrome cSs5was found in the DOC-supernatant from normal chromatophores, the DOC-supernatant from green chromatophores contained considerable amounts of cytochrome cSsS. The cytochrome-containing fraction from green chromatophores (after DOC extraction and Bio-Gel Al.5 chromatography) typically had a cytochrome c,,,:cytochrome cSSsratio near 5. The cytochrome cj,,:cytochrome czs:,ratio in the preparations from green chromatophores was generally somewhat less than 1. The chromatophore memFIG. 2. a-band reduced wcilzus oxidized difference branes from the green and normal cells spectrum of partially purified low-potential C. vi~osum apparently differ enough so that deoxychocytochromes. The cytochrome-containing fraction late releases cytochrome csSjfrom the former after Bio-Gel Al.5 chromatography of a preparation from normal chromatophores in the presence of 1 mM but not from the latter. Difference spectra hydroquinone was treated as described in Fig. 1. of the 300,OOOgpellet obtained after DOC treatment confirm that the majority (almost 100% in the case of chromatophores from sulfate fraction, but considerable cyto- normal C. vinosum cells) of the cytochrome chrome remained in the 40% ammonium cSSzis not released by this detergent treatsulfate supernatant. The cytochrome recov- ment. Neither DOC preparations from normal ery at this step could be increased by increasing the ammonium sulfate concentra- nor green chromatophores contained any tion but only at the cost of a higher bacterio- detectable cytochrome c’ (13). The nonheme chlorophyll:cytochrome ratio. The recovery iron content (9) of the preparations was of cytochromes csz3and b,,, was 1.2% and variable. Electron paramagnetic resonance an eightfold purification (based on bacterio- spectroscopy at liquid helium temperatures of dithionite-reduced preparations did not chlorophyll) was obtained. TABLE PARTIAL PURIFICATION OFC.
Preparation Chromatophores DOC-supernatant ZO-40% ammonium sulfate precipitate Bio-Gel Al.5 chromatography
WNOSUM
I MEMBRANE-BOUND
Cytochrome cgjZh (nmol) 202 (100%) 26.2 (13%) 2.88 (1.4%) 2.46 (1.2%)
CYTOCHROMES~
Bacteriochlorophyll
kmol) 30.9 (100%) 1.3 (4.2%) 0.097 (0.30%) 0.046 (0.14%)
BacteriochlorophylV cytochrome cSS8 153 50 34 19
n Chromatophores from normal C. vinosum cells were treated as described in the text. p Cytochrome bjsa was present in amounts essentially equimolar to cytochrome cSag, as judged by equal a-band absorbances in the DOC supernatant and at all subsequent stages of the procedure.
Chromatium
vinosum
CYTOCHROMES
reveal the presence of any iron-sulfur centers in preparations from normal chromatophores. As spectroscopy at cryogenic temperatures is often a useful tool for resolving cytochromes that are difficult to distinguish at room temperature (1,14), we have determined the absorbance characteristics at 77°K of the C. vinosum cytochromes obtained after Bio-Gel chromatography of the material solubilized from green chromatophores by deoxycholate. The ascorbate reduced minus oxidized difference spectrum of this preparation at 77”K, shown in Fig. 3, exhibits a peak at 552.5 nm and a distinct shoulder at 548 nm. Since spectra at 300°K indicate that only cytochrome csjsis reduced under these conditions, it can be concluded that the 555nm a-band absorbance of this cytochrome shifts to shorter wavelength and splits at liquid nitrogen temperatures.
b AND c
161
Such splitting has previously been observed for otherc-type cytochromes (1,15). Figure 4 shows the dithionite-reduced minus ascorbate-reduced difference spectrum for this preparation at 77°K. Such a spectrum would be expected to show the a-bands of cytochrome es53and cytochrome b,,, and two bands can be clearly seen in the difference spectrum. The band at 549-550 nm can be assigned to cytochrome es53while that at 556-557 nm can be assigned to cytochrome b,,,. Similar blue shifts in the a-band maxima of b-type cytochromes at liquid nitrogen temperatures have been observed in chloroplasts (1, 15) and mitochondria (14). We have also characterized the Soret region bands of these three C. vinosum cytochromes at 77°K. Figure 5 shows that that reduced cytochrome cs3j (ascorbate-reducible) has a Soret band at 420 nm at 77°K (shifted from 422 nm at 300°K). The two dithionitereducible cytochromes also exhibit spectral shifts at 77”K, with the Soret band of cytochrome cj53 shifted to 421 nm (compared to 424 nm at 300°K) and that of cytochrome
552 5
FIG. 3. n-band reduced minus oxidized difference spectrum of cytochrome cjss at 77°K. To the cytochrome-containing fraction after Bio-Gel Al.5 chromatography of a preparation from green chromatophores, glycerol was added to give a final glycerol concentration of 60%. The material was placed in the low-temperature cuvette of the Aminco DW-2a spectrophotometer and sodium ascorbate (final concentration, 5 mM) was added to the sample side. The spectrum was obtained at liquid nitrogen temperature.
FIG. 4. a-band reduced minus oxidized difference spectrum of low-potential C. vinosum cytochromes at 77°K. Conditions as in Fig. 3 except that both sample and reference cuvettes contained 5 mM sodium ascorbate and the sample cuvette contained, in addition, sodium dithionite.
KNAFF
ET AL.
contains a membrane-bound b-type cytochrome. The spectral characteristics of this cytochrome, as well as those of the two membrane-bound c-type cytochromes found in C. vinosum, have been determined at both physiological and cryogenic temperatures. Some progress has been made in purifying these cytochromes but the complete separation of the cytochromes from bacteriochlorophyll and carotenoids has not yet been achieved. It is of considerable interest that deoxycholate treatment preferentially releases cytochromes b,,, and es53from C. vinosum chromatophores and releases little cyto. chrome css5from the membrane. The results reported above are quite different from those reported by Kennel and Kamen (8), 440 430 410 410 who found that sodium cholate solubilized Wavelength (“ml, a complex containing cytochromes cjjB and css5in a 2:l ratio but containing no cytoFIG. 5. Soret band reduced minus oxidized difchrome b. Presumably, these differences ference spectra of C. vinosum cytochromes at 77°K. arise from the fact that Kennel and Kamen Conditions as in Figs. 3 and 4. extracted all the pigments with acetone b,,, shifted to 427 nm (compared to 430 nm prior to detergent treatment and used conat 300°K). The spectral characteristics of siderably higher concentrations of detergent. The fact that cytochrome b,,, and cytothe three cytochromes are summarized in chrome cjjs are found in all fractions from Table II. normal C. vinosum cells in essentially equimolar ratios raises the possibility that a DISCUSSION complex of the two cytochromes exists. The data presented above clearly confirm Further purification studies are required the earlier report, based on protoheme to determine whether a true cytochrome analyses and in situ a-band spectra, that c. cytochrome b complex occurs in C. vinothe purple sulfur bacterium C. vinosum sum but the existence of such complexes in higher plants (16) and respiratory electron transfer chains (1’7, 18) suggests the TABLE II possibility of such a complex in photosynSPECTRALPROPERTIESOFC.VINOSUM CYTOCHROMES" thetic bacteria. -.
a-band maximum
Sore&band maximum
300°K
77°K
300°K
‘77°K
552.5 (548) 549.5 556.5
422
420
424 430
421 427
Cytochrome
cjs5
555
Cytochrome Cytochrome
css3 b,,,
553 560
a The wavelength maxima (nm) were measured on the deoxycholate-solubilized material from green chromatophores after ammonium sulfate fractionation and Bio-Gel Al.5 chromatography. The 300°K values are identical to those in situ. The 77°K values were measured in a 60% glycerol:40% water glass.
ACKNOWLEDGMENTS The authors would like to thank Mr. Don Carlson and Mr. Bud Faris for growing the C. vinosum cells and Professor Graham Palmer for the use of his epr facilities. This investigation was supported in part by National Science Foundation PCM76-24131 and Robert A. Welch Foundation (D-710) grants to David B. Knaff and a National Institutes of Health grant (GM-207511 to Richard Malkin. REFERENCES 1. BOARDMAN,N. K. (1968)Adv. Enzymol. 30,149. 2. KNAFF, D. B. (1978) Coord. Chem. Rev. 26, 47-70.
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CYTOCHROMES
3. KNAFF, D. B., AND BUCHANAN, B. B. (1975) Biochim. Biophys. Acta 376, 549-560. 4. FOWLER, C. (1974) Biochim. Biophys. Acta 357, 327-331. 5. OLSON, J. M., GIDDINGS. T. H., AND SHAW, E. K. (1976) Biochim. Biophys. Acta 449, 197-208. 6. CASE, G. D., ANDPARSON, W. W. (1973)Biochim. Biophys. Acta 325, 441-453. 7. CLAYTON, R. K. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A., and Vernon, L. P., eds.), p. 498, Antioch Press, Yellow Springs, Ohio. 8. KENNEL, S. J., AND KAMEN, M. D. (1971) Biochim. Biophys. Acta 253, 153-166. 9. MILLER, R. W., AND MASSEY, V. (1965) J. Biol. Chem. 240, 1453-1465. 10. PARSON, W. W. (1969) Biochim. Biophys. Acta 189, 397-403.
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11. CUSANOVICH, M. A., AND KAMEN, M. D. (1968) Biochim. Biophys. Acta 153, 376-396. 12. CASE, G. D., AND PARSON, W. W. (1971) Biochim. Biophys. Acta 253, 187-202. 13. BARTSCH, R. G., AND KAMEN, M. D. (1960) J. Biol. Chem. 235, 825-831. 14. SATO, N., WILSON, D. F., AND CHANCE, B. (1971) Biochim. Biophys. Acta 253, 88-97. 15. BOARDMAN, N. K., AND ANDERSON, J. M. (1967) Biochim. Biophys. Acta 143, 187-203. 16. NELSON, N., AND NEUMANN, J. (1972) J. Bid. Chem. 247, 1817-1824. 17. HATEFI, Y., HAAVIK, A. G., AND GRIFFITHS, D. E. (1962) J. Biol. Chem. 237, 1681-1685. 18. ZAUGG, W. S., AND RIESKE, J. S. (1962) Biochem. Biophys. Res. Commun. 9, 213-217.
