- Email: [email protected]
Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex
Vol.
179,
August
No. 30,
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
INHIBITORY EFFECTS OF DICYCLOHEXYLCARBODIIMIDE CYTOCHROME b6-f COMPLEX
507-511
ON SPINACH
Liang-Bi Li, Linda Yu, and Chang-An Yu Departmentof Biochemistry, OAES, OklahomaStateUniversity Stillwater, OK 74078
Received
July
15,
1991
The electron transferactivity of purified cytochrome bfj-fCOmpkX of spinachchloroplastis inhibited by dicyclohexylcarbodiimide (DCCD) in a concentrationand incubationtime dependent manner. The maximum inhibition of 75 % is observedwhen 300 mole of DCCD per mole of protein (basedon cytochromej) is incubatedwith cytochrome bg-fcomplex at room temperature for 40 min. The inhibition of the complex is not due to the formation of crosslinks between subunitsbut due to the modification of carboxyls. The amountof DCCD incorporation is directly proportional to the activity loss,suggestingthat somecarboxyl groupsin the complex are directly or indirectly involved in the catalytic function. The incorporatedDCCD is locatedmainly at cytochrome bg protein. The partially inhibited complex showsthe sameH+/e- ratio asthat of the intact complex when embeddedin phospholipidvesicles. 0 1991Academic Press,Inc.
The cytochrome be-f complex, which catalyzeselectron transfer from plastoquinolto plastocyanin, is a segmentof the photosyntheticelectron transfer chain [l-7]. It is similar to the cytochrome b-cl complex of the mitochondrialrespiratory chain in both function and essential redox components[7,8]. The oxidation of plastoquinolis coupled with the generationof a proton gradient acrossthe thylakoid membranehaving a H+/e- ratio approaching2 [9, lo], which is compatiblewith the Q-cycle mechanism.According to the Q-cycle hypothesis,protons are translocatedby a ligand conduction mechanismin which plastoquinolis the only proton carrier. Someof the available experimentalevidencearguesagainsta Q-cycle mechanismin the cytochrome b&f complex. The uncertainty of the fast electron transferbetweenthe two cytochromesbg andthe similarredox potentialsand spectralcharacteristicsof thesetwo cytochromesare not predictedby the Q-cycle hypothesis. Other proton transfer mechanisms,such asredox linked proton pumping, have beenproposed[ 111. The detection of a specific DCCD binding site in yeast cytochrome b-cl complex and the inhibition of proton transfer, but not of electron transfer, by DCCD seemto supportthe proton pumping mechanism[ 121. In the caseof the cytochrome b6-f complex, DCCD inhibits both proton andelectron transferactivities, and the inhibition is directly proportional to the uptake of DCCD by cytochrome bg protein, suggestingthe involvement of a carboxyl group of the cytochrome bg protein in both electron and proton transport.
507
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in an! form reserved.
Vol.
179, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS The lipid- andplastoquinone-deficientcytochrome b&f complex waspreparedand assayed asreported [131. This isolatedcomplex contained,in nanmoles/milligramof protein: cytochromef ,lO.l; cytochrome b6.19.8 and plastoquinone6.1. The specific activity of the complex was45 j.tmolescytochrome c reducedper mg per hour after being replenishedwith glycolipids isolated from chloroplasts[l3]. The cytochrome be-f complex phospholipidvesicleswere preparedessentiallyaccordingto the methodof Leung and Hinkle [ 141,except a phospholipid-to-proteinratio of 50 and potassium phosphatebuffer, pH 7.0, were used. The assaymediumfor proton ejection contained 1.35 mL of 0.1 M KCl, 50 ll.L cytochrome b&f vesicle (5 FM cyt, f), 4 pL cytochrome c (2 mM) and 2 pL valinomycin (1 mg/mL). The reaction was startedby the addition of 5 pL of 10 mM PQzH2. P&H2 [ 151and Decanoy-N-methylglucamide(DMG) [ 161were syntheziedaccordingto the reported methods. Incorporation of [14C]DCCD into cytochrome bg-f complex protein andinhibition of enzymatic activity were determinedby a methodsimilarto that usedto measurethe incorporationof photoactivated azido-Q [ 131. One hundredl.tL of cytochrome b&f complex, 10 l.tM cytochromef, in 50 mM Tris-succinatebuffer, pH 7.0, containing 0.4 % DMG, 1 % sodiumcholate, and 10 % glycerol, were incubatedwith 5 pL of ethanolic solutioncontainingthe indicated amountof DCCD at room temperaturefor 40 min. After incubation,the mixtures were diluted with 3.5 volumesof 50 mM Tris-succinatebuffer containing 1 % sodiumcholate and onevolume of asolectinsolution (2 m&L) in 0.5 % DMG. The mixture was then further incubatedat 0” C for 30 min before enzymatic activity wasdetermined. To determinethe incorporation of DCCD into protein, 5 p.1of DCCD treated samples(at the end of 40 min. incubation) were withdrawn and spottedon Whatman paper No. 3. After all sampleswere spottedand dried, the paper was developedwith chlorofom-nmethanol(2: 1) (v/v). Denaturedproteinsremainedat the origin while unreactedDCCD moved up with the solvent. The protein spotswere cut out andplacedin scintillation vials containing 5 ml of Insta-gel. Radioactivity was determinedin a PackardTri-Carb Scintillation Analyzer, 1900 CA. Distribution of radioactivity amongthe subunitsof the cytochrome b-f complex was estimatedby the reported method [ 131. The [14C]DCCD treated sampleswere dialyzed against water and extracted with organic solvent, asdescribedpreviously [ 171. The extracted samples were redissolvedin 50 mM IVNa-phosphatebuffer, pH 7.0, to a protein concentrationof 1 mg/ml. SDS-PAGE and radioactivity determinationwere performed accordingto Doyle et al. [131.
RESULTS AND DISCUSSION Inhibition of cvtochrome bA,f complex bv DCCD: As shownin the Figure 1, the inhibition of the cytochrome b&f complex dependson the concentrationof DCCD used. Maximum inhibition of 75 % is observedwhen 300 mole of DCCD per mole of protein (basedon cytochromefl is incubatedwith cytochrome b6-f complex at room temperaturefor 40 min. The stock solution of DCCD was freshly madein absoluteethanol. The concentrationof solvent in the sampleswaskept constantat 5%, while various concentrationsof DCCD were used. In the control experiment, in the presenceof solvent andabsenceof DCCD, the complex lost about 10 % activity during incubation. Figure 2 showsthe incubation time dependentinhibition of cytochrome be-f complex by DCCD. At a constantconcentrationof DCCD, the activity of the cytochrome b6-f complex decreasesin direct proportion to the incubation time. A maximuminhibition is reachedat 40 min and with a TIE of 10 min.
508
Vol.
179, No. 1, 1991
100
BIOCHEMICAL
200
Concentration,
300
400
mol DCCD/mol
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
500
02
f
Time, min
Fig. 1. DCCDconcentration-dependent inhibitionof thecytochromeb6-f complex.Thecomplex (10 pM cytochromefi,in 50mM Tris-succinate buffer, pH 7.0, containing1%sodium cholateand10%glycerol,wastreatedwith theindicatedethanolicDCCD solutionfor 40 minat roomtemperature (-0-). Thesampleprepared underidenticalconditionswithout DCCDwasusedasa referencefor thecalculationof theremainingactivity. 100%activity represents 41 pmolescytochromec reducedpernmolecytochromefperhour. Thecurve with triangles(A) represents DCCDincorporationinto thecytochromebg-f complexwhen treatedwith [t4C] DCCDunderthesameconditions. Fig. 2. Effect of DCCDincubationtimeon theinhibitionof thecytochromeb6zf complex.The experimental conditionswerethe sameasin Fig. 1, exceptthata constantamountof DCCD,300mol/molcytochromebg-f complex,wasusedandincubationtimeswere varied. The crosssymbols(-x-) showthesampletreatedwith DCCD;thecircles(-0-) representcontrolexperiments in whichonly solventwasaddedto thecomplex
Correlation betweenactivitv inhibition andincornoration of DCCD: When cytcchrome be-f complex is treated with [14C]DCCD at various concentrationsfor 40 mitt, the incorporation of DCCD into protein is proportional to the concentrationof DCCD used. As indicated in Figure 1, the amountof DCCD incorporation is directly proportional to the activity loss,suggestingthat somecarboxyl groupsin the hydrophobic region of the complex are directly or indirectly involved in the catalytic function of the cytochrome bg-fcomplex. If the activity lossdue to DCCD resulted from intra-subunit or inter-subunitcrosslinking, no direct correlation betweenDCCD uptakeand activity losswould be expected. Also, the formation of an inter-subunit crosslink shouldbe readily detectableby SDS-PAGE of the DCCD-treated complex. The SDS-PAGE patternsof treatedand untreatedcytochrome bg-f complex are identical (datanot shown)clearly indicating that no cross-linking occursbetweensubunits. From the specificradioactivity of DCCD it was calculatedthat when 0.7 mole of DCCD is incorporatedinto one mole of cytochrome be-f complex, there is 40 % activity loss. This suggeststhat one or two molesof carboxyls per mole of complex are modified by DCCD. Bindine of IuClDCCD to cvtochrome b4: Figure 3 showsthe distribution of radioactivity among the subunitsof the [14C]DCCD treated complex. DCCD is bound specifically to the cytochrome bg protein. Very little radioactivity is found in the other subunits. Occasionally someradioactivity is observedat the top of the gel due to aggregationof the cytochrome bg protein. The appearanceof radioactivity at the top of the gel correlateswith a decreasein the radioactivity in the cytochrome bg protein, suggestingthat part of the latter is aggregatedunder the conditionsused. This correlation 509
Vol. 179, No. 1, 1991
03
0.0
BIOCHEMICAL
0.2
0.4
0.6
Relative
Mobllity
0.8
AND BIOPHYSICAL
1.0
RESEARCH COMMUNICATIONS
t 04 PQlHl
Fig. 3. The distributionof [14C]DCCDamongthesubunitsof the cytochrome66-fcomplex. The cytochromeb(j-f COmpleX wastreatedwith 300molarexcessof [14C]labeledDCCD, 10 nCi/molDCCD, atroomtemperature for 40min underthe conditionsgivenin Fig. 1. After incubation,thesamplewasdialyzedextensivelyagainstwaterandextractedwith organicsolvent. Theextractedsamplewaslyophilized,redissolved in 50mM K/Na phosphate buffer to a proteinconcentration of 1mg/mLandsubjected to SDS-PAGEafter treatmentwith 1 % SDSand1 % P-mercaptoethanol at 37 ‘C for 2 hrs. The conditionsfor electrophoresis andthe estimation of radioactivitydistributionwerethesameasreported previously(13). The Arabic numerals1to 4 representsubunitsI to IV of the cytochrome b6-f Complex. Fig. 4. Protonejectionof DCCDtreatedanduntreatedcytochromeb6-f complexinlaidin phospholipid vesicles.Thepreparationof cytochromeb6-fcomplex-phospholipid vesicles is detailedin thetext. Curve 1 isthe untreatedcomplexandcurve2 (67 % inhibition)isthe DCCDtreatedsample.
also suggeststhat the lossof activity during treatmentwith DCCD is due to modification of carboxyl groupsin the cytochrome bg protein, which renderscytochrome bg protein lesssoluble. Since the only carboxyl groupsthat will be labeledby DCCD are thoselocatedin the hydrophobic environment lacking active hydrogen groupsin the vicinity, the carboxyl groups could either be Asp-155 or Glu-166 Thesetwo amino acid residuesare located in the fourth transmembranehelice of the five transmembranehelice modelof cytochrome be. In the four transmembranehelice model, theseresiduesare locatedon the lumen sideof the thylakoid membrane(18). DCCD inhibition of proton ejection from cvtochrome bc-f uhosuholinidvesicles: Figure 4 shows the proton ejection by cytochrome b&f complex embeddedin phospholipidvesiclesusing cytochrome c asthe electron acceptorand PQ2H2asthe electron donor. The ratio of H+/e- for intact cytochrome b&f vesiclesis about 1.9. The DCCD treatedcomplex showsa similarH+/eratio when incorporatedinto phospholipidvesiclesdespitethe significant decreasein proton ejection rate. This decreasein proton ejectionrate is proportional to the decreasein activity of the complex after treatmentwith DCCD. In contrast to yeastcytochrome b-cl complex [12], in which DCCD inhibits proton translocationbut haslittle effect on electron transferactivity, the electron transfer activity of the cytochrome b&f complex is directly linked to proton translocation. It shouldbe mentionedthat in intact thylakoid membrane,the electron acceptorplastocyaninis located on the lumen sideandprotons are transportedin. In the reconstitutedvesicle system,with cytochrome c asthe electron acceptor,only thosecomplexesarrangedwith cytochromeffacing outward will be accessibleto cytochrome c and active in electron transfer. The cytochrome b&f
510
Vol.
179,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
complexes which have the opposite topological arrangement will not catalyzes electron transfer or proton translocation.
ACKNOWLEDGMENTS Journal Article No.J-6050 of the Agricultural Experiment Station, Oklahoma State University. This work was supported by grants from the USDA (AMD 9101532) . We thank Dr. Roger Koeppe for his critical review of this manuscript.
REFERENCES 1.
2. 3. 4. 5. 6. 7. ;: 10. 11. 12. 13. 14. 1.5. 16. 17. 18.
Nelson, N., and Neumann, J. (1972) J. Biol. Chem., 247, 1917-1924. Wood, P. M., and Bendall, D. S. (1976) Eur. J. Biochem., .& 337-344. Hurt, E., and Hauska, G. (1981) Eur. .I. Biochem., 117, 591-599. Clark, R. D., and Hind, G. (1983) J. Biol. Chem., 528, 10348-10354. Doyle, M. F., and Yu, C. A. (1985) Biochem. Biophys. Res. Communi., 131, 700-706. Black, M. T., Widger, W. R., and Cramer, W. A. (1987) Arch. Biochem. Biophys., 252, 655-661. Cramer, W. A., Black, M. F., Widger, W. R., and Girvin, M. E. (1987) in “The Light Reactions” (J. Barber, ed.,) pp. 447-493. Elsevier Sci Publ., Amsterdam. Hurt, E. C., and Hauska, G. (1982) J. Bioenerg. Biomembr., 14, 405-423. Hurt, E. C., Gabellini N, Shahak, Y., Lockau, W.,and Hauska, G. (1983) Arch Biochem Biophys 225, 879-885. Willms, I., Malkin, R., and Chain, R. K. (1987) Arch. Biochem. Biophys., 258. 24% 258. Papa. S., Guerrieri, F., Lorusso, M.,Izzo, G., and Capuano, f. (1982) in “Function of Quinone in Energy Conserving Systems” (Trunpower, B. L., ed.) Academic Press, New York, pp. 527-540. Beattie, D. S., and Cleian, L. (1982) FEBS Lett. 149, 245-248. Doyle, P. M., Li, L-B., Yu, L., and Yu, C. A. (1989) J. Biol. Chem., 264. 1387-1392. Leung, K.H., and Hinkle, P. (1975) J. Biol. Chem., 250, 8667-8671. Yu, C. A., Yu, L. (1982) Biochem. 21,4096-4101. Hildreth, J. E. K. (1982) Biochem. J. 207, 363-366. Yu, L., Yang, F-D., and Yu., C. A. (1985) J. Biol. Chem., 260, 963-973. Szczepaniak, A and Cramer, W. A. (1990) J. Biol. Chem., 265, 17720-17726.
511
179,
August
No. 30,
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
INHIBITORY EFFECTS OF DICYCLOHEXYLCARBODIIMIDE CYTOCHROME b6-f COMPLEX
507-511
ON SPINACH
Liang-Bi Li, Linda Yu, and Chang-An Yu Departmentof Biochemistry, OAES, OklahomaStateUniversity Stillwater, OK 74078
Received
July
15,
1991
The electron transferactivity of purified cytochrome bfj-fCOmpkX of spinachchloroplastis inhibited by dicyclohexylcarbodiimide (DCCD) in a concentrationand incubationtime dependent manner. The maximum inhibition of 75 % is observedwhen 300 mole of DCCD per mole of protein (basedon cytochromej) is incubatedwith cytochrome bg-fcomplex at room temperature for 40 min. The inhibition of the complex is not due to the formation of crosslinks between subunitsbut due to the modification of carboxyls. The amountof DCCD incorporation is directly proportional to the activity loss,suggestingthat somecarboxyl groupsin the complex are directly or indirectly involved in the catalytic function. The incorporatedDCCD is locatedmainly at cytochrome bg protein. The partially inhibited complex showsthe sameH+/e- ratio asthat of the intact complex when embeddedin phospholipidvesicles. 0 1991Academic Press,Inc.
The cytochrome be-f complex, which catalyzeselectron transfer from plastoquinolto plastocyanin, is a segmentof the photosyntheticelectron transfer chain [l-7]. It is similar to the cytochrome b-cl complex of the mitochondrialrespiratory chain in both function and essential redox components[7,8]. The oxidation of plastoquinolis coupled with the generationof a proton gradient acrossthe thylakoid membranehaving a H+/e- ratio approaching2 [9, lo], which is compatiblewith the Q-cycle mechanism.According to the Q-cycle hypothesis,protons are translocatedby a ligand conduction mechanismin which plastoquinolis the only proton carrier. Someof the available experimentalevidencearguesagainsta Q-cycle mechanismin the cytochrome b&f complex. The uncertainty of the fast electron transferbetweenthe two cytochromesbg andthe similarredox potentialsand spectralcharacteristicsof thesetwo cytochromesare not predictedby the Q-cycle hypothesis. Other proton transfer mechanisms,such asredox linked proton pumping, have beenproposed[ 111. The detection of a specific DCCD binding site in yeast cytochrome b-cl complex and the inhibition of proton transfer, but not of electron transfer, by DCCD seemto supportthe proton pumping mechanism[ 121. In the caseof the cytochrome b6-f complex, DCCD inhibits both proton andelectron transferactivities, and the inhibition is directly proportional to the uptake of DCCD by cytochrome bg protein, suggestingthe involvement of a carboxyl group of the cytochrome bg protein in both electron and proton transport.
507
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in an! form reserved.
Vol.
179, No. 1, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS The lipid- andplastoquinone-deficientcytochrome b&f complex waspreparedand assayed asreported [131. This isolatedcomplex contained,in nanmoles/milligramof protein: cytochromef ,lO.l; cytochrome b6.19.8 and plastoquinone6.1. The specific activity of the complex was45 j.tmolescytochrome c reducedper mg per hour after being replenishedwith glycolipids isolated from chloroplasts[l3]. The cytochrome be-f complex phospholipidvesicleswere preparedessentiallyaccordingto the methodof Leung and Hinkle [ 141,except a phospholipid-to-proteinratio of 50 and potassium phosphatebuffer, pH 7.0, were used. The assaymediumfor proton ejection contained 1.35 mL of 0.1 M KCl, 50 ll.L cytochrome b&f vesicle (5 FM cyt, f), 4 pL cytochrome c (2 mM) and 2 pL valinomycin (1 mg/mL). The reaction was startedby the addition of 5 pL of 10 mM PQzH2. P&H2 [ 151and Decanoy-N-methylglucamide(DMG) [ 161were syntheziedaccordingto the reported methods. Incorporation of [14C]DCCD into cytochrome bg-f complex protein andinhibition of enzymatic activity were determinedby a methodsimilarto that usedto measurethe incorporationof photoactivated azido-Q [ 131. One hundredl.tL of cytochrome b&f complex, 10 l.tM cytochromef, in 50 mM Tris-succinatebuffer, pH 7.0, containing 0.4 % DMG, 1 % sodiumcholate, and 10 % glycerol, were incubatedwith 5 pL of ethanolic solutioncontainingthe indicated amountof DCCD at room temperaturefor 40 min. After incubation,the mixtures were diluted with 3.5 volumesof 50 mM Tris-succinatebuffer containing 1 % sodiumcholate and onevolume of asolectinsolution (2 m&L) in 0.5 % DMG. The mixture was then further incubatedat 0” C for 30 min before enzymatic activity wasdetermined. To determinethe incorporation of DCCD into protein, 5 p.1of DCCD treated samples(at the end of 40 min. incubation) were withdrawn and spottedon Whatman paper No. 3. After all sampleswere spottedand dried, the paper was developedwith chlorofom-nmethanol(2: 1) (v/v). Denaturedproteinsremainedat the origin while unreactedDCCD moved up with the solvent. The protein spotswere cut out andplacedin scintillation vials containing 5 ml of Insta-gel. Radioactivity was determinedin a PackardTri-Carb Scintillation Analyzer, 1900 CA. Distribution of radioactivity amongthe subunitsof the cytochrome b-f complex was estimatedby the reported method [ 131. The [14C]DCCD treated sampleswere dialyzed against water and extracted with organic solvent, asdescribedpreviously [ 171. The extracted samples were redissolvedin 50 mM IVNa-phosphatebuffer, pH 7.0, to a protein concentrationof 1 mg/ml. SDS-PAGE and radioactivity determinationwere performed accordingto Doyle et al. [131.
RESULTS AND DISCUSSION Inhibition of cvtochrome bA,f complex bv DCCD: As shownin the Figure 1, the inhibition of the cytochrome b&f complex dependson the concentrationof DCCD used. Maximum inhibition of 75 % is observedwhen 300 mole of DCCD per mole of protein (basedon cytochromefl is incubatedwith cytochrome b6-f complex at room temperaturefor 40 min. The stock solution of DCCD was freshly madein absoluteethanol. The concentrationof solvent in the sampleswaskept constantat 5%, while various concentrationsof DCCD were used. In the control experiment, in the presenceof solvent andabsenceof DCCD, the complex lost about 10 % activity during incubation. Figure 2 showsthe incubation time dependentinhibition of cytochrome be-f complex by DCCD. At a constantconcentrationof DCCD, the activity of the cytochrome b6-f complex decreasesin direct proportion to the incubation time. A maximuminhibition is reachedat 40 min and with a TIE of 10 min.
508
Vol.
179, No. 1, 1991
100
BIOCHEMICAL
200
Concentration,
300
400
mol DCCD/mol
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
500
02
f
Time, min
Fig. 1. DCCDconcentration-dependent inhibitionof thecytochromeb6-f complex.Thecomplex (10 pM cytochromefi,in 50mM Tris-succinate buffer, pH 7.0, containing1%sodium cholateand10%glycerol,wastreatedwith theindicatedethanolicDCCD solutionfor 40 minat roomtemperature (-0-). Thesampleprepared underidenticalconditionswithout DCCDwasusedasa referencefor thecalculationof theremainingactivity. 100%activity represents 41 pmolescytochromec reducedpernmolecytochromefperhour. Thecurve with triangles(A) represents DCCDincorporationinto thecytochromebg-f complexwhen treatedwith [t4C] DCCDunderthesameconditions. Fig. 2. Effect of DCCDincubationtimeon theinhibitionof thecytochromeb6zf complex.The experimental conditionswerethe sameasin Fig. 1, exceptthata constantamountof DCCD,300mol/molcytochromebg-f complex,wasusedandincubationtimeswere varied. The crosssymbols(-x-) showthesampletreatedwith DCCD;thecircles(-0-) representcontrolexperiments in whichonly solventwasaddedto thecomplex
Correlation betweenactivitv inhibition andincornoration of DCCD: When cytcchrome be-f complex is treated with [14C]DCCD at various concentrationsfor 40 mitt, the incorporation of DCCD into protein is proportional to the concentrationof DCCD used. As indicated in Figure 1, the amountof DCCD incorporation is directly proportional to the activity loss,suggestingthat somecarboxyl groupsin the hydrophobic region of the complex are directly or indirectly involved in the catalytic function of the cytochrome bg-fcomplex. If the activity lossdue to DCCD resulted from intra-subunit or inter-subunitcrosslinking, no direct correlation betweenDCCD uptakeand activity losswould be expected. Also, the formation of an inter-subunit crosslink shouldbe readily detectableby SDS-PAGE of the DCCD-treated complex. The SDS-PAGE patternsof treatedand untreatedcytochrome bg-f complex are identical (datanot shown)clearly indicating that no cross-linking occursbetweensubunits. From the specificradioactivity of DCCD it was calculatedthat when 0.7 mole of DCCD is incorporatedinto one mole of cytochrome be-f complex, there is 40 % activity loss. This suggeststhat one or two molesof carboxyls per mole of complex are modified by DCCD. Bindine of IuClDCCD to cvtochrome b4: Figure 3 showsthe distribution of radioactivity among the subunitsof the [14C]DCCD treated complex. DCCD is bound specifically to the cytochrome bg protein. Very little radioactivity is found in the other subunits. Occasionally someradioactivity is observedat the top of the gel due to aggregationof the cytochrome bg protein. The appearanceof radioactivity at the top of the gel correlateswith a decreasein the radioactivity in the cytochrome bg protein, suggestingthat part of the latter is aggregatedunder the conditionsused. This correlation 509
Vol. 179, No. 1, 1991
03
0.0
BIOCHEMICAL
0.2
0.4
0.6
Relative
Mobllity
0.8
AND BIOPHYSICAL
1.0
RESEARCH COMMUNICATIONS
t 04 PQlHl
Fig. 3. The distributionof [14C]DCCDamongthesubunitsof the cytochrome66-fcomplex. The cytochromeb(j-f COmpleX wastreatedwith 300molarexcessof [14C]labeledDCCD, 10 nCi/molDCCD, atroomtemperature for 40min underthe conditionsgivenin Fig. 1. After incubation,thesamplewasdialyzedextensivelyagainstwaterandextractedwith organicsolvent. Theextractedsamplewaslyophilized,redissolved in 50mM K/Na phosphate buffer to a proteinconcentration of 1mg/mLandsubjected to SDS-PAGEafter treatmentwith 1 % SDSand1 % P-mercaptoethanol at 37 ‘C for 2 hrs. The conditionsfor electrophoresis andthe estimation of radioactivitydistributionwerethesameasreported previously(13). The Arabic numerals1to 4 representsubunitsI to IV of the cytochrome b6-f Complex. Fig. 4. Protonejectionof DCCDtreatedanduntreatedcytochromeb6-f complexinlaidin phospholipid vesicles.Thepreparationof cytochromeb6-fcomplex-phospholipid vesicles is detailedin thetext. Curve 1 isthe untreatedcomplexandcurve2 (67 % inhibition)isthe DCCDtreatedsample.
also suggeststhat the lossof activity during treatmentwith DCCD is due to modification of carboxyl groupsin the cytochrome bg protein, which renderscytochrome bg protein lesssoluble. Since the only carboxyl groupsthat will be labeledby DCCD are thoselocatedin the hydrophobic environment lacking active hydrogen groupsin the vicinity, the carboxyl groups could either be Asp-155 or Glu-166 Thesetwo amino acid residuesare located in the fourth transmembranehelice of the five transmembranehelice modelof cytochrome be. In the four transmembranehelice model, theseresiduesare locatedon the lumen sideof the thylakoid membrane(18). DCCD inhibition of proton ejection from cvtochrome bc-f uhosuholinidvesicles: Figure 4 shows the proton ejection by cytochrome b&f complex embeddedin phospholipidvesiclesusing cytochrome c asthe electron acceptorand PQ2H2asthe electron donor. The ratio of H+/e- for intact cytochrome b&f vesiclesis about 1.9. The DCCD treatedcomplex showsa similarH+/eratio when incorporatedinto phospholipidvesiclesdespitethe significant decreasein proton ejection rate. This decreasein proton ejectionrate is proportional to the decreasein activity of the complex after treatmentwith DCCD. In contrast to yeastcytochrome b-cl complex [12], in which DCCD inhibits proton translocationbut haslittle effect on electron transferactivity, the electron transfer activity of the cytochrome b&f complex is directly linked to proton translocation. It shouldbe mentionedthat in intact thylakoid membrane,the electron acceptorplastocyaninis located on the lumen sideandprotons are transportedin. In the reconstitutedvesicle system,with cytochrome c asthe electron acceptor,only thosecomplexesarrangedwith cytochromeffacing outward will be accessibleto cytochrome c and active in electron transfer. The cytochrome b&f
510
Vol.
179,
No.
1, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
complexes which have the opposite topological arrangement will not catalyzes electron transfer or proton translocation.
ACKNOWLEDGMENTS Journal Article No.J-6050 of the Agricultural Experiment Station, Oklahoma State University. This work was supported by grants from the USDA (AMD 9101532) . We thank Dr. Roger Koeppe for his critical review of this manuscript.
REFERENCES 1.
2. 3. 4. 5. 6. 7. ;: 10. 11. 12. 13. 14. 1.5. 16. 17. 18.
Nelson, N., and Neumann, J. (1972) J. Biol. Chem., 247, 1917-1924. Wood, P. M., and Bendall, D. S. (1976) Eur. J. Biochem., .& 337-344. Hurt, E., and Hauska, G. (1981) Eur. .I. Biochem., 117, 591-599. Clark, R. D., and Hind, G. (1983) J. Biol. Chem., 528, 10348-10354. Doyle, M. F., and Yu, C. A. (1985) Biochem. Biophys. Res. Communi., 131, 700-706. Black, M. T., Widger, W. R., and Cramer, W. A. (1987) Arch. Biochem. Biophys., 252, 655-661. Cramer, W. A., Black, M. F., Widger, W. R., and Girvin, M. E. (1987) in “The Light Reactions” (J. Barber, ed.,) pp. 447-493. Elsevier Sci Publ., Amsterdam. Hurt, E. C., and Hauska, G. (1982) J. Bioenerg. Biomembr., 14, 405-423. Hurt, E. C., Gabellini N, Shahak, Y., Lockau, W.,and Hauska, G. (1983) Arch Biochem Biophys 225, 879-885. Willms, I., Malkin, R., and Chain, R. K. (1987) Arch. Biochem. Biophys., 258. 24% 258. Papa. S., Guerrieri, F., Lorusso, M.,Izzo, G., and Capuano, f. (1982) in “Function of Quinone in Energy Conserving Systems” (Trunpower, B. L., ed.) Academic Press, New York, pp. 527-540. Beattie, D. S., and Cleian, L. (1982) FEBS Lett. 149, 245-248. Doyle, P. M., Li, L-B., Yu, L., and Yu, C. A. (1989) J. Biol. Chem., 264. 1387-1392. Leung, K.H., and Hinkle, P. (1975) J. Biol. Chem., 250, 8667-8671. Yu, C. A., Yu, L. (1982) Biochem. 21,4096-4101. Hildreth, J. E. K. (1982) Biochem. J. 207, 363-366. Yu, L., Yang, F-D., and Yu., C. A. (1985) J. Biol. Chem., 260, 963-973. Szczepaniak, A and Cramer, W. A. (1990) J. Biol. Chem., 265, 17720-17726.
511