Photosynthesis by isolated chloroplasts X. Dependence of carbon dioxide assimilation on the photochemical reactions of chloroplasts

THE Joun~a . OF BIOLOGICAL CHEMISTRY Vol. 234, NO. 11, No embe 1959 P in ed in U.S.A. Pho osyn hesis by Isola ed Chlo oplas s X. DEPENDENCE OF CARBON DIOXIDE ASSIMILATION ON THE PHOTOCHEMICAL REACTIONS OF CHLOROPLASTS* A. V. TREBST, M. LOSADA, AND DANIEL I. ARNON~ F om he Labo a o y o Plan Physiology, Uni e si y o Cali o nia, Be keley, Cali o nia (Recei ed o publica ion, June 30, 1959) Un il ecen ly, he only expe imen ally documen ed pho o- chemical ac i i y o isola ed chlo oplas s was he Hill eac ion (3) in which illumina ed chlo oplas s e ol e oxygen in acco dance wi h Equa ion 1: A + Hz0 + AH2 + $0, (1) whe e A cp cscn s a nonphysiological elec on o hyd ogen accep o such as e icyanide o benzoquinone (4). Recen expe imen s (5-8) ha e shown ha he Hill eac ion is a agmen o a pho osyn he ic phospho yla ion o he non- cyclic ype (9). Fo example, wi h e icyanide as he elec on accep o , oxygen e olu ion is coupled wi h ATP o ma ion, in acco dance wi h Equa ion 2.* 4Fe3+Cy + 2HzO + 2AnP + 2Pi -+ 4Fez+Cy + 02 + 2ATP + 4H+ (‘I The ecogni ion o he Hill eac ion as an uncoupled pho o- phospho yla ion was pa alleled (5-S) by he iden i ica ion o he physiological coun e pa o Reac ion 2, he noncyclic pho o- phospho yla ion eac ion in which oxygen e olu ion and ATP o ma ion a c linked wi h TPN educ ion (Equa ion 3). 2TPN + 2HeO + 2ADP + 2P< -- 2TPNHz + 02 + PATP (3) Isola ed chlo oplas s ha e also been ound o o m ATP by a cyclic pho ophospho yla ion (5-8) in which ATP is he sole p oduc o he pho ochemical eac ion (Equa ion 4). Al1P + Pi -+ ATP (4) In addi ion o pho osyn he ic phospho yla ion ecen wo k has also p o ided di ec expe imen al e idence (10-14) o he o en asse ed bu ne e p e iously demons a ed capaci y o isola ed chlo oplas s o assimila e CO2 pho osyn he ically o he le el o ca bohyd a es. CO2 assimila ion by isola ed chlo o- plas s was ound o be a da k p ocess (15) dependen on a pho ochemically gene a ed “assimila o y powe ” comp ising wo componen s: TPNH2 and ATP o med by he noncyclic and cyclic pho ophospho yla ion eac ions (Equa ions 3 and 4). Noncyclic pho ophospho yla ion (Equa ion 3) p o ides all he h ee expec ed p oduc s o he ligh phase o pho osyn hesis: 02, TPNH2, and ATP. Cyclic pho ophospho yla ion (Equa ion * P elimina y epo s o his wo k ha e been published p e i- ously (1) 2). Aided byg an s om he Na ,ional Ins i u eso Heal h, IJni ed S a es Public-Heal h Se ice, and he O ice o Na al Resea ch. 1 The abb e ia ions used a e: FUN, la in mononucleo ide; Pi, o hophosphn e. 4) supplies only ATP and he pa icipa ion o his eac ion in CO2 assimila ion would bc needed only i he ATP o med in Reac ion 3 we e insu icien o COz assimila ion o he le el o ca bohyd a e. The pu pose o his a icle is o p esen e idence ha in pho osyn hesis by isola ed chlo oplas s, assimila ion o CO2 o he le el o suga phospha es equi es a p ope ly balanced pa ici- pa ion o bo h ligh eac ions: cyclic and noncyclic pho ophos- pho yla ion (Equa ions 4 and 3). In he p esen in es iga ion he balance be ween he wo ligh eac ions was main ained by egula ing he concen a ion o co ac o s o cyclic pho ophos- pho yla ion (16, 17). EXPERIMENTAL Me hods-B oken chlo oplas s om spinach (16) o suga bee lea es (18) we e used in all he expe imen s desc ibed he ein. The b oken chlo oplas s we e p epa ed wi h asco ba e (Cl, pa icles) as p e iously desc ibed (16). Chlo oplas ex ac was p epa ed wi h 0.035 M NaCl and used wi hou dialysis. COz ixa ion was ca ied ou a 20” in ec angula Wa bu g manome e essels, lushed wi h a gon gas be o e u ning on he ligh (app oxima ely 23,000 lux). The pe iod o illumina ion was 30 minu es. The eac ion was s opped by adding o each essel 0.1 ml o glacial ace ic acid. To al COz ixa ion was measu ed by pipc ing aliquo s om each ea men on s ainless s eel planche s, e apo a ing o d yness, and coun ing Cl4 wi h a hin window Geige -Miillc coun e . Fo he iden i ica ion o he p oduc s o CO% ixa ion he con en s o he Wa bu g essels we e cen i uged, and aliquo s o he supc na an liquid we e subjec ed o wo-dimensional pape ch oma og aphy (on Wha man No. 41 pape ) using as sol en s (a) 80 phenol-20 wa e and (b) a mix u e o 52 pa s n-bu anol, 14 pa s glacial ace ic acid and 35 pa s wa e . The adioac i i y in he indi idual compounds, loca ed on he pape s by adioau og aphy, was de e mined by coun ing on he d ied pape s. The indi idual compounds we e iden i ied by elu ion and subsequen coch oma og aphy wi h samples o au hen ic compounds. Suga phospha es we e u he iden i ied by dephospho yla ion wi h phospha asc (Polidase) and cch oma- og aphy wi h he co esponding au hen ic suga s. RESULTS Expe imen al documen a ion o he pa icipa ion o cyclic pho ophospho yln ion (Equa ion 4) in COz assimila ion by chlo oplas s equi ed use o a sys em di e en om he one desc ibed be o e in which Tl’KI& and ATP we e al eady supplied a FAC BIOLOGIA/BIBLIOTECA on May 21, 2017h p://www.jbc.o g/Downloaded om 3056 CO2 Assimila ion by Chlo oplas s Vol. 234, No. 11 in excess, and a second eac ion o gene a ing ATP would be supe luous (cj. T ebs e al. (15), Table I). In he expe imen s o be desc ibed p esen ly, e idence o he pa icipa ion o cyclic pho ophospho yla ion in CO2 assimila ion o isola ed chlo o- plas s was ob ained in a “ca aly ic” sys em, i.e. one in which, as in an in ac cell, TPNH, and ATP we e o med in ca aly ic amoun s and CO2 ixa ion was he e o e possible only in he ligh while TPNHz and ATP we e being con inuously egene a ed a he expense o abso bed ligh ene gy. E$ec o FMN on CO2 Fixa ion-As shown in Table I, when he exogenous supply o ATP (2 /Imoles) was eplaced by a “ca aly ic” sys em con aining 0.5 pmole o ADP, o hophos- pha e, and 0.3 pmole o TPN, CO% ixa ion was sha ply educed (T ea men B). Howe e , he addi ion o he eac ion mix u e o an ex emely minu e quan i y o FMN (0.001 pmole) g ea ly inc eased o al COZ ixa ion (T ea men C). In he p esence o his minu e amoun o FMN he capaci y o chlo oplas s o ix COZ was equal o, i no g ea e han, ha in T ea men A in which 2 pmoles o exogenous ATP we e supplied. TABLE I E ec oj ibojla in phospha e (FMN) on CO, ixa ion dependen on egene a ion o ATP in ligh Each essel con ained in a inal olume o 2.5 ml : b oken chlo o- plas s (Cl.) con aining 0.5 mg o chlo ophyll; chlo ophyll ex ac (CE) equi alen o 2 mg o chlo ophyll; and he ollowing in wmoles: is(hyd oxyme hyl)aminome hane pH 7.5, 80; MgCL, 5; MnC12, 2; sodium asco ba e, 10; sodium phospha e, 5; educed glu a hione, 5; ibose 5-phospha e, 0.3; sodium ca bona e-W, 10. In addi ion o he indica ed ATP and ADP supplemen s, T ea - men A included 2 moles o TPN and each o T ea men s B and C, 0.3 pmole o TPN. T ea men To al C”00a ixed c.p.m. A. Con ol, 2 amoles o ATP. 232,000 B. 0.5 @mole o ADP.. 109,ooo C. 0.5 pmole o ADP, 0.001 Hmole o FMN. . 265,000 TABLE II E$ec o FMN concen a ion on CO2 $xa ion by illumina ed chlo oplas agmen s om suga bee s The eac ion mix u e was he same as ha desc ibed o T ea - men B in Table I excep ha glu a hione was omi ed and each essel con ained, in addi ion o he indica ed concen a ion o FMN, 0.3 mole o glucose l-phospha e ins ead o ibose li-phos- pha e. Final olume 3.0 ml. T ea - men FMN added jmlle/3 ml c.p.m. % % A 0 110,000 93 3 0.0002 105,000 32 65 0.0005 220,000 34 63 B 0.001 208,000 20 80 0.005 231,000 10 85 0.01 208,000 14 77 0.1 275,000 60 35 C 0.5 164,000 75 24 * Suga mono- and diphospha es and dihyd oxyace one phos- pha e. To al C” ixed as To al Co? ixed Phospho&e i Suga phospha es* The e ec o FMN on COz ixa ion was in es iga ed in g ea e de ail by a ying he concen a ion o his co ac o o cyclic pho ophospho yla ion. In addi ion o an inc ease in o al COz ixa ion, a s iking co ela ion was obse ed be ween he con- cen a ion o added FMN and he pa e n o ca bon compounds o med. Table II shows ha , depending on he concen a ion o FMN, he p oduc s o CO2 assimila ion we e ei he p edomi- nan ly suga phospha es, which a e aken he e as a measu e o a educ i e (pho osyn he ic) assimila ion pa e n, o phospho- glyce ic acid. Phosphoglyce ic acid was he chie p oduc o CO2 assimila ion when ei he no FMN (T ea men A) o a ela i ely la ge amoun o FMN (0.5 pmole) (T ea men C) was added o he eac ion mix u e. A a ange o low FMN concen a ion, om abou 0.001 o 0.01 c mole pe 3 ml, suga phospha es we e he p edominan p oduc s o CO2 assimila ion (T ea men B). These e ec s o FMN concen a ion on he pa e n o CO2 assimila ion a e illus a ed in Figs. 1 o 3. E ec o Vi amin KS and Phenazine Me hosulja e on CO2 Fixa ion-Simila e ec s on o al CO2 ixa ion and he pa e n o compounds o med we e also obse ed by adding wo o he co ac o s o cyclic pho ophospho yla ion: i amin K and phenasine me hosul a e. Typical esul s a e shown in Table III. A he same mola concen a ion, FMN, i amin K3, o phenasine me hosul a e p oduced compa able e ec s on o al CO2 ixa ion and on he ela ion be ween phosphoglyce a e and suga phospha es. The addi ion o small amoun s o any one o he h ee co ac o s o cyclic phospho yla ion (0.01 pmole pe 3 ml) inc eased o al CO2 ixa ion se e al imes and ga e suga phospha es as he main p oduc s o pho osyn hesis. A a concen a ion o 0.3 pmoles pe 3 ml he o ma ion o suga phospha e was ma kedly dec eased and phosphoglyce ic acid appea ed as he p incipal p oduc o CO2 ixa ion. DISCUSSION The esul s o his in es iga ion show ha he addi ion o one o he co ac o s o cyclic pho ophospho yla ion, FMN, i amin FIG. 1. Radioau og aph o a ch oma og am showing p oduc s o pho osyn he ic CWn assimila ion by illumina ed chlo oplas s in he absence o added ibo la in phospha e (FMN). O he con- di ions as gi en in Table 2. a FAC BIOLOGIA/BIBLIOTECA on May 21, 2017h p://www.jbc.o g/Downloaded om No embe 1959 A. V. T ebs , M. Losada, and D. I. A wn 3057 FIG. 2. Radioau og aph o a ch oma og am showing p oduc s o pho osyn he ic CY402 assimila ion by illumina ed chlo oplas s FIG. 3. Radioau og aph o a ch oma og am showing p oduc s supplied wi h 0.001 PM ibo la in phospha e (FMN). O he con- o pho osyn he ic CIQ assimila ion by illumina ed chlo oplas s di ions as gi en in Table 2. supplied wi h 0.5 PM ibo la in phospha e (FMN). O he condi- ions as gi en in Table 2. K, o phenazine me hosul a e, has a ma ked e ec on COZ TABLE III assimila ion in a “ca aly ic” sys em in which CO2 assimila ion E$ec o concen a ion o co ac o s o cyclic pho ophospho yla ion by chlo oplas s depends on egene a ion by ligh o assimila o y on CO2 $xa ion by illumina ed spinach chlo oplas s powe , i.e. on TPNHz and ATP. Since he e is no e idence o Expe imen al condi ions as in Table II, excep o he indica ed indica e ha minu e amoun s o FMN, i amin K, o phenazine addi ions o FMN, i amin K3, and phenazine me hosul a e. me hosul a e in luence di ec ly he enzyma ic eac ions esponsi- ble o COZ assimila ion, i seems easonable o seek an explana- ion o he obse ed esul s in he p e iously obse ed e ec s o hese co ac o s o cyclic pho ophospho yla ion on he cou se o he ligh eac ions (5, 17). Co ac o added To al C’“Oz ixed pmole C.). n. 79,000 318,066 415,060 530,000 390,090 310,660 267,666 Wi hou he addi ion o one o he co ac o s o cyclic pho o- None phospho yla ion, he ligh eac ions o isola ed chlo oplas s a e FMN limi ed o noncyclic pho ophospho yla ion (Equa ion 3). The a io o TPNHz o ATP o med is 1: 1. Adding a minu e amoun Vi amin K1 o FMN, i amin KS, o phenazine me hosul a e o a noncyclic pho ophospho yla ion sys em inc eases ATP o ma ion wi hou Phenazine app eciably dep essing oxygen e olu ion and he co esponding me hosul a e TPNHz accumula ion (c . A non e al. (17), Tables 3 and 4). The a io o ATP o TPNH o med becomes g ea e han 1. I appea s likely ha unde hese condi ions cyclic pho ophos- pho yla ion (Equa ion 4) is supe imposed on he noncyclic p ocess (Equa ion 3) and con ibu es addi ional ATP. The shi om phosphoglyce a e o suga phospha es as he main p oduc s o CO2 assimila ion (compa e Fig. 1 and Fig. 2) is explained by he addi ional ATP o med in he ligh by cyclic pho ophospho yla ion as a esul o adding one o i s co ac o s. Wi hou his addi ion he ligh eac ion is limi ed o noncyclic pho ophospho yla ion (Equa ion 3) and ails o p o ide su i- cien ATP o he educ ion o CO2 o suga s. I is concluded ha he 1:l a io o ATP o TPNHs which cha ac e izes Reac- ion 3 is insu icien o he educ ion o COZ o he le el o ca bohyd a e. The need o mo e ATP han TPNHz in CO2 assimila ion is consis en wi h he iew ha he e a e wo si es o phospho yla- ion bu only one si e o educ ion in he o ma ion o ca bo- hyd a es, The wo phospho yla ion eac ions a e: he phospho ibulokinase eac ion (Equa ion 5) (19-22) and he phos- - I % % 0.01 0.3 0.01 0.3 0.01 0.3 15 69 65 32 16 77 92 6 8 86 70 27 * Suga mono- and diphospha es and dihyd oxyace one phos- pha e. To al C”Oz ixed as Phospho- glyce a e Suga phospha es* phoglyce a e kinase eac ion (Equa ion 6). TPNHz is used only in he iosephospha e dehyd ogenase eac ion (Equa ion 7). All h ee enzymes conce ned ha e been ound in he chlo oplas p epa a ions used in hese expe imen s.2 Ribulose 5-phospha e + ATP phospho ibulokinase , (5) ibulose 1,5-diphospha e + ADP 3-phosphoglyce ic acid + ATP phosphoglyce a e kinase b (6) 1,3-diphosphoglyce ic acid + ADP 1,3-diphosphoglyce ic acid + TPNHz iosephospha e dehyd ogenase > (7) glyce aldehyde 3-phospha e + H3POd + TPN 2 M. Losada, A. V. T ebs , and D. I. A non, manusc ip sub- mi ed o publica ion. a FAC BIOLOGIA/BIBLIOTECA on May 21, 2017h p://www.jbc.o g/Downloaded om 3058 CO2 Assimila ion by Chlo oplas s Vol. 234, No. 11 When, in he absence o added co ac o s o cyclic pho ophos- pho yla ion, he ligh eac ions o chlo oplas s a e limi ed o noncyclic pho ophospho yla ion (Equa ion 3), he ATP o med is insu iicicn o bo h phospho yla ing si es. As he ATP o med by Reac ion 3 is used in he phospho yla ion o ibulose 5.phospha e (Equa ion 5), he 1: 1 a io be ween ATP andTPNH:! ncedcd o he educ ion o phosphoglycc a c (Equa ions 6 and 7) begins o dcc msc. E en ually, in a “ca aly ic” sys em, his would b ing he educ ion o phosphoglycc a c o a hal . Phos- phoglycc a c would hen end o accumula e (Fig. 1) as he ibulosc diphospha e o med in Reac ion 5 ac s as a CO2 accep o and is subsequen ly clea ed by he ca boxylase eac ion (21-24). The p edominance o phosphoglycc ic acid among he p oduc s o COZ assimila ion when a la ge amoun o one o he ac o s o cyclic pho ophospho yla ion is added o he eac ion mix u e (Fig. 3) is also explained by he p e iously obsc cd e ec s o FMN, i amin K, o phenazinc mc hosul a e on noncyclic pho ophospho yla ion (5, 17). When one o hese co ac o s is added o a noncyclic pho ophospho yla ion sys em a he highe concen a ions shown in Fig. 3 (also in Tables II and III), he noncyclic pho ophospho yla ion is con e ed o he cyclic ype (5, 17). Oxygen e olu ion and he accumula ion o educed TPN a e supp essed, phospho yla ion is sha ply inc eased, and he p incipal p oduc o he ligh eac ion is ATI’ (cj. A non e al. (17), Figs. 7 and 8). Unde hese condi ions he educ ion o phosphoglycc a c acid could no occu , since he ioscphospha e dchyd ogenasc eac ion (Equa ion 7) would be blocked by a lack o he educ an TPNH2. I appea s, he e o e, ha he nonoccu ence o a educ i e (pho osyn he ic) COz assimila ion esul ing in he o ma ion o suga phospha e can be caused ei he by a sho age o ATP (Fig. 1) o o TPNHz (Fig. 3). In bo h cases phosphoglyce a e would appea as he p edominan p oduc o CO* assimila ion because i s u he assimila ion would be blocked. Al hough on he basis o p esen e idence he wo si es o ATP ac ion in COZ assimila ion appea o be he phospho- ibulosc and he phosphoglyce a c kinase eac ions (Equa ions 5 and 6), he e is a possibili y ha he second si e o ATP ac ion in he phospho yla ion may be no he phospho yla ion o phosphoglyce a c bu ha o some uns able 6 ca bon com- pound, which, in ai o, unde goes phospho yla ion be o e educ- ion by TPSHz wi hou b eaking up in o 2 moles o phospho- glyce a c (25, 26). In he expe imen s epo ed he e he o ma ion o suga, phospha es which is aken as a mcasu c o pho osyn he ic CO2 assimila ion occu ed only when a p ope balance was main ained bc wccn cyclic and noncyclic pho ophospho yla ion. In isola ed chlo oplas s his balance was main ained by adding di e en amoun s o one o he ca alys s o cyclic pho ophospho yla ion. I is assumed ha he in ac ccl1 has sui able physiological cguln o y mechanisms o keeping he wo cnc ions in balance. SUMhl.QRy CO? assimila ion in isola ed chlo oplas s was inycs iga. cd in a “ca aly ic” sys em unde h ee condi ions: (I) when he pho o- chemical phase was limi ed o noncyclic pho ophospho yla ion, (2) when he pho ochcmical phase was limi ed o cyclic pho o- phospho ~ln iol1, and (3) when he pho ochemical phase included bo h cyclic and noncyclic pl~o ophospho yla ions. Unde Condi ions 1 o 2, CO2 assimila ion was limi ed almos en i ely o he o ma ion o phosphoglyce a e. Suga phos- pha es we e he p edominan p oduc s o CO2 assimila ion only in Condi ion 3. These esul s a e in e p e ed as ha ing been caused by a sho age o adenosine iphospha e (ATP) in Condi- ion 1 and o educed iphosphopy idine nuclco ide (TPNH2) in Condi ion 2; only in Condi ion 3 was a p ope balance es ab- lished be ween ATP and TPNHz o med a he expense o ligh ene gy, o make he o ma ion o suga phospha es possible. 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A non CHLOROPLASTS DIOXIDE ASSIMILATION ON THE PHOTOCHEMICAL REACTIONS OF Pho osyn hesis by Isola ed Chlo oplas s: X. DEPENDENCE OF CARBON 1959, 234:3055-3058.J. Biol. Chem. h p://www.jbc.o g/con en /234/11/3055.ci a ion Access he mos upda ed e sion o his a icle a Ale s: When a co ec ion o his a icle is pos ed• When his a icle is ci ed• o choose om all o JBC's e-mail ale sClick he e h p://www.jbc.o g/con en /234/11/3055.ci a ion. ull.h ml# e -lis -1 This a icle ci es 0 e e ences, 0 o which can be accessed ee a a FAC BIOLOGIA/BIBLIOTECA on May 21, 2017h p://www.jbc.o g/Downloaded om