Photosynthetic phosphorylation and molecular oxygen

1314 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. he modynamic o ces owa d he mos p obable o m, na i e ibonuclease. Some o he less likely possibili ies men ioned abo e can only be igo ously excluded upon comple ion o cu en expe imen s on he na u e o he pai ing o hal -cys ine esidues du ing he lag phase. The au ho s wish o hank M s. Juani a Cooke o he expe assis ance in many o hese expe imen s. * P esen add ess: Massachuse s Gene al Hospi al, Bos on 14, Massachuse s. On lea e o absence om The Weizmann Ins i u e o Science, Reho o h, Is ael. 1 Sela, M., F. H. Whi e, J ., and C. B. An insen, Biochim. e Biophys. Ac a, 31, 417 (1959). 2 Whi e, F. H., J ., J. Biol. Chem., 236, 1353 (1961). 3An insen, C. B., and E. Habe , ibid., 236, 1361 (1961). 4Aq is , S. E. G., and C. B. An insen, ibid., 234, 1112 (1959). 6 Pe e son, E. A., and H. A. Sobe , J. Am. Chem. Soc., 78, 751 (1956)-. 6 Hi s, C. H. W., S. Moo e, and W. H. S ein, J. Biol. Chem., 200, 493 (1953). 7 Spackman, D. H., W. H. S ein, and S. Moo e, Anal. Chem., 30, 1190 (1958). 8 An insen, C. B., R. R. Red ield, W. L. Choa e, J. Page, and W. R. Ca oll, J. Biol. Chem., 207, 201 (1954). 9 C es ield, A. M., Smi h, K. C., and F. W. Allen, ibid., 216, 185 (1956). 10 Richa ds, F. M., Comp . end. a . lIb. Ca lsbe g, Se . Chim., 29, 315 (1955). 11 Boye , P. D., J. Am. Chem. Soc., 76, 4331 (1954). 12 B ay, G. A., Anal. Biochem., 1, 279 (1960). 13 Sela, M., and C. B. An insen, Biochim. e Biophys. Ac a, 24, 229 (1957). 14 Sela, M., C. B. An insen, and W. F. Ha ing on, ibid., 26, 502 (1957). - Habe , E., M. Sela, and C. B. An insen, Fede a ion P oc., 20, Pa I, 217 (1961). 16 Expe imen al wo k is now in p og ess o de e mine he na u e o he pai ing o hal -cys ine esidues a a ious imes du ing he ea ly s ages o eoxida ion. P elimina y esul s indica e ha pai ing is qui e andom and ha "inco ec ly" o med bonds a e p esen . 17 Ha ing on, W. F., and J. A. Schellman, Comp . end. a . lib. Ca lsbe g, Se . Chim., 30, 21 (1956). 18 Sluy e man, L. A. A., Biochim. e Biophys. Ac a, 48, 429 (1961). 19 Habe , E., and C. B. An insen, unpublished da a. PHOTOSYNTHETIC PHOSPHORYLATION AND MOLECULAR OXYGEN* BY DANIEL I. ARNON M. LOSADA, F. R. WHATLEY, H. Y. TsuJIMOTO, D. 0. HALL, AND A. A. HORTON DEPARTMENT OF CELL PHYSIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY Communica ed July 3, 1961 Oxygen and pho osyn hesis we e i s linked abou 200 yea s ago when bo h we e disco e ed almos simul aneously. The ea lies concep o pho osyn hesis was ha o plane a y en ila ion in which illumina ed plan s exchanged CO2 o "bad ai " o 02 o " i al ai " (see his o ical e iew1). A mechanism o his gas exchange was p oposed in 1796 by Ingenhousz.2 G een plan s, he sugges ed, abso b om "ca bonic acid in he sunshine, he ca bon, h owing ou a ha ime he oxygen alone, and keeping he ca bon o i sel as nou ishmen ." 2 Fo o e a hund ed yea s a e wa d, he iew ha CO2 assimila ion always in- ol ed a libe a ion o oxygen gas was so i mly en enched ha i was e en ex- ended o he da k CO2 assimila ion by chemosyn he ic bac e ia.3-5 The idea VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1315 o pho osyn hesis wi hou oxygen e olu ion seemed a con adic ion o e ms un il he conclusi e wo k o an Niel6' 7wi h pho osyn he ic bac e ia i mly es ablished ha , basically, he assimila ion o CO2 a he expense o ligh ene gy does no de- pend on he libe a ion o oxygen o on he p esence o oxygen. Bac e ial pho o- syn hesis occu s unde s ic ly anae obic condi ions and includes o ganisms ha a e obliga e anae obes ( o example, Chlo obium and Ch oma ium6, 7). Al hough molecula oxygen was shown o be unnecessa y and o en e en de i- men al o bac e ial pho osyn hesis, i s ole in he ene gy ans o ma ions ha occu du ing pho osyn hesis in g een plan s has gained mo e a en ion wi h ime. In 1939, Hill pos ula ed8 ha he consump ion o molecula oxygen is necessa y o pho osyn hesis o g een plan s in o de o supply addi ional ene gy o CO2 assimi- la ion. He en isaged " i s a educ ion o a subs ance by chlo oplas s gi ing oxygen and hen a eoxida ion o he educed subs ances by pa o he oxygen oge he wi h a simul aneous educ ion o he necessa y equi alen o ca bon di- oxide." 9 In 1951 Bu k and Wa bu g'0 epo ed he sepa a ion o pho osyn hesis in o a ligh eac ion and a da k eac ion on he basis o manome ic measu emen s wi h in ac Chlo ella cells. In he wo ds o Wa bu g, "in he ligh eac ion, one molecule o 02 will de elop pe molecule o chlo ophyll ... whe eas du ing he da k pe iod ollowing he end o illumina ion i can be obse ed manome ically ... ha wo- hi ds o he oxygen gas de eloped du ing he ligh pe iod unde goes a back eac ion." '1 This da k consump ion o oxygen is, in Wa bu g and Bu k's iew, a pho osyn he ically induced espi a ion ha supplies wo- hi ds o he needed ene gy and is, he e o e, indispensable o CO2 assimila ion in pho osyn hesis. As summed up ecen ly by Wa bu g, "Keine (CO2) Fixie ung ohne A mung." 12 A biochemical model o oxygen consump ion in pho osyn hesis in acco dance wi h he iews o Hill and Wa bu g was p oposed by Vishniac and Ochoa.13 They sugges ed ha he ATP used o CO2 assimila ion in pho osyn hesis is o med by a collabo a ion be ween chlo oplas s and mi ochond ia. In hei model, chlo o- plas s educed py idine nucleo ide in ligh and ATP was o med when mi ochond ia eoxidized he educed py idine nucleo ide wi h molecula oxygen, by he da k p ocess o oxida i e phospho yla ion. A simila model o ATP o ma ion has been used in se e al gene al schemes o pho osyn hesis, as, o example, in he one p oposed in 1951 by Holze l4 and in 1954 by Cal in's g oup ( e . 1a, Fig. 7). In all hese schemes, oxida i e phospho yla ion by mi ochond ia, and hence oxygen consump ion, was an essen ial ea u e o he mechanism o pho osyn hesis. A mechanism o pho osyn hesis ha includes a consump ion o molecula oxygen canno apply o he s ic ly anae obic bac e ial pho osyn hesis and leads, he e- o e, o he in e ence ha he mechanism o ene gy con e sion in bac e ial pho o- syn hesis is basically di e en om ha in plan pho osyn hesis. Howe e , he disco e y o pho osyn he ic phospho yla ion in isola ed spinach chlo oplas s16 and, sho ly he ea e , in cell- ee p epa a ions o Rhodospi illum ub um, 17 has poin ed o mechanisms o ATP o ma ion a he expense o ligh ene gy ha a e common o g een plan s and pho osyn he ic bac e ia. A uni ied concep o pho osyn hesis in plan s and bac e ia, based on pho osyn he ic phospho yla ion as he common denomina o is now possible and has been p esen ed elsewhe e. 18-20 A key p emise in his concep is ha , in g een plan s as in baq e ia, pho osyn he ic phospho yla ion is undamen ally independen o molecula oxygen. Since his 1316 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. has ecen ly been ques ioned12' 21 we ha e e-examined he ela ion o oxygen o pho osyn he ic phospho yla ion. The pu pose o his a icle is o b ing oge he and add o he e idence which con i ms and ex ends ou ea lie conclusions ha pho osyn he ic phospho yla ion, bo h in g een plan s and pho osyn he ic bac e ia, is basically independen o molecula oxygen and hence o espi a ion. Cyclic and Noncyclic Pho ophospho yla ion.-Pho osyn he ic phospho yla ion (pho ophospho yla ion) is a e m coined in 1954 o desc ibe a ligh -induced ATP o ma ion by isola ed chlo oplas s, wi hou he aid o mi ochond ia and wi hou he consump ion o oxygen.16 The o e -all eac ion o pho osyn he ic phospho yla- ion is ep esen ed by equa ion (1): ligh ADP + P ATP (1) Reac ion 1, in which he sole p oduc is ATP, was subsequen ly designa ed cyclic pho ophospho yla ion, o dis inguish i om a second pho ophospho yla ion eac ion by isola ed chlo oplas s (equa ion (2)) which was ound a ew yea s la e 22 and named noncyclic pho ophospho yla ion.18 ligh TPN + 2H+ + ADP + P + 20H- ) TPNH2 + ATP + H20 + '/202 (2) The e ms "cyclic" and "noncyclic" e e o he elec on low mechanisms which ha e been p oposed'8' 19 o hese wo eac ions ha now join ly cons i u e he p ocess o pho osyn he ic phospho yla ion. In cyclic pho ophospho yla ion all o he biochemically e ec i e ligh ene gy is used o ATP o ma ion. In non- cyclic pho ophospho yla ion only a po ion o he biochemically e ec i e ligh ene gy is used o he o ma ion o ATP; he emainde is used o he o ma ion o a educ an , TPNH2, and he exc e ion (e olu ion) o oxygen. A nonphysiologi- cal a ian o noncyclic pho ophospho yla ion ( eac ion 2) is eac ion 3, in which TPN is eplaced by e icyanide22' 23 ( ep esen ed he e by Fe3+). ligh 2Fe3+ + ADP + P + 20H- - 2Fe2+ + ATP + H20 + 1/202 (3) The signi icance o cyclic and noncyclic pho ophospho yla ion o he o e -all mechanism o pho osyn hesis in g een plan s is ha hey supply-aside om oxygen as he exc e ed by-p oduc - he wo p oduc s, ATP and TPNH2, which alone su ice o con e ing C02 in o ca bohyd a es in he da k.24 25 Bo h ATP and TPNH2 (also oxygen) a e o med by noncyclic pho ophospho yla ion (equa ion (2)) bu cyclic pho ophospho yla ion (equa ion (1)), which supplies only ATP, is also needed o C02 assimila ion because he ATP o med in noncyclic pho ophos- pho yla ion is insu icien o con e CO2 o ca bohyd a e.26 Chlo oplas agmen s ("g ana") do no espi e27' 28 and hence, acco ding o Wa bu g, would no be expec ed o ix CO2 ("G ana a men nich und ixie ien nich " 12). Howe e , by ac iona ing isola ed chlo oplas s we ha e shown ha g ana ail o assimila e C02 no because hey a e unable o espi e bu because hey lack he equisi e chlo oplas enzymes. These a e wa e -soluble and a e eadily leached ou in he p epa a ion o "g ana."29 The soluble chlo oplas enzymes, ei he in si u o when ex ac ed om chlo oplas s, assimila e CO2 o he le el o ca bohyd a es and do so solely24 a he expense o ATP and TPNH2- VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1317 he wo compounds o med in chlo oplas s by cyclic and noncyclic pho ophos- pho yla ion (equa ions (1) and (2)) and no by espi a ion. CO2 assimila ion p ope in pho osyn hesis, whe he in whole cells30 o in isola ed chlo oplas s,24' 25 consis s o he same exclusi ely da k, enzyma ic eac ions3' ha a e now known o ope a e in nonpho osyn he ic cells.32-34 The e is, so a as we know, no alid expe imen al basis, o pos ula ing in pho osyn hesis a peculia pho ochemically p oduced " educing powe " ha is capable o di ec educ ion o CO2 o he le el o ca bohyd a e by some special mechanism unknown elsewhe e in biochemis y (see, o example, e . 35 and 36). I we abandon he no ion o an unspeci ied, hypo he ical " educing powe " and in e p e pho osyn hesis in e ms o known biochemical mechanisms, hen he unique ea u e o pho osyn hesis as a biological p ocess is no CO2 assimila ion-a da k p ocess d i en, in bo h pho osyn he ic and nonpho osyn he ic cells, by ATP and educed py idine nucleo ide-bu he o ma ion o hese wo compounds by cyclic and noncyclic pho ophospho yla ion. The disco e y o pho osyn he ic phospho yla ion was pa o a coinciden , b oade inding'6' 27 ha isola ed chlo oplas s a e capable o ca ying ou a com- ple e ex acellula pho osyn hesis, i.e., a con e sion o CO o s a ch and suga , a physiological empe a u es and wi h no ene gy supply excep isible ligh . The iew ha chlo oplas s a e he si es o comple e pho osyn hesis was once widely held wi hou he suppo o c i ical expe imen al e idence and was la e abandoned because o e idence o he con a y, only o be o mula ed anew on he basis o imp o ed me hods o isola ing unc ional chlo oplas s om lea es (see e iew'). The disco e y o cyclic and noncyclic pho ophospho yla ion by isola ed chlo o- plas s (also o CO2 assimila ion, see e iew') was con i med and ex ended in o he labo a o ies, no ably hose o Jagendo ,37-40 Wessels,41 42 Vennesland,43 44 and Hill. Mos o he wo k has been done wi h spinach chlo oplas s bu ecen ly Wha ley e al.46 ha e also demons a ed cyclic and noncyclic pho ophospho yla ion (and CO2 assimila ion) in chlo oplas s isola ed om se e al o he species o plan s.47 O he wo pho ophospho yla ion eac ions (equa ions (1) and (2)), he cyclic ype (equa ion (1)) appea ed he mo e basic om he s andpoin o a gene al mechanism o ene gy ans o ma ion in pho osyn hesis since his eac ion was ound in ep esen a i es o all he di e en g oups o pho osyn he ic o ganisms. Williamsw ound pho osyn he ic phospho yla ion in he obliga ely anae obic pho osyn he ic bac e ia, Ch oma ium and Chlo obium. Cyclic pho ophospho yla- ion in algal p epa a ions was ound by Thomas and Haans6' and Pe ack and Lipmann.52 (Fo a mo e comple e e iew o li e a u e see e . 53.) Special Fea u es o Cyclic and Noncyclic Pho ophospho yla ion.-The unique ea u es o cyclic pho ophospho yla ion a e ha ATP is o med wi h no added elec on dono and no added elec on accep o . Cyclic pho ophospho yla ion con- sumes nei he espi a o y subs a e no molecula oxygen. Bo h a e eplaced by ligh 53 which p o ides he ee ene gy equi ed o he syn hesis o py ophospha e bonds o ATP. This seemed a i s no o be he case in pho osyn he ic bac e ia. F enkel's cell- ee p epa a ions o R. ub um became subs a e-dependen a e washing; he a e o phospho yla ion was doubled on adding a-ke oglu a a e.17 Bu in subsequen expe imen s F enkel54 uled ou he dependence on an added chemical subs a e and once his undamen al poin was cla i ied, he equi alence 1318 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. o cyclic pho ophospho yla ion in chlo oplas s and bac e ial pa icles seemed p obable.55 The independence o pho osyn he ic phospho yla ion om a espi a o y sub- s a e is con i med by he independence o his p ocess om CO2 assimila ion. Pho osyn he ic phospho yla ion occu s when he enzymes o he educ i e ca bon cycle a e emo ed by washing he chlo oplas s, CO2 is no supplied o he eac ion mix u e, and he eac ion essels con ain KOH in he cen e well. This would no exclude a possible ca aly ic pa icipa ion o CO2 in pho ophospho yla ion (c . Wa bu g e al.12 and Vennesland e al.56). Wha can be de ini ely excluded is ha subs a e amoun s o ca bon compound(s) a e i s syn hesized in ligh om CO2 and a e hen used as elec on dono s o he o ma ion o ATP. While cyclic pho ophospho yla ion was ecognized as a common denomina o o all pho osyn heses, noncyclic pho ophospho yla ion as ep esen ed by equa ion (2)-a eac ion ha libe a es oxygen-seemed a i s des ined o emain a special ea u e o plan pho osyn hesis, since oxygen is no e ol ed in bac e ial pho o- syn hesis. Howe e , Losada e al.57 (c . also Duysens e al.58) ha e ecen ly sepa- a ed eac ion 2 in o wo dis inc pho ochemical eac ions (a) a pho ooxida ion o hyd oxyl ions ha yields oxygen (equa ion (4)) and (b) a pho ophospho yla ion eac ion p ope in which he pho o educ ion o iphosphopy idine nucleo ide is coupled wi h he o ma ion o adenosine iphospha e (equa ion 5). ligh 2A + 20H- > 2A- + H2O + 1/202 (4) ligh TPN + 2H+ + ADP + P + 2A- >-TPNH2+ ATP + 2A (5) ligh Sum: TPN+2H++ADP+P+2OH--- TPNH2 + ATP + H20 + 1/202 (2) Only he pho ooxida ion o hyd oxyl ions (equa ion (4)) is peculia o g een plan s since only g een plan s seem capable o using OH- as an elec on dono wi h he esul an exc e ion o molecula oxygen. Reac ion 5, he noncyclic pho ophospho yla ion eac ion p ope , occu s also in pho osyn he ic bac e ia.59 Since hey use ino ganic o o ganic elec on dono s, such as hiosul a e o succina e ins ead o OH-, oxygen e olu ion does no occu in he eac ion. Figu e 1 depic s he o e -all scheme o noncyclic pho ophospho yla ion in g een plan s ha we now en isage. The in e media e A in Figu e 1 is bo h he elec on accep o o he i s ligh eac ion and he elec on dono o he second ligh eac- ion (c . equa ions (4) and (5)). We ha e used indophenol dyes as an expe imen al de ice o sepa a e he wo pho ochemical eac ions. The na u al in e media es wi h which he dyes ha e in e ac ed ha e no been iden i ied. They may include cy och ome componen s60 o a quinone.61, 62 The e minal physiological elec on accep o in noncyclic pho ophospho yla ion (B in Fig. 1) is py idine nucleo ide. Ou o iginal scheme o noncyclic pho ophospho yla ion ( e . 22, Fig. 3) en- isaged he phospho yla ion s ep as occu ing be ween he unknown educ an (H), o med by he pho olysis o wa e and TPN, whe eas ou p esen scheme, based on he elec on low heo y,18' 53 links he phospho yla ion s ep wi h he oxida ion o cy och ome by pho oac i a ed chlo ophyll. VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1319 (?e,-- B - e Chi ~+) Cy - A I e ! LIGHT pigmen J(4, OH O - H20 [OH] LIGHT 4°2 FIG. 1.-Scheme o noncyclic pho ophospho yla ion o he g een plan ype. In he i s ligh eac ion, he pigmen molecule (see ex ) becomes exci ed by he abso p ion o a quan um o ligh . The exci ed pigmen dona es i s high-ene gy elec on (e -) o an in e media e elec on accep o (A) and accep s an elec on om an hyd oxyl ion. The oxida ion p oduc (OH) o he hyd oxyl ion is he p ecu so o molecula oxygen. In he second ligh eac ion, he chlo ophyll molecule (Chl), exci ed by he abso p ion o a quan um o ligh , dona es i s high-ene gy elec on (e-) o he e minal elec on accep o (B) and accep s, ia he cy och ome sys em (Cy ), an elec on om he educed in e media e (A-) o med in he i s ligh eac ion. The phospho yla ion s ep is linked wi h he ans e o he elec on om cy och ome o chlo ophyll. (.e-,. B [g cz~ e- e- A P. LIGHT FIG. 2. -Scheme o noncyclic pho ophospho yla ion o he bac e ial ype. The chlo ophyll molecule (Chl) becomes exci ed by he abso p ion o a quan um o ligh . The exci ed chlo- ophyll dona es i s high ene gy elec on (e-) o he elec on accep o (B) and accep s, ia he cy och ome sys em (Cy ), an elec on om an ex e nal elec on dono (A -). The phospho yla ion s ep is linked wi h he ans e o he elec on om cy och ome o chlo o- phyll. TPN educ ion in noncyclic pho ophospho yla ion ( eac ion 2), al hough no - mally coupled wi h, may also be uncoupled om, ATP o ma ion. T ebs e al. ( e . 63, Table 1) ha e shown ha ammonia64 supp esses almos comple ely he o ma ion o ATP in eac ion 2 in ligh wi hou inhibi ing he o ma ion o TPNH2. These esul s speak agains he sugges ion o Chance and Olson65 ha he pho o- educ ion o TPN may be d i en by ATP, o med pe haps by cyclic pho ophos- pho yla ion (c . also Ma 6 and Fo i66). The con a y is, in ac , he case. Elec- 1320 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. /00 02 p oduced 80 ~~~~~~~~/00 02 p oduced % 60 80 Xi 40 - /- 60 _a TPN//2 ~~~ o med I 'a,~~~~~~~~~~~~~~~~~~q z, ,00 40 0_A_ oxygene olu onand TPNH educ io med o 20 4~~~~~~0 Oxygen was measu ed manome iATP o med 0630644 66/ 677 699 FIG. 3.-E ec i eness o monoch oma ic 66 6 6 6 ligh , in he ed egion o he spec um, on oxygen e olu ion and TPN educ ion, occu - (m ing as sepa a e chlo oplas eac ions.57 Oxygen was measu ed manome ically when FIg. e olu ionwss o dsc ibed he pho ochemical eac ion was limi ed o Fig. 4ig e ec aegin o mo nc h oma o he pho ooxida ion o OH- (equa ion (4)) ligh n m h ed egio o hespe um do in a eac ion mix u e which included, in a oxgn ou ion andlume cylc oophosp ho la- inal olume o 3 ml, chlo oplas agmen s ioyen, u igassep n s mcls op as eaciens. (Cae) con aining 1 mg. o chlo ophyll and Oxgn e olu i on meason as dylesc p ibed he ollowing in Mmoles: is/ace a e bu - ih Fu e,3. AP o ma ion by; cyclic mpho o e , pH 8, 40; po assium e icyanide, 15; phospho yla (eque ean in () was measu ed and 2, 6 - dichlo ophenol indophenol 0i ap llel e imn inal ea io mi (DCPIP), 0.2. TPN educ ion (measu ed wi inclue ina ial oum ae o m spec opho ome ically a 340 mM) was chlo oplas agmen s (C18) con aining 1 mg ca ied ou in a pa allel expe imen in which chlo ophyll and he ollowing in Mmoles: is! he pho ooxida ion o wa e was blocked by HCl bu e , pH 8.3, 80; MgSO4, 5; ADP, 10; he omission o chlo ide and he addi ion K5)). 10;i an d ian K0 (mnaedione) o e 2ecin X e un Mo CMU The ele o dono 0.3.s qiaen o010Ml P e sys em euc cons do 100 on he o dina e scale is equi alen o moles asco ba e and 0.05 moles o 2,3',6- a oms oxygen e ol ed pe ichlo ophenol indophenol (equa ion (5)). quan a o ligh abso bed. 60 on he o dina e The eac ions we e un o 15 mm a 15'C. scale is equi alen o 0.10 ,umole ATP o med Gas phase, ni ogen. pe /mole quan a o ligh abso bed. 100 on he o dina e scale is equi alen o 0.15 MAa oms oxygen e ol ed pe M~mole quan a o ligh abso bed. 58 on he o dina e scale is equi alen o 0.09 M~moles TPNH2 o med pe /Amole quan a o ligh abso bed. on anspo , esul ing in TPN educ ion, may p oceed wi hou ATP o ma ion, bu no pho ophospho yla ion can occu unless i is coupled wi h elec on anspo . Expe imen ally, i is possible o con e noncyclic pho ophospho yla ion in chlo oplas s (equa ion (2) and Fig. 1) o a "bac e ial" ype (equa ion (5) and Fig. VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1321 2) by eplacing OH- wi h an exogenous elec on dono a he le el o A-. Reduc- ion o py idine nucleo ide is hen coupled wi h he oxida ion o he exogenous elec- on dono and a simul aneous ATP o ma ion bu wi hou oxygen e olu ion.57 We ha e no de ini ely iden i ied he "pigmen " shown in Figu e 1 ha is in- ol ed in he pho ooxida ion o hyd oxyl ions. Recen expe imen s on he e - ec i eness o monoch oma ic ligh on he pho ochemical eac ions o chlo oplas s sugges ha his may be a special pho osyn he ic pigmen such as chlo ophyll b o ano he accesso y pigmen ha is ound only in oxygen-e ol ing o ganisms, i.e. highe plan s and algae, a he han chlo ophyll a (o he closely ela ed bac e io- chlo ophyll) ound in all pho osyn he ic cells. (See e iew o ela ed li e a u e in e . 57.) In he ed egion o he spec um he pho ooxida ion o OH- (equa ion (4)) was mos e ec i e a ound 644 miu, which co esponds o he maximum abso p- ion peak67 in he ed o chlo ophyll b (Figs. 3 and 4). The e ec i eness o mono- ch oma ic ligh in he ed egion on pho o educ ion o TPN (equa ion (5)) and cyclic pho ophospho yla ion (equa ion (1)) was dis inc ly di e en om ha on oxygen e olu ion (equa ion (4) and Figs. 3 and 4), hus suppo ing he iew ha hese a e di e en ligh eac ions. The expe imen s wi h monoch oma ic ligh a o he conclusion ha he pho ochemical eac ion which p oduces oxygen is di e en om he o he pho ochemical eac ions which p oduce ATP and educed py idine nucleo ide. The ela ion o oxygen p oduced o ATP o ma ion in noncyclic pho o- phospho yla ion will now be examined in mo e de ail. Oxygen as an Exc e ed By-P oduc o Noncyclic Pho ophospho yla ion.-The es- sence o noncyclic pho ophospho yla ion, bo h in plan s and in pho osyn he ic bac e ia, appea s o be he o ma ion o ATP which is coupled wi h an elec on anspo om an ex e nal elec on dono o py idine nucleo ide ac ing as he elec on accep o . As al eady men ioned, in plan s, as in pho osyn he ic bac e ia, oxygen is no consumed in hese essen ial aspec s o noncyclic pho ophospho yla ion. Oxygen may be libe a ed in his p ocess bu only in he speci ic ins ance, ha is cha ac e is ic o plan bu no o bac e ial pho osyn hesis, when OH- is he ex- e nal elec on dono . This concep is suppo ed by he expe imen al indings ha ATP o ma ion g ea ly inc eases he a e o oxygen e olu ion and he concomi an educ ion o he elec on accep o , whe he i is e icyanide o TPN22 23, 39, 68, 69 (equa ions (2) and (3)). These esul s un coun e o Hill's o iginal hypo hesis ha in chlo oplas s " he educed p oduc o med in he ligh migh be eoxidized by molecula oxygen o gi e a phospho yla ing sys em simila o ha in chemosyn he ic bac e ia." 45 As Hill and Bonne ha e ecen ly poin ed ou , "In such a hypo hesis one would expec ha addi ion o ino ganic phospha e and ADP o he sys em would esul in dec eased a es o (H) accep o educ ion and o oxygen p oduc ion" 60 (i alics ou s). Hill has accep ed he new expe imen al indings and concluded ha " he abo e ... hypo hesis has o be abandoned." 60 Wa bu g,'2 howe e , has e ained he hy- po hesis o oxygen consump ion in ene gy ans o ma ions in pho osyn hesis and ques ioned he new expe imen al ac s. Speci ically, Wa bu g e al.12 ound ha wi h naph hoquinone sul ona e as he hyd ogen accep o , he s oichiome y o oxygen e olu ion by chlo oplas s is no a ec ed by phospho yla ion. Wa bu g e al.12 sugges ed ha he inc ease in pho op oduc ion o oxygen as a co sequence o 1322 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. pho ophospho yla ion- i s obse ed by o he in es iga o s22' 68 wi h e icyanide (equa ion (3))-was he esul o an uncon olled change in acidi y. Since he inc eased p oduc ion o oxygen ha accompanied pho ophospho yla ion in he e icyanide sys em was ound in solu ions ha we e s ongly bu e ed wi h is (hyd oxyme hyl) aminome hane bu e ,23 i seemed unlikely ha he ob- se ed e ec was he esul o uncon olled pH changes as sugges ed by Wa bu g e al.'2 Mo eo e , Da enpo 69 has ecen ly ound ha he a e o pho o educ ion o TPN, he physiological hyd ogen accep o in noncyclic pho ophospho yla ion (equa ion (2)), was also g ea ly inc eased a wo di e en pH's when a phospha e- accep ing sys em was p esen . We ha e now ein es iga ed he e ec o pho ophospho yla ion on oxygen e olu ion by isola ed chlo oplas s a di e en hyd ogen ion concen a ions, using ei he TPN o e icyanide as elec on accep o s. The pH alue o each eac ion mix u e was checked a he end o he expe imen . The esul s a e shown in Figu es 5 and 6. Wi h ei he TPN o e icyanide as he elec on accep o , we obse ed a signi i- TPN # (ADP+ P) qj TPN I 7.6 7.8 80 8.2 84 8.6 pH FIG. 5.-E ec o pho ophospho yla ion a a ious pH alues on oxygen e olu ion accom- panying TPN educ ion. The eac ion was ca ied ou in Wa bu g manome e essels a 150C. Illumina ion, 25,000 Lux o 6 min.; gas phase, ni ogen. In all essels he eac- ion mix u e con ained, in a inal olume o 3 ml, 200 Mimoles is-HCl bu e , 4 ,moles TPN, pu i ied TPN- educ ase om spinach, and b oken chlo oplas s (Pi,) con aining 0.2 mg chlo ophyll. To he phospho yla ing se ies [TPN plus (ADP plus P)] we e added 10 Mmoles MgSO4, 10 Mmoles po assium phospha e and 10 /Amoles ADP. T is bu e , po as- sium phospha e, and ADP we e each adjus ed o he selec ed pH; he TPN was adjus ed o pH 7.5. The pH shi , as de e mined a he end o he eac ion, was ound no o ex- ceed 0.1 uni . VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1329 o a ela i ely high concen a ion o chlo oplas ma e ial sugges s ha he anae obic FMN and i amin K sys ems equi e mo e chlo oplas ac o (s) han he pseudo- cyclic oxygen-dependen sys em. No simila equi emen s we e obse ed o he anae obic phenazine me hosul a e sys em. Despi e he simila i y o he o e -all eac ions (equa ions (1) and (8)), he anae o- bic, cyclic pho ophospho yla ions ca alyzed by i amin K and FMN can be dis in- guished expe imen ally om hei oxygen-dependen , pseudocyclic analogues. The la e depend on he pho op oduc ion o oxygen (equa ions (6), (7), and 8)) which e- qui es chlo ide", 80 and a e sensi i e o wo well-known inhibi o s o oxygen e olu ion in pho osyn hesis, CMU8' (p-chlo ophenyldime hyl u ea) and o-phenan h oline.80 Table 1 shows ha , unde expe imen al condi ions a o able o each sys em, TABLE 1 EFFECT OF CHLORIDE ON PHOTOPHOSPHORYLATION IN NITROGEN OR AIR pmoles ATP Fo med- T ea men Minus chlo ide Plus chlo ide Ni ogen, FMN 5.1 5.7 Ni ogen, i amin K, 9.7 9.9 Ai , FMN 0.5 6.1 Ai , i amin K3 0.4 5.5 In he ni ogen se ies he illumina ion was 2,000 Lux o 30 min, and he eac ion mix u e included, in a inal olume o 3 ml, chlo oplas agmen s (Ci.) con aining 2.5 mg chlo ophyll and 0.3 moles o FMN o i amin Ks. In he ai se ies, he illumina ion was 50,000 Lux o 30 min and he eac ion mix u e included, in a inal olume o 3 ml, chlo oplas agmen s (Ci,) con aining 0.2 mg chlo ophyll and 0.003 pumoles o FMN o i amin Ka. O he componen s o he eac ion mix u e we e, in pmoles: is bu e , pH 8.3, 80; K H32PO4, 15; ADP, 15; and MgSO4, 5. Chlo oplas s we e p epa ed in 0.5 M suc ose and chlo ide- ee eagen s we e used. In he plus chlo ide se ies 10 jsmoles NaCl we e added. Ni ogen gas was pu i- ied by passing h ough a zinc-me hylene blue solu ion (A. T. Jagendo , pe sonal com- munica ion). he omission o chlo ide had sca cely an e ec on he anae obic cyclic pho ophos- pho yla ion wi h FMN o i amin K, bu se e ely inhibi ed hei oxygen-de- penden , pseudocyclic coun e pa s. Likewise, CMU (also o-phenan h oline53) inhibi ed he oxygen-dependen , pseudocyclic pho ophospho yla ion bu had ela i ely li le e ec on hei anae obic, cyclic coun e pa s (Table 2). TABLE 2 EFFECT OF CMU ON PHOTOPHOSPHORYLATION IN NITROGEN AND AIR WITH HIGH CHLOROPHYLL AT Low LIGHT INTENSITY pmoles ATP Fo med T eamn - Ni ogen Ai - T ea men Con ol CMU Con ol CMU FMN 7.0 6.6 4.2 1.7 Vi amin K3 8.1 7.6 3.3 1.3 PMS 2.7 2.4 2.9 2.8 The eac ion mix u e included, in a inal olume o 3.0 ml, chlo oplas agmen s (Ci.) con aining 2.5 mg chlo ophyll and, in moles: is bu e , pH 8.3, 80; MgSO4, 10; K2H32PO4, 15; ADP, 15; and, whe e indica ed, FMN, 0.3; i amin Ks, 0.3, o , phenazine me hosul a e (PMS), 0.1. The inal concen a ion o CQMU was 2 X 10-6 M. The eac ion was un o 30 min a an illumina ion o 2,000 Lux. The ni ogen gas was pu i ied as desc ibed in Table 1. When some expe imen al condi ions a e un a o able o anae obic, cyclic pho o- phospho yla ion, he high a ini y o educed FMN o i amin K, o molecula oxygen may s ill gi e ise o a pseudocyclic, oxygen-dependen pho ophospho yla- ion. Thus, we ha e obse ed in he p esence o 10-4 M FMN o i amin K3 a a high ligh in ensi y, and wi h a ela i ely low concen a ion o chlo oplas ma e ial (0.25 mg chlo ophyll/3 ml) ha pho ophospho yla ion became oxygen- dependen , e en when he eac ion was s a ed in pu i ied ni ogen gas ha was 1330 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. TABLE 3 EFFECT OF CMU ON PHOTOPHOSPHORYLATION IN NITROGEN AND AIR WITH Low CHLOROPHYLL AT HIGH LIGHT INTENSITY - yjmoles ATP Fo med Ni ogen Ai T ea men Con ol CMU Con ol C 1[U FMN 5.6 1.5 5.4 1.1 Vi amin K3 8.6 4.7 5.3 1.0 PMS 9.3 9.9 8.9 8.6 Expe imen al condi ions as in Table 2 excep ha less chlo oplas ma e ial (Ci.) was used (0.25 mg chlo ophyll) and he illumina ion was inc eased o 20,000 Lux. Reac ion ime, 15 min. ea ed o emo e oxygen impu i ies (Table 3). Phospho yla ion became oxygen- dependen when he ca aly ic amoun s o FMN o i amin K? we e pho ochemically educed by chlo oplas s. An equi alen amoun o oxygen was hen libe a ed which was su icien o sus ain an oxygen-dependen pho ophospho yla ion (c . T ebs and Eck75). The oxygen dependence o he sys em was de e mined by blocking he oxygen e olu ion eac ion wi h CMU. ATP o ma ion hen became s ongly inhibi ed. (I seems likely ha a simila dependence on pho ochemically gene a ed oxygen accoun s o he esul s epo ed by Vennesland e al. in e . 56, Table 2.) Pho ophospho yla ions ca alyzed by phenazine me hosul a e we e esis an o inhibi ion by CMU and o-phenan h oline,53 ega dless whe he he eac ion was ca ied ou unde condi ions a o ing he anae obic cyclic, o he oxygen-dependen pseudocyclic pa hway (Tables 2 and 3). Concluding Rema ks.-F om he s andpoin o cellula physiology, he ole o oxygen in ATP o ma ion in pho osyn hesis p o ides an in e es ing con as o he ole o oxygen in ATP o ma ion in espi a ion. The high he modynamic e iciency o espi a ion, and i s supe io i y o e e men a ion in using he ee ene gy o he deg ada ion o oods u s o syn hesis o he py ophospha e bonds o ATP, is made possible by he key ole ha molecula oxygen plays as he e - minal elec on accep o in oxida i e phospho yla ion by mi ochond ia. Only wi h oxygen can he oxida ion o oods u s be comple e ( o CO a ld wa e ) and hei ee ene gy eleased in ull o me abolic pu poses. In he las se en yea s biochelnical in es iga ions o pho osyn hesis in cell- ee sys ems e ealed no such ole o oxygen in ATP o ma ion by chlo oplas s and bac e ial pa icles. He e, ATP o ma ion occu s in cyclic pho ophospho yla ion unde s ic ly anae obic condi ions whe e he pa icipa ion o oxygen is excluded. I i noncyclic pho o- phospho yla ion, oxygen may eplace TPN as a e minal elec on accep o , bu , as discussed la e , his esul s in a loss ins ead o a gain in he yield o physiologically use ul ene gy. The s ic ly anae obic cha ac e o cyclic pho ophospho yla ion in bac e ial p epa a ions needs no elabo a ion since pho osyn hesis in. bac e ia is i mly es- ablished as an anae obic p ocess.7 In isola ed chlo oplas s, anae obic cyclic pho ophospho yla ion has been demons a ed wi h phenazine me hosul a e as a ca alys , and also wi h i amin K3 and FMN. Wi h he la e , a a mo e igid con ol o expe imen al condi ions was equi ed han wi h phenazine me hosul a e. Special expe imen al sa egua ds a e needed o demons a ing in chlo oplas s an anae obic cyclic pho ophospho yla ion ca alyzed by i amin K3 and FMN, because hese subs ances, when educed' pho ochemically, eadily eac wi h small concen a ions o molecula oxygen. Phenazine me hosul a e also eac s eadily VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1331 wi h oxygen, bu i s a ini y o oxidized cy och omes82 seems o be g ea e han o oxygen; i is hus able o ca alyze a cyclic elec on low (Fig. 8) e en in he p esence o molecula oxygen. Noncyclic pho ophospho yla ion consis s o ATP o ma ion ha is coupled wi h he educ ion o py idine nucleo ide and he oxida ion o an exogenous elec- on dono . The p ocess, which was o iginally ound in isola ed chlo oplas s,22 has now also been demons a ed in bac e ial pa icles.59 The di e ence be ween noncyclic pho ophospho yla ion in he wo cases cen e s on he elec on dono . Fo isola ed chlo oplas s he elec on dono s a e hyd oxyl ions which on oxida ion o m oxygen; his accoun s o oxygen e olu ion in plan pho osyn hesis. Pho o- syn he ic bac e ia canno use hyd oxyl ions bu only such elec on dono s as o ganic acids o educed sul u compounds6' 7; his accoun s o he absence o oxygen e olu ion in bac e ial pho osyn hesis. Ins ead o oxygen, oxidized bac- e ial elec on dono s a e o med, which may be u he me abolized o may ac- cumula e and yield, o example, elemen al sul u .6 The ole o oxygen in pho osyn hesis is, he e o e, only inciden al and no i al o he ene gy ans o ma ion eac ions, as i is in espi a ion. Oxygen is e ol ed because i is an exc e ed p oduc o he oxida ion o hyd oxyl ions- he elec on dono in noncyclic pho ophospho yla ion by chlo oplas s. Expe imen ally, when ano he elec on dono is subs i u ed o hyd oxyl ions, noncyclic pho o- phospho yla ion in chlo oplas s becomes con e ed o he bac e ial ype: ATP o ma ion is coupled wi h py idine nucleo ide educ ion bu no oxygen is e ol ed.57 In a cell- ee sys em, oxygen e olu ion by chlo oplas s will be obse ed mano- me ically only unde ce ain condi ions. Fi s , he accep o o he elec ons "expelled" om chlo ophyll in he p ima y pho ochemical eac ion mus no eac p e e en ially wi h he componen s o he pho osyn he ic elec on anspo chain (c . T ebs and Eck83). I i does eac , as in he case o phenazine me hosul a e, hen cyclic pho ophospho yla ion, in which oxygen is no e ol ed, will esul . Second, he elec on accep o mus no be easily oxidized by molecula oxygen. This is o en, bu no always, ela ed o edox po en ial. Thus, TPN wi h a edox po en ial, a pH 7, o E'o = -324 mV sha es his p ope y wi h e icyanide (E'o = 360 mV) and p-benzoquinone (E'o = 293 mV). In he in ac plan , oxygen is no mally e ol ed du ing pho osyn hesis because he TPNH2 o med in non- cyclic pho ophospho yla ion is no eoxidized by he oxygen o med, bu is used up in ca bon assimila ion. A special case o noncyclic pho ophospho yla ion by isola ed chlo oplas s occu s when oxygen ac s as he e minal elec on accep o and becomes educed o hyd o- gen pe oxide. The pho o educ ion o oxygen by isola ed chlo oplas s, disco e ed by Mehle ,72 has been in es iga ed by B own and Good84 and Good and Hill,76 who sugges ed ha i is "an a i ac " 84 and ha " he di ec educ ion o oxygen does no o dina ily occu o a signi ican ex en in i o." 76 In a cell- ee sys em con aining mic oca aly ic amoun s o FMN o i amin K3, oxygen may eplace TPN as he elec on accep o in noncyclic pho ophospho yla- ion by chlo oplas s and gi e ise o an oxygen-dependen , pseudocyclic pho o- phospho yla ion. The subs i u ion o oxygen o TPN is analogous o he sub- s i u ion o e icyanide o TPN. ATP is s ill o med in each case, bu he e- duc an p oduced is no longe TPNH2 bu ei he e ocyanide, o , when oxygen 1332 BIOCHEMISTRY: ARNON ET AL. PROC. N. A. S. is he elec on accep o , H202 o wa e . Thus, he in e en ion o oxygen in noncyclic pho ophospho yla ion esul s in a loss o biochemically use ul ene gy which would ha e no mally been used o o ming a s ong educ an , i.e. TPNH2. When oxygen is he elec on accep o and wa e is he inal p oduc o oxygen educ ion (equa ions (6) and (7)) he ene gy loss is equi alen o a edox po en ial o 1139 mV (di e ence be ween. Eo' -815 mV o he wa e /oxygen sys em an(d Eo' = -324 mV o he TPN/TPNH2 sys em). I hyd ogen pe oxide is allowed o accumula e as he inal p oduc o he oxygen educ ion, he compu ed ene gy loss would be equi alen o a edox po en ial o 1006 mV. * The ollowing abb e ia ions a e used: ATP, adenosine iphospha e; ADP, adenosine di- phospha e; P, o hophospha e; TPN, TPNH2, oxidized and educed o ms o iiphosphopy idine nucleo ide; FMN, ibo la in phospha e ( la in mononucleo ide); PMS, phenazine me hosul a e. Aided by g an s om he Na ional Ins i u es o Heal h and he O ice o Na al Resea ch. 1 A non, D. I., Bull. To ey Bo an. Club, 88 (1961). 2 Ingenhousz, J., Essay on he ood o plan s and he eno a ion o soils, London (1796). 3Lebede , A., Biochem. Z., 7, 1 (1907). 4Lebede , A., Be . deu . bo an. 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I., Na u e, 184, 10 (1959). 19 A non, D. I., M. Losada, M. Nozaki, and K. Tagawa, ibid., 190, 601 (1961). 20 S anie , R. Y., Bac e iol. Re ., 25, 1 (1961). 21 K ogmann, D. W., and B. Vennesland, J. Biol. Chem., 234, 2205 (1959). 22 A non, D. I., F. R. Wha ley, and M. B. Allen, Science, 127, 1026 (1958). 23 A non, D. I., F. R. Wha ley, and M. B. Allen, Biochim. e Biophys. Ac a, 32, 47 (1959). 24 T ebs , A. V., H. Y. Tsujimo o, and D. I. A non, Na u e, 182, 351 (1958). 25 Losada, M., A. V. T ebs , and D. I. A non, J. Biol. Chem., 235, 832 (1960). 28 T ebs , A. V., M. Losada, and D. I. A non, ibid., 234, 3055 (1959). 27 A non, D. I., in Enzymes: Uni s o Biological S uc u e and Func ion, ed. 0. H. Gaeble (New Yo k: Academic P ess, Inc., 1956), p. 279. 28 A non, D. I., M. B. Allen, and F. R. Wha ley, Biochim. e Biophys. Ac a, 20, 449 (1956). 29 Wha ley, F. R., M. B. Allen, L. L. Rosenbe g, J. B. Capindale, and D. I. A non, ibid., 20, 462 (1956). 30 Cal in, M., P oc. In e n. Cong . Biochem., 3 d Cong ., B ussels, 1955 (New Yo k: Academic P ess, Inc., 1956), p. 211. 31 Vishniac, W., B. L. Ho ecke , and S. Ochoa, Ad ances in Enzymol., 19, 1 (1957). 32 T udinge , P. A., Biochem. J., 64, 274 (1956). 33 Aube , J. P., G. Milhaud, and J. Mille , Ann. ins . Pas eu , 92, 515 (1957). 34 McFadden, B. A., and D. E. A kinson, A ch. Biochem. Biophys., 66, 16 (1957); McFadden, B. A., J. Bac e iol., 77, 339 (1959). VOL. 47, 1961 BIOCHEMISTRY: ARNON ET AL. 1333 '5 S ehle , B. L., A ch. Biochem. Biophys., 43, 67 (1953). 36 Ga on, H., in Au o ophic Mic oo ganisms, ed. B. A. F y and J. L. Peel (Camb idge, Eng- land: Camb idge Uni e si y P ess, 1954), p. 163. 37 A on, M., and A. T. Jagendo , Na u e, 179, 428 (1957). 38 A on, M., A. T. Jagendo , and M. E ans, Biochim. e Biophys. Ac a, 26, 262 (1957). 39 Jagendo , A. T., in The Pho ochemical Appa a us, B ookha en Symposia in Biology, No. 11 (1958). 40 Jagendo , A. T., and M. A on, J. Biol. Chem., 231, 277 (1958). 41 Wessels, J. S. C., Biochim. e Biophys. Ac a, 25, 97 (1957). 42 Ibid., 29, 113 (1958). 43 Chow, C. T., and B. Vennesland, Plan Physiol., 32 (Supp.), i (1957). 44 Nakamo o, T., D. W. K ogmann, and B. Vennesland, J. Biol. Chem., 234, 2783 (1959). 46 Hill, R., and D. A. Walke , Plan Physiol., 34, 240 (1959). 46 Wha ley, F. R., M. B. Allen, A. V. T ebs , and D. I. A non, ibid., 35, 188 (1960). 47 O he accoun s o he disco e y o pho osyn he ic phospho yla ion and CO2 assimila ion by isola ed chlo oplas s a e some imes ound in he li e a u e. Thus in 1956, J. A. Bassham and M. Cal in, in Cu en s in Biochemical Resea ch, ed. D. B. G een (New Yo k: In e science, 1956), asc ibed he disco e y o CO2 assimila ion by isola ed chlo oplas s o Boychenko and Ba ano .48 In 1959, Cal in ( e . 49, p. 152) asc ibed he disco e y o bo h CO2 assimila ion and ATP syn- hesis by isola ed chlo oplas s o his own labo a o y. 48 Boychenko, E. A., and V. I. Ba ano , Doklady Akad. Nauk S.S.S.R., 95, 1025 (1954); Chem. Abs ., 48, 8881 (1954). 49 Cal in, M., Re . Mode n Phys., 31, 147 (1959); also in Biophysical Science-A S udy P o- g am, ed. J. C. Oncley (New Yo k: John Wiley and Sons, 1959), p. 152. 50 Williams, A. M., Biochim. e Biophys. Ac a, 19, 570 (1956). 51 Thomas, J. B., and A. M. Haans, ibid., 18, 286 (1955). 52 Pe ack, B., and F. Lipmann, in Ligh and Li e, ed. W. D. McEl oy and B. Glass (Bal imo e: Johns Hopkins P ess, 1961), p. 621. 6 A non, D. I., ibid., p. 489. 64"F enkel, A. W., J. Biol. Chem., 222, 823 (1956). 56 Gelle , D. M., "Pho ophospho yla ion by Rhodospi illum ub um p epa a ions," Doc o al Disse a ion, Di . Med. Sci., Ha a d Uni e si y (1957). 66 Vennesland, B., T. Nakamo o, and B. S e n, in Ligh and Li e, ed. W. D. McEl oy and B. Glass (Bal imo e: Johns Hopkins Uni e si y P ess, 1961), p. 609. 67 Losada, M., F. R. Wha ley, and D. I. A non, Na u e, 190, 606 (1961.) 58 Duysens, L. N. M., J. Amesz, and B. M. Kamp, ibid., 190, 510 (1961). 69 Nozaki, M., K. Tagawa, and D. I. A non, hese PROCEEDINGS, 47, 1334 (1961). 60 Hill, R., and W. D. Bonne , in Ligh and Li e, ed. W. D. McEl oy and B. Glass (Bal imo e: Johns Hopkins Uni e si y P ess, 1961), p. 424. 61 C ane, F. L., Plan Physiol., 34, 128 (1959); Bishop, N. I., hese PROCEEDINGS, 45, 1696 (1959). 62 K ogmann, D.. W., Biochem. Biophys., Resea ch Commun., 4, 275 (1961). 63T ebs , A. V., M. Losada, and D. I. A non, J. Biol. Chem., 235, 840 (1960). 64 K ogmann, D. W., A. T. Jagendo , and M. A on, Plan Physiol., 34, 272 (1959). 65 Chance, B., and J. M. Olson, A ch. Biochem. Biophys., 88, 54 (1960). " Ma b, E., and E. Fo i, Science, 126, 976 (1957). 67 F ench, C. S., in Handbuch de P anzenphysiolqgie, ed. W. Ruhland (Heidelbe g: Sp inge , 1960), ol. 5, pa 1, p. 252. 68 A on, M., D. W. K ogmann, and A. T. Jagendo , Biochim. e Biophys. Ac a, 30, 144 (1958). 69 Da enpo , H. E., Biochem. J., 77, 471 (1960). 70 A non, D. I., pape p esen ed a he Cell Symposium, Ame . Assn. o he Ad ancemen o Sci., Be keley Mee ing (1954); Science, 122, 9 (1955). 71 A non, D. I., M. B. Allen, and F. R. Wha ley, Biochim. e Biophys. Ac a, 20, 449 (1956). 72 Mehle , A. H., A ch. Biochem. Biophys., 33, 65 (1951). 73 Ibid., 34, 339 (1951). 74 Mehle , A. H., and A. H. B own, ibid., 38, 365 (1952). 75 T ebs , A., and H. Eck, Z. Na u o sch. (in p ess). 1334 BIOCHEMISTRY: NOZAKI ET AL. PROC. N. A. S. 76 Good, N., and R. Hill, A ch. Biochem. Biophys., 57, 355 (1955). 77 K ogmann, D. W., J. Biol. Chem., 235, 3630 (1960). 78 A non, D. I., in Handbuch de P lanzenphysiologie, ed. W. Ruhland (Heidelbe g: Sp inge , 1960), ol. 5, pa 1, p. 773. 79 Bal sche sky, H., S ensk Kemisk Tidsk i , 72, 4 (1960). 'I Wa bu g, O., in Hea y Me al P os he ic G oups and Enzyme Ac ion (Ox o d, England: Cla en- don P ess, 1949), p. 213. 81 Wessels, J. S. C., Biochim. e Biophys. Ac a, 19, 548 (1956). 82 Massey, V., ibid., 34, 255 (1959). 83T ebs , A., and H. Eck, Z. Na u o sch., 16b, 44 (1961). 84 B own, A. H., and N. Good, A ch. Biochem. Biophys., 57, 340 (1955). NONCYCLIC PHOTOPHOSPHORYLATION IN PHOTOSYNTHETIC BACTERIA BY M. NOZAKI, K. TAGAWA, AND DANIEL I. ARNON* LABORATORY OF CELL PHYSIOLOGY, UNIVERSITY OF CALIFORNIA, BERKELEY Communica ed Augus 2, 1961 Noncyclic pho ophospho yla ion is he o ma ion o adenosine iphospha e in a ligh -induced elec on low om an ex e nal elec on dono o an elec on accep o such as py idine nucleo ide. In pho osyn hesis o g een plan s he elec on dono s a e he hyd oxyl ions o wa e and hence he o e -all eac ion also p oduces, aside om ATP and PNH2,l molecula oxygen, which is libe a ed when OH- is pho o- oxidized (see Fig. 1 in p eceding a icle2). Pho osyn he ic bac e ia canno use OH- as an elec on dono o pho osyn hesis bu use a a ie y o educ an s s onge han OH-, among hem hyd ogen gas, succina e, o hiosul a e. Wi h hyd ogen gas no inpu o ligh ene gy is equi ed o he educ ion o py idine nucleo ide, since hyd ogen gas, wi h he aid o bac- e ial hyd ogenase, can educe py idine nucleo ide in he da k.3 The con ibu ion o ligh o bac e ial pho osyn hesis is hen limi ed o he o ma ion o ATP by cyclic pho ophospho yla ion.4 I Wi h o he elec on dono s such as, o example, succina e, he addi ional inpu o ene gy ha is necessa y o educe py idine nucleo- ide is p o ided by ligh . Thus, in bac e ial pho osyn hesis wi h succina e as he elec on dono , ligh ene gy se es a dual pu pose: i supplies ATP by cyclic pho ophospho yla ion and, by pho ooxidizing he elec on dono , i also p o ides elec ons o educing py idine nucleo ides by a noncyclic elec on low mechanism. Pho o educ ion o py idine nucleo ide (DPN) by cell- ee p epa a ions o Rhodo- spi illum ub um6 I and Ch oma ium3 has been obse ed bu a emp s o ind a simul aneous ATP o ma ion, analogous o he noncyclic pho ophospho yla ion in chlo oplas s, led o nega i e esul s.7' 8 As poin ed ou by Ve non and Ash,8 a demons a ion o ATP o ma ion by a noncyclic elec on low mechanism is mo e di icul in ch oma opho es han in chlo oplas s because in ch oma opho es i canno be expe imen ally dis inguished om a simul aneous ATP o ma ion by cyclic pho ophospho yla ion. In ch oma opho es, a igo ous cyclic pho ophos- pho yla ion occu s unde anae obic condi ions and wi hou added co ac o s,3 whe eas in chlo oplas s cyclic pho ophospho yla ion is always unde expe imen al