photophosphorylation associated with photosystem ii

Plant Physiol. (1977) 59, 33-37

Photophosphorylation Associated with Photosystem II

I. PHOTOSYSTEM II CYCLIC PHOTOPHOSPHORYLATION CATALYZED BY p-PHENYLENEDIAMINE

Received for publication June 17, 1976 and in revised form August 8, 1976

CHARLES F. YOCUM AND JAMES A. GUIKEMADepartment ofCell and Molecular Biology, Division ofBiological Sciences, The University ofMichigan, AnnArbor, Michigan 48109

ABSTRACT

Incubation of spinach chloroplast membranes for 90 mnutes in thepresence of 50 mM KCN and 100 FtM HgCI2 produces an inhibition ofphotosystem I activity which is stable to wshing and to storage of thechloroplasts at -70 C. Subsequent exposure of these preparations toNH20H and ethylenediaminetetraacetic acid destroys 02 evolution andflow of electrons from water to oxidized p-phenylenediamine, but twotypes of phosphorylating cyclc electron flow can still be observed. In thepresence of 3-(3,4-dichlorophenyl)-1,1'-dimethylurea, phenazineme-thosuffate catalyzes ATP synthesis at a rate 60% that observed inuninhibited chloroplasts. C-Substituted p-phenylenediamines will alsosupport low rates of photosystem I-catalyzed cyclic photophosphoryla-tion, butp-phenylenediamine is completely inactive. When photosystemH is not inhibited, p-phenylenediamine will catalyze ATP synthesis atrates up to 90 Fmol/hr mg chlorophyll. This reaction is unaffected byannerobiosis, and an action spectrum for ATP synthesis shows a peak at640 m. These results are interpreted as evidence for the existence ofphotosystem H-dependent cyclic photophosphorylation in these chloro-plast preparations.

A large body of experimental evidence supports the hypothe-sis that the noncyclic pathway of electron transport in chloro-plasts contains two sites of photophosphorylation. Noncyclicelectron transport from water to photosystem I acceptors such asferricyanide is inhibited by KCN, which inactivates plastocyanin(15), or by the quinone antagonist DBMIB1 (10, 17). Electrontransport in inhibited chloroplasts is restored by addition of p-phenylenediimines or p-benzoquinones, and the accompanyingphosphorylation has a P/e2 approaching 0.5 (15, 17). This par-tial reaction, involving photosystem II, is termed site II. Whenphotosystem II activity is blocked by addition of DCMU, pro-ton/electron donors such as DAD will catalyze photosystem I(site I)-dependent electron transport to an acceptor such as MV.The P/e2 of this reaction is about 0.5 when assays are carried outin the presence of superoxide dismutase to correct for apparent02 uptake due to the formation of superoxide ion (4, 14).Summation of the efficiencies (P/e2) at sites I and II obtained byassay of site-specific partial reactions produces a value near 1.0,which is the value observed for noncyclic electron transport from

1 Abbreviations: MV: methylviologen; P/e2: ratio of ATP synthesis toelectron pairs transferred; E.T.: rate of electron transport; DBMIB: 2,4-dibromo-3-methyl-6-isopropyl-p-benzoquinone; PD: p-phenylenedi-amine; PD..: p-phenylenediimine; DAD: 2,3,5,6-tetramethyl-p-phenylenediamine; DAT: 2,5-diaminotoluene; STN: 0.4 M sucrose-20mM Tricine (pH 8)-15 mm NaCI; PMS: phenazinemethosulfate; Asc:ascorbate.

water to ferricyanide, MV, or ferredoxin/NADP in uninhibitedchloroplast preparations (16).

In addition to noncyclic pathways of energy-conserving elec-tron flow, cyclic electron transport involving photosystem I willsupport ATP synthesis. In the presence of DCMU, p-phenylene-diamines, PMS, or ferredoxin catalyze cyclic photophosphoryla-tion (6). Recent experiments in several laboratories have pro-vided evidence that ATP synthesis may also occur by means ofphotosystem II cyclic reactions. Izawa (8) reported that additionof catechol to chloroplasts inhibited by DBMIB and EDTA plusNH20H produced a low rate of ATP synthesis which was as-cribed to a photosystem II cyclic reaction. Ben-Hayyim et al. (2)investigated the properties of diaminobenzidine as a photosys-tem II donor in tris-washed chloroplasts, and found that itsupported ATP synthesis which was partially insensitive to KCNinhibition of photosystem I activity. Although the DCMU sensi-tivity of this reaction was not reported, the data of Ben-Hayyimet al. (2) suggest the occurrence of a photosystem II cyclicreaction. Yocum (19) has shown that PD restored ATP synthesisin chloroplasts inhibited with DBMIB and NH20H plus EDTA.At low concentrations of PD (<65 gM), the reaction was com-pletely sensitive to DCMU, while at higher catalyst concentra-tions, DCMU sensitivity was partially lost. These results wereinterpreted to indicate the existence of an artificial cyclic path-way of ATP synthesis involving only photosystem II at low PDconcentrations.

Previous experiments on photosystem II cyclic reactions haverelied on DBMIB, which may participate in these reactions, toblock activity between the photosystems. In addition, theDCMU sensitivity of reactions in tris-washed, KCN-inhibitedchloroplasts has not been reported. We have, therefore, contin-ued our investigations into cyclic phosphorylating electron trans-port reactions involving photosystem II with a view towardestablishing the mechanism of this reaction, and assessing itsutility in examining the over-all mechanism of photosyntheticenergy transduction. In this communication, we report the meth-ods for preparation of chloroplasts strongly and irreversiblyinhibited at or near plastocyanin, which have been further in-hibited by deletion of 02 evolution. In spite of these drasticconditions of inhibition, such chloroplast preparations retaincyclic photophosphorylation activity with PMS and p-phenylene-diamines, and under the appropriate conditions catalyze ATPsynthesis which appears to be due entirely to a photosystem IIcyclic reaction.

MATERIALS AND METHODS

Broken chloroplasts were isolated from market spinach bygrinding depetiolated leaves in STN according to the procedureof Guikema and Yocum (5). Chlorophyll concentrations weredetermined by Amon's (1) procedure. Electron transport andphotophosphorylation assays were carried out in a 1.6-ml reac-

33

YOCUM AND GUIKEMA

tion mixture containing 50 mM Tricine (pH 8), 50 mm NaCl, 3mM MgCl2, 1 mm ADP, 5 mm NaH232P04 (106 cpm/ml) and 20to 30 ,ug Chl. Noncyclic activities were assayed in a thermostat-ted cuvette fitted with an 02 electrode. White light (5 x 105 ergs

cm-2sec-') was provided by a microscope lamp. The illuminationtime was 1 min unless otherwise noted. When electron transportfrom water to ferricyanide was assayed, the concentration offerricyanide was 2.4 mM; PD (0.25 mM) was also present whensite II alone was assayed. When site I activity was assayed, Asc(3.0 mM), DAD (0.25 mM), MV (0.125 mM) and DCMU (9/.M) were added to the reaction mixture. Correction for super-

oxide ion formation during photoinduced electron transport insite I assays was done by computing the ratio of electron trans-port activities in the presence and absence of 200 ,ug/ml ofbovine erythrocyte superoxide dismutase. The average ratio(+dismutase/-dismutase) determined from several experimentswas 0.69, and agreed with results obtained in other laboratories(S. Izawa, personal communication). When site I activity was

assayed in the absence of superoxide dismutase, the rate ofelectron transport obtained in such experiments was multipliedby 0.69.

Cyclic ATP synthesis was assayed in test tubes placed in a

thermostatted (25 C) water bath illuminated with white light(106 ergs cm-2sec-5) from photoflood lamps. The illuminationtime, unless otherwise indicated, was 1 min. The reaction mix-ture was the same as that described above for noncyclic activityand the concentrations of electron carriers used in these assaysare noted in the tables and figures under "Results." All reactions(cyclic and noncyclic) were terminated by addition of 0.2 ml of30% trichloroacetic acid. The amount of light-induced ATPsynthesis was determined by gas flow counting after extraction ofunreacted phosphate as described earlier (19).The effect of anaerobiosis on photosystem II cyclic photo-

phosphorylation was examined by purging cyclic reaction mix-tures with either air or O2-free helium for 1 or 5 min; at the endof these time periods, the reaction tubes were quickly stopperedand subjected to illumination. For the measurement of actionspectra, the intensity of light from an Oriel model 6325 lightsource was adjusted to 4,000 ergs cm-2sec-1 through each of a

series of Oriel and Balzers interference filters (575, 600, 640,700, and 720 nm, all with half-bandwidths of 10 nm). Lightintensity was varied with a rheostat, and measured by means of a

YSI model 65 radiometer.All chemicals used in these studies were of the purest grades

commercially available. Crystalline bovine superoxide dismutasewas a gift of J. Fee. Tricine, HEPES, ADP, PD, and PMS were

obtained from Sigma. NaH232P04 was purchased from New

England Nuclear. DAD was obtained from Research Organics/Inorganics, and DAT was purchased from Eastman OrganicChemicals. All of the p-phenylenediamines were recrystallizedaccording to the procedure of Gould (4), and stock solutions (20mM) in water were prepared immediately prior to assay.

RESULTS

Inhibition of Photosystem I Activity with KCN and Hg. The

mechanism by which KCN inhibits photosystem I is known to

involve inactivation of plastocyanin (15). We discovered that

centrifugation and resuspension of KCN-inhibited chloroplastsin STN containing 0.2% BSA caused a partial restoration of

photosystem I activity. An attempt was made to modify the KCNinhibition procedure to prevent the subsequent restoration of

activity by either washing of inhibited chloroplasts or cold stor-

age at -70 C for long periods of time. It has been reported that

HgCl2 is a potent inhibitor of photosystem I activity (12), and

that this inhibition is caused by inactivation of plastocyanin. Aseries of preliminary experiments were done to see whether

HgC12 could be combined with KCN to produce an irreversible

inhibition of photosystem I activity. Since Hg is known to dam-age photophosphorylation, it was necessary to determine theminimum concentration of HgCl2 which would, in combinationwith KCN, produce the desired inhibition. A concentration ofHgCl2 (1 mol/3 mol Chl) which, by itself was incapable ofproducing a strong inactivation of photosystem I (12), would,when included in an incubation mixture with the appropriateconcentration of KCN, lead to an irreversible inhibition of pho-tosystem I activity.

Based on these findings, a modified photosystem I inhibitionprotocol was devised which consisted of incubating chloroplasts(300 ,ug Chl/ml) for 90 min at 4 C in 0.1 M sucrose, 50 mMHEPES (pH 8), 15 mm NaCl, 50 uM ferricyanide, 0.2% BSA,and 50 mm KCN (pH 8) plus or minus 100 ,uM HgCl2. Incuba-tion was terminated by centrifugation of the suspensions at5,000g and resuspension of the pellets in STN plus 0.2% BSA toa final Chl concentration of 2 to 3 mg/ml. These suspensionswere assayed for activity and then frozen in 0.5-ml aliquots at-70 C for assay at a later time (48 hr). The conditions ofincubation reported here led to maximal photosystem I inhibi-tion in 60 to 90 min, and cold storage of the resuspendedmaterial for periods up to six weeks did not affect the extent ofinhibition.The results of a typical series of incubation experiments with

KCN alone and in the presence of HgCl2 are presented in TableI. Incubation with KCN in the absence and presence of HgCl2inhibits activities which require either ferricyanide (sites II and I)or Asc, DAD and MV (site I) for electron transport activity. Theextent of these inhibitions is on the order of 90%. Assays of siteII (H20 -- PDOX) show substantial retention of activity in agree-ment with the results of other investigators (15). Examination ofTable I shows that when the incubation mixture was centrifuged,resuspended, and reassayed, the preparation incubated in KCNalone regained some site I (Asc, DAD -- MV) electron trans-port activity. The activity of the preparation incubated in thepresence of HgCl2 did not show a comparable increase. Data arealso included in this table which indicate that both preparationsretained site I inhibition and site II activity after freezing andthawing. Although the data in Table I do not exclude the possi-bility that HgCl2 has damaged the energy-conserving apparatusin these chloroplasts, the rates of electron transport and associ-ated photophosphorylation observed in the presence of HgC12

TABLE I

Inhibition of Photosystem I by KCN Alone or in Combination with HgC12

IncubationConditions Activity

H20-Fe(CN)6j H20+PDox Asc/DAD-*MVE.T.1 ATP2 Pie2 E.T.1 ATP2 P/e2 E.T.1'3 ATP2 P/e2

-KCN,-Hg 600 292 0.97 1000 248 0.50 1446 332 0.46+KCN,-Hg 40 20 1.00 820 156 0.38 155 40 0.51+KCN,+Hg 50 18 0.72 921 164 0.36 106 35 0.66

Centri fugation,Resuspension, of:


48 hr ColdStorage of:


1,pequiv. transferred/hr-mg Chl21imol synthesized/hr-mg Chl3Corrected for superoxide ion formation

34 Plant Physiol. Vol. 59, 1977

PHOTOSYSTEM II PHOTOPHOSPHORYLATION

show little deviation from the results obtained with KCN byitself. Of more importance is the observation that inclusion ofHgCl2 during incubation has produced an inhibition of photosys-tem I electron transport activity which is not readily reversed bycentrifugation and resuspension.

Inhibition of Oxygen Evolution in KCN-Hg-treated Chloro-plasts. Previous investigations (19) showed that elimination of02 evolution increased the ATP yield of a photosystem II cyclicreaction catalyzed by PD. It was therefore necessary to inhibit02 evolution in KCN-Hg-treated chloroplasts. Chloroplasts werefirst inhibited with KCN and HgCl2 as described above, andcentrifuged and resuspended (1-2 mg Chl/ml) in a mediumconsisting of STN, 0.2% BSA, and 2 mM MgCl2. This suspen-sion was then exposed at 4 C to 5 mm NH2OH and 1 mm EDTAaccording to the procedure of Ort and Izawa (13). Maximalinhibition of 02 evolution was obtained within 20 min, afterwhich the suspension was centrifuged and resuspended in STNcontaining 0.2% BSA and frozen at -70 C in 0.5-ml aliquots.The results of a representative experiment (Table II) clearlyshow that this procedure did not appreciably reverse the photo-system I inhibition of electron transport caused by prior expo-sure to KCN and Hg. Data presented in Table II also show thatKCN-Hg-NH2OH-inhibited chloroplasts retain inhibitions ofelectron transport after cold storage at -70 C.ATP Synthesis by KCN-Hg-NH2OH-inhibited Chloroplasts.

Ouitrakul and Izawa (15) showed that PMS concentrations be-tween 100 and 200 ,tM could partially bypass the site of KCNinhibition in photosystem I, while concentrations of DAD ashigh as 0.6 mm could not. Similar experiments were carried outwith KCN-Hg-NH2OH-inhibited chloroplasts and uninhibitedpreparations in the presence of 9 aM DCMU. The results of atypical set of experiments are presented in Table III. Whileabout 60% of the photophosphorylation activity catalyzed byPMS is retained in inhibited chloroplasts, the catalytic activitiesof DAD and DAT, even at high concentrations, are more than95% inhibited. p-Phenylenediamine, which in untreated chloro-plasts produces a rate of photophosphorylation 10% that of thecorresponding PMS rate, is completely incapable of bypassingthe site of KCN-Hg inhibition. These results demonstrate thatthe KCN-Hg inhibition of photosystem I is similar to the inhibi-tion obtained with KCN alone, and the results with PMS showthat a considerable capacity for ATP synthesis is retained by ourinhibited preparations, in spite of the drastic conditions used toeliminate noncyclic electron transport activity.The effect of DCMU on PD-catalyzed photophosphorylation

in KCN-Hg-NH2OH-inhibited chloroplasts was next examined.The results of an experiment in which the concentration of PDwas varied in the absence and presence of 9 ,uM DCMU areshown in Figure 1. When the reducing side of photosystem II isinhibited by DCMU, ATP synthesis is inhibited at all PD con-centrations shown. In the absence of DCMU, however, ATP

TABLE II

Failure of NHzOH and EDTA to Reverse Photosystem IInhibition Caused by KCN and HgCl2

Electron transport assays were carried out as described in"Materials and Methods" in the absence of ADP and NaH232P04.

Treatment Activity

H20-PDox Asc, DAD-MVieq transferred/hr *mg_ Chl

None 1100 1550+KCN,+Hg 800 50+KCN,+Hg,+NH2OH 5 60Same, stored 48 hr at -70 C 5 55

35

TABLE III

Photosystem I-Dependent Cyclic ATP Synthesis byUntreated and KCN-Hg-NH2OH Inhibited Chloroplasts

Catalyst Rate of ATP Synthesis % of Untreated Rate

Untreated KCN-Hg-NH20HChloroplasts Inhibited Chloroplasts

imol synthesized/hr-mg ChlNone 2 0PMS(125 uM) 800 475 59DAD(1250 jIM) 435 18 4DAT(1250 ufq) 215 5 2 -

PD(1250 PM) 80 0 0

0

0

ob

E

Co

I-

0.

zn

I--

4

(PD), pMFIG. 1. Effect of PD concentration and DCMU on ATP synthesis by

KCN-Hg-NH20H-inhibited chloroplasts. When present, the concentra-tion of DCMU was 9 ,UM.

synthesis increases sharply when the PD concentration is raisedfrom 0 to 100 /.M. Note that in the absence of DCMU and PD,there is residual activity amounting to approximately 7 to 10,umol ATP synthesized/hr- mg Chi. The effect of increasingconcentrations of PD on phosphorylation activity is complex.Levels of PD in excess of 100 AM continue to stimulate activity,but the increase in phosphorylation is much less dramatic than atPD concentrations below 100 ,M. Even at a PD concentrationof 1250 ,M, the reaction has not reached saturation. The bi-phasic nature of the rate-concentration curve shown in Figure 1suggests that a complex mechanism is involved in the PD-cata-lyzed reaction.The sensitivity of the reaction shown in Figure 1 to DCMU

demonstrates that this activity requires electron carriers whichfunction on the reducing side of photosystem II. It was ofinterest to see whether conditions affecting the oxidizing side ofphotosystem II would affect PD-catalyzed activity. A series ofexperiments were therefore done in which PD-catalyzed ATPsynthesis was assayed both before and after the elimination of 02evolution by NH2OH exposure. Ferricyanide was not included in

Plant Physiol. Vol. 59, 1977

YOCUM AND GUIKEMA

these assays, ruling out the possibility of noncyclic electrontransport. The results of such an experiment are shown in TableIV. In O2-evolving preparations, ATP synthesis is depressed;elimination of this activity stimulates the rate of phosphorylationmore than 8-fold, showing that water and PD compete for acommon electron donation site. It is thus apparent that PD-catalyzed ATP synthesis in KCN-Hg-NH2OH-inhibited chloro-plasts requires access to electron donor and acceptor sites onboth the oxidizing and reducing sides of photosystem II, which inturn indicates that the activity observed with PD arises fromcyclic electron transport.

Effect of Anaerobiosis on PD-catalyzed ATP Synthesis byKCN-Hg-NH2OH-inhibited Chloroplasts. When inhibited chlo-roplasts were illuminated with PD in an 02 electrode cuvette,neither evolution nor consumption of 02 could be detected.Although this result is in accord with a cyclic reaction, we wereconcerned that in the presence of 02, H202 might be formed onillumination and subsequently participate as an electron donor(7) at low concentrations during our assays. This possibility waseliminated by carrying out assays of activity on reaction mixtureswhich had been purged with either air or 02-free helium. Theresults (Table V) of these experiments show that purging witheither air or helium causes some decline in activity, but thesimilarity in extent of the inactivations suggests that the purgingoperation rather than 02 depletion is responsible for loss ofactivity. In view of the results in Table I, it is extremely unlikelythat H202 is participating in the photosystem II cyclic reaction.

Action Spectra of Photosystem II- and Photosystem I-cata-lyzed Cyclic ATP Synthesis. If the activity catalyzed by PD inKCN-Hg-NH2OH-inhibited chloroplasts was, in fact, due tophotosystem II rather than photosystem I activity, then theaction spectrum of this reaction should differ from that of areaction dependent on photosystem I activity. We thereforecompared the action spectra for PD-catalyzed activity in in-hibited preparations and DAD-catalyzed activity in untreatedchloroplasts in the presence of DCMU (9.0 uM). The results ofthese experiments are presented in Figure 2; ATP synthesis isadjusted to a value of 1.00 at the wavelength of peak activity for

TABLE IV

Stimulation of PD-Catalyzed ATP Synthesis in KCN-HgInhibited Chloroplasts by Incubation with NH2OH and EDTA

The concentration of PD was 250 PM throughout, and ferri-cyanide was excluded from all assays shown.

Inhibition by Addition to Assay ATP Synthesis

KCN, HgKCN, HgKCN, Hg, NH2OHKCN, Hg, NH2OH

None9 i'M DCMUNone9 iM DCMU

umol synthesized/hr-mg Chl

80

650

TABLE V

Effect of Air and Helium Purging on PD-CatalyzedATP Synthesis by KCN-Hg-NH20H Inhibited Chloroplasts

The concentration of PD was 250 1M in all assays.

Reaction MixturePurged with ATP Synthesis % of Control Rate

limol synthesized/hr-mg Chl75 100

Air (1 min) 72 96Air (5 min) 65 87Helium (1 min) 68 91Helium (5 min) 63 84

.00

0.80C

cD

(0Co

znwI9--z

U)

a.I-

0.60

0.40

0 .20

0

560 580 600 620 640 660 680 700 720

WAVELENGTH (nm)FIG. 2. Action spectra for photosystem II (PD-catalyzed) and photo-

system I (DAD-catalyzed) cyclic photophosphorylation reactions. Chlconcentrations were 24 ,ug/ml (KCN-Hg-NH20H-inhibited) for the PDreaction and 24.6 ,ug/ml (untreated) for the DAD reaction. In addition,the DAD-catalyzed reaction was carried out in the presence of 9 ,UMDCMU. The light intensity at all wavelengths was 4000 ergs cm-2sec-1,and the illumination time was 5 min. The figure presents rates of ATPsynthesis normalized to values of 1.00 for the activity at peak wave-

lengths. The actual activities were 10.8 ,umol ATP/hr * mg Chl at 640 nmfor the PD system and 5 umol ATP/hr - mg Chl for the DAD system.

each assay system. The PD-catalyzed system shows peak activityat 640 nm, whereas the DAD-catalyzed reaction peaks at 700nm. Although our results are not as precise as other actionspectra determinations (11) owing to the availability and qualityof interference filters, the observed differences in our actionspectra clearly indicate that the PD-catalyzed reaction is associ-ated with photosystem II, whereas the DAD-catalyzed reactionis, as expected, dependent on photosystem I activity.

DISCUSSION

Inhibition of Noncyclic Electron Transport. Incubation ofchloroplasts with KCN and HgCl2 irreversibly inhibits photosys-tem I electron transport. Use of this procedure in concert withsubsequent exposure to NH2OH and EDTA provides a conven-

ient means of preparing chloroplasts in which noncyclic electrontransport reactions have been eliminated. Although Bradeen etal. (3) and Izawa and Good (9) have shown that Hg can act as aninhibitor of photophosphorylation, and our data (Table I) can-not exclude this possibility from our preparations, comparison ofsite II activity before elimination of 02 evolution in KCN andKCN-Hg-inhibited preparations reveals that inclusion of Hg hasnot dramatically affected the efficiency of site II energy conver-sion.

Cyclic ATP Synthesis by KCN-Hg-NH2OH-inhibited Chloro-plasts. The finding that PMS can bypass the KCN-Hg inhibitionsite in our inhibited chloroplasts while p-phenylenediaminescannot is in accord with the results of previous investigations(15) which used KCN alone to inhibit photosystem I activity.The observation that PD was completely inactive as a catalyst ofphotophosphorylation in our preparations when DCMU was

PD CATALYZEDACTIVITY

DAD CATALYZEDACTIVITY

I I I I6o

36 Plant Physiol. Vol. 59, 1977

k

PHOTOSYSTEM II PHOTOPHOSPHORYLATION

present permitted the use of this compound as a catalyst ofelectron transport in the absence of DCMU to demonstrate theexistence of photosystem TI-dependent photophosphorylation ininhibited chloroplasts. The inhibition of this reaction by DCMUor the presence of functional O2-evolving activity, its insensitiv-ity to anaerobiosis, and its action spectrum led us to concludethat PD-catalyzed activity in KCN-Hg-NH2OH-inhibited chloro-plasts represents photosystem II cyclic photophosphorylation.The rate concentration curve shown in Figure 1 suggests that theprocess of catalysis of activity by PD is complex, consisting oftwo reactions, one of which saturates below 100 ,tM PD, andanother which is not fully saturated at 1,250 tLM.

The ability of PD to catalyze DCMU-sensitive photosystem IIactivity is a consequence of its ability to donate electrons tophotosystem II, while at the same time exhibiting little affinityfor electron donation past the KCN-Hg site of inhibition. Ya-mashita and Butler (18) used PD and Asc to restore electrontransport in tris-washed chloroplasts, and their results indicatedthat PD was preferentially oxidized by photosystem II. Ort andIzawa (14) compared the electron donation efficiencies of sev-

eral phenylenediamines to photosystem I, and found that PDwas the least efficient of this class of compounds. Thus, PD aloneamong the p-phenylenediamines appears to be sufficiently ineffi-cient as a photosystem I donor to permit its use as a catalystwhere measurements of pure photosystem II activity are re-

quired.Our data provide no clue as to the mechanism of PD-catalyzed

photosystem II cyclic ATP synthesis. The observation that a lowrate of photophosphorylation can be obtained in our prepara-

tions without added catalyst indicates that native carriers in thechloroplast membrane must be participating in activity. Thehigher rates of ATP synthesis obtained with PD need not,

however, use the same electron transport pathway. The simplestexplanation for the activity of PD is that it acts in a manner

similar to PMS in photosystem I cyclic reactions, undergoingphotooxidation inside the membrane and photoreduction on theoutside to establish an artificial shuttle of protons and electronsfrom outside to inside the membrane. While this is the most

reasonable explanation for the activity of PD, we cannot excludethe interesting possibility that the photosystem II cycle operates

by means of native carriers in the membrane such as plastoqui-none and/or Cyt b559. Experiments are now underway to deter-mine more precisely the pathway of electron flow which supports

photosystem II cyclic ATP synthesis.

37

Acknowledgment - We thank H. Ikuma for many helpful suggestions in the course of this

investigation.

LITERATURE CITED

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2. BEN-HAYYIM, G., Z. DRECHSLER, J. GOFFER, AND J. NEUMANN. 1975. Diaminobenzidineas an electron donor to photosystem I and photosystem II in chloroplasts. Eur. J.Biochem. 52: 135-141.

3. BIADEEN, D., G. WINGET, J. M. GOULD, AND D. R. ORT. 1973. Site-specific inhibition ofphotophosphorylation in isolated spinach chloroplasts by mercuric chloride. Plant Physiol.52: 680-682.

4. GOULD, J. M. 1975. The phosphorylation site associated with the oxidation of exogenousdonors of electrons to photosystem I. Biochim. Biophys. Acta 387: 135-148.

5. GUIKEMA, J. A. AND C. F. YOCUM. 1976. The mechanism of quinonediimine acceptoractivity in photosynthetic electron transport. Biochemistry 15: 362-367.

6. HAUSKA, G., S. REIMER, AND A. TREBsr. 1974. Native and artificial energy-conservingsites in cyclic photophosphorylation systems. Biochim. Biophys. Acta 357: 1-13.

7. INOUE, H. AND M. NISHIMURA. 1971. Electron flow from hydrogen peroxide in photosys-tem II-catalyzed reactions of spinach chloroplast fragments. Plant Cell Physiol. 12: 739-747.

8. IzAwA, S. 1975. Energy coupling at photosystem II. Plant Physiol. 56: S-55.9. IzAwA, S. AND N. E. GOOD. 1969. Effect of p-chloromercuribenzoate and mercuric ion on

chloroplast photophosphorylation. Prog. Photosynthesis Res. 3: 1288-1298.10. IZAWA, S., J. M. GOULD, D. R. FELKER, AND N. E. GOOD. 1973. Electron transport and

photophosphorylation in chloroplasts as a function of the electron acceptor. III. Adibromothymoquinone-insensitive phosphorylation reaction associated with photosystemII. Biochim. Biophys. Acta 305: 119-128.

11 . JOLIOT, P., A. JOLIOT, AND B. KOK. 1968. Analysis of the interactions between the twophotosystems in isolated chloroplasts. Biochim. Biophys. Acta 153: 635-652.

12. KIMimULA, M. AND S. KATOH. 1972. Studies on electron transport associated with photo-system I. I. Functional site of plastocyanin: inhibitory effects of HgCl2 on electrontransport and plastocyanin in chloroplasts. Biochim. Biophys. Acta 283: 279-292.

13. ORT, D. R. AND S. IZAwA. 1973. Studies on the energy coupling sites of photophosphoryla-tion. II. Treatment of chloroplasts with NH2OH plus EDTA to inhibit water-oxidationwhile maintaining energy coupling efficiencies. Plant Physiol. 52: 595-600.

14. ORT, D. R. AND S. IzAwA. 1974. Studies on the energy coupling sites of photophosphoryla-tion. V. Phosphorylation efficiencies (P/e2) associated with aerobic photooxidation ofartificial electron donors. Plant Physiol. 53: 370-376.

15. OurrRAKUL, R. AND S. IzAwA. 1973. Electron transport and photophosphorylation as a

function of the electron acceptor. II. Acceptor-specific inhibition by KCN. Biochim.Biophys. Acta 305: 105-1 18.

16. SAHA, S., R. OUTRAKUL, S. IzAwA, AND N. E. GOOD. 1971. Electron transport andphotophosphorylation in chloroplasts as a function of the acceptor. J. Biol. Chem. 246:3204-3209.

17. TaxBsT, A. AND S. REIMER. 1973. Properties of photoreductions by photosystem II inisolated chloroplasts. An energy conserving step in the photoreduction of benzoquinonesby photosystem II in the presence of dibromothymoquinone. Biochim. Biophys. Acta 305:129-139.

18. YAMASaITA, T. AND W. L. BUTLER. 1968. Photoreduction and photophosphorylation withtris-washed chloroplasts. Plant Physiol. 43: 1978-1986.

19. Yocum, C. F. 1976. Photosystem II-mediated cyclic photophosphorylation. Biochem.Biophys. Res. Commun. 68: 828-835.

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photophosphorylation associated with photosystem ii

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