formation the fe-s cluster in lysed chloroplasts' · pdf...

Plant Physiol. (1991) 95, 97-1 030032-0889/91 /95/0097/07/$01 .00/0

Received for publication July 9, 1990Accepted September 13, 1990

Formation of the Fe-S Cluster of Ferredoxin in LysedSpinach Chloroplasts'

Yasuhiro Takahashi*, Akira Mitsui2, and Hiroshi Matsubara

Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560, Japan

ABSTRACT

In vitro formation of the 3S-labeled Fe-S cluster of ferredoxin(Fd) has been achieved by incubating apo-Fd and [35S]cysteinewith osmotically lysed chloroplasts of spinach (Spinacia olera-cea). Correct integration of the 35S-labeled Fe-S cluster into Fdwas verified on the basis of the following: (a) Under nondenatur-ing conditions, 35S-labeled holo-Fd showed the same electropho-retic mobility as authentic holo-Fd; (b) 35S-labeled holo-Fd showedan ability to bind Fd-NADP+ reductase; (c) the 35S-labeled moietywas removed from the Fd polypeptide by TCA treatment but notby 2-mercaptoethanol treatment; (d) extemally added pea 11 apo-Fd was converted to 35S-labeled holo-Fd. This reconstitution wasdependent on both ATP and light, and formation of the 35S-labeledFe-S cluster was observed upon addition of ATP or when an ATPgeneration-system was constructed in the light. In contrast, ATP-consuming systems abolished the Fe-S cluster formation. A non-hydrolyzable ATP analog was unable to serve as an ATP substi-tute, indicating the requirement of ATP hydrolysis for clusterformation. GTP was able to substitute for ATP, but CTP and UTPwere less effective. Fe-S cluster formation in lysed chloroplastswas stimulated by light even in the presence of added ATP. Lightstimulation was inhibited by DCMU or methyl viologen but not byNH4+. NADPH was able to substitute for light, indicating that lightenergy is required for the production of reducing compoundssuch as NADPH in addition to the generation of ATP. These resultsconfirm the requirement of light for the Fe-S cluster formationobserved previously in intact chloroplasts.

maturation. Due to the instability of the Fe-S cluster moiety,the formation of the Fe-S cluster during Fd biosynthesis hasnot been characterized. Although apo-Fd can be reconstitutedto holo-Fd by chemical reaction with iron and sulfide in thepresence of thiols (10, 11, 13), it is uncertain whether an

analogous reaction is involved in the in vivo formation of theFe-S cluster ( 14).To investigate the biological formation of the Fe-S cluster

of Fd, we previously developed an intact chloroplast systemin which [35S]sulfide derived from [35S]cysteine is incorporatedinto the Fe-S cluster of Fd (19). Thus, the Fe-S cluster isassembled into Fd within the chloroplast. In this intact chlo-roplast system, light illumination was a prerequisite for for-mation of the Fe-S cluster of Fd, although an energy require-ment has not been found in the chemical reconstitutionsystems. In this study we developed a system using osmoticallylysed chloroplasts in which formation of the 35S-labeled Fe-Scluster of Fd was examined using [35S]cysteine as a sulfursource. This system had the advantage of permitting more

extensive studies on Fe-S cluster assembly and provided evi-dence that both ATP and NADPH are required for thereaction. A nonhydrolyzable ATP analog (AMP-PNP3) was

unable to serve as a substitute for ATP, indicating the require-ment of ATP hydrolysis for Fe-S cluster formation.

MATERIALS AND METHODS

Fd in higher plants is localized in chloroplasts and transferselectrons from photosystem I to a wide variety ofbiochemicalreactions, including NADP+ reduction, sulfite reduction, ni-trite reduction, glutamate synthesis, and thioredoxin reduc-tion (2). The oxidation-reduction center of chloroplast-typeFd is an Fe-S cluster which consists of two iron atoms andtwo labile sulfur atoms carried by four cysteinyl residues in a

polypeptide (5). Fd is encoded by a nuclear gene (7, 9),synthesized as a larger precursor in the cytoplasm (8, 16),imported into chloroplasts with concomitant processing tomature size (17), and functionally located in the stroma (15).The precursor Fd is possibly made as apo-Fd, an iron- andsulfur-free form, and the Fe-S cluster is then inserted during

'This work was supported in part by Grants-in-Aid for Encour-agement ofYoung Scientists (No. 61740389 and 62740390) from theMinistry of Education, Science and Culture of Japan.

2 Present address: Basic Research Laboratory, Ajinomoto Co., Ltd.,Suzuki-chou, Kawasaki-ku, Kawasaki 210, Japan.

97

Materials and Reagents

Radioactive L-[35S]cysteine (>600 Ci/mmol) was purchasedfrom Amersham International. Spinach (Spinacia oleracea)leaves were obtained from local markets. Fd was purified asdescribed previously (18). Apo-Fd was prepared anaerobicallyby TCA-denaturation (12). All chemicals were of reagentgrade and purchased from commercial sources.

Formation of 35S-Labeled Fe-S Cluster of Fd in LysedChloroplasts

Intact chloroplasts were isolated from spinach leaves byPercoll gradient centrifugation (19), and washed twice with amedium composed of 50 mm Tricine-KOH (pH 8.0) and 0.33M sorbitol. The chloroplast pellet was then resuspended in 5mM Tricine-KOH (pH 8.0) at a concentration of 2 mg Chl/mL. This resuspension caused immediate lysis as judged byphase-contrast microscopy. Chl was determined according to

'Abbreviations: AMP-PNP, 5'-adenylyl imidodiphosphate; FNR,Fd-NADP reductase.

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Arnon (1). The reaction mixture for formation of the 35S-labeled Fe-S cluster of Fd contained 50 mM Tricine-KOH(pH 8.0), 0.33 M sorbitol, 5 mm ATP, 10 ,ug holo-Fd, 10 ,ugapo-Fd, 5 ,ICi L-[35S]cysteine and broken chloroplasts (equiv-alent to 100 gg of Chl) in a total volume of 100 ,uL. Sampleswere illuminated with filtered red light at about 8000 lux orwrapped with aluminum foil (dark control), and incubated at25°C with gentle shaking for 90 min. Intact chloroplasts wereincubated with [35S]cysteine under the conditions describedpreviously (19).

Electrophoresis and Autoradiography

Following the incubation, the samples were chilled on iceand centrifuged at 15,000g, for 10 min. The supernatantfractions containing the stromal proteins were desalted byrapid gel filtration on syringe-type columns of SephadexG-25 equilibrated with 50 mm Tris-HCl buffer (pH 7.2)containing 10 mm 2-mercaptoethanol. Aliquots were sub-jected to conventional PAGE (17.5%) under nondenaturingconditions with the buffer system (pH 8.0 in the separationgel) of Williams and Reisfeld (21) using vertical gel slabs. Thegels were fixed at 4°C for 30 min with prechilled 20% ethanolto prevent denaturation of Fd, and then dried under vacuum.The 35S-labeled holo-Fd was analyzed by autoradiography(19). For quantification of 35S-labeled holo-Fd in the gel,polyacrylamide gel was prepared with ethylene diacrylate as across-linking agent instead of N,N'-methylenebisacrylamideto dissolve the gel in alkaline solution after electrophoresis(3). After the PAGE and subsequent autoradiography, thebands corresponding to holo-Fd were cut out of the dried gel.The gel slices were solubilized in 2 mL of 1 N NaOH solutionand their radioactivities were determined using a scintillationcounter (Beckman, LS 4000) with Aquasol 2 (New EnglandNuclear). In each case, the background radiation in the gelwas determined using an equal-sized piece of gel which hadbeen excised from a blank region of the gel. The blank valuewas subtracted from the experimental values. This correctionamounted to 20 to 40% for the 35S-labeled holo-Fd.

Nucleotides

In some reactions for Fe-S cluster formation, GTP, CTP,UTP, dATP, AMP-PNP, ADP, or AMP was used at a con-centration of 5 mm in place of ATP. Demonstration of theATP dependence of Fe-S cluster formation was done with anATP-consuming system or an ATP-regenerating system. TheATP-consuming system contained 5 units/mL potato apyrase(Sigma, grade VIII) or 5 units/mL yeast hexokinase (OrientalYeast Co., Japan) with 10 mM D-Glc and 5 mM MgCl2. TheATP-regenerating system was composed of 20 ,g/mL rabbitmuscle phosphocreatine kinase (Sigma), 10 mM phosphocrea-tine, and 2 mM MgC12.

RESULTS AND DISCUSSION

Formation of 35S-Labeled Holo-Fd in Lysed Chloroplasts

Freshly isolated intact chloroplasts were osmotically lysedby suspending them in a hypotonic solution, and the resultingbroken chloroplasts were incubated with apo-Fd and [35S]

cysteine. The radioactively labeled, soluble proteins were ana-lyzed by PAGE under nondenaturing conditions, by which agood separation between holo- and apo-Fd was achieved (Fig.1). Apo-Fd migrated on the gel as two major bands and anumber of minor ones, probably due to the formation ofrandom disulfide bonds between cysteine residues of intra-and intermolecule. In this gel system, spinach holo-Fd I andII (18) migrated the same distance on the gel and the holo-Fdcould be separated from the other stromal proteins, thusallowing for easy detection of 35S-labeled holo-Fd on anautoradiogram of the gel.The formation of 35S-labeled holo-Fd was markedly facili-

tated under light illumination in both intact and brokenchloroplasts (Fig. 1). Addition of apo-Fd and ATP was nec-essary for the efficient formation of 35S-labeled holo-Fd in thelysed chloroplast system, probably due to the dilution of thesoluble components upon chloroplast lysis. The amount of35S-labeled holo-Fd in broken chloroplasts was equal to orgreater than that in intact chloroplasts when compared on thebasis of Chl content.

apo-Fd

I,....^..IH-F ....J

Stainrnci

0L

4-

0T

._*

t.-torad'.i.c r. pr

Figure 1. Analysis of radioactively labeled holo-Fd in intact and lysedchloroplasts. Intact chloroplasts were prepared from spinach leavesand incubated with [35S]cysteine under the conditions describedpreviously (19). A portion of intact chloroplasts was lysed by sus-pending them in a hypotonic solution, and the resulting brokenchloroplasts (equivalent to 0.1 mg of Chi) were incubated with [35S]cysteine, 10 igg apo-Fd, 10 ug holo-Fd, and 5 mm ATP in a finalvolume of 0.1 mL at 250C for 90 min. Radioactively labeled solubleproteins were analyzed by PAGE under nondenaturing conditionsand autoradiography. Lane 1, holo-Fd (5 Ag) purified from spinachleaves; lane 2, apo-Fd (20 Ag) prepared by TCA treatment of holo-Fd; lanes 3 and 4, 35S-labeled holo-Fd in intact chloroplasts incubatedin the light and in the dark, respectively; lanes 5 and 6, 35S-labeledholo-Fd in lysed chloroplasts incubated in the light and in the dark,respectively. Aliquots of stromal proteins derived from chloroplasts(50 ,gg Chl) were subjected to PAGE. CT, chloroplasts.

TAKAHASHI ET AL.98



Fe-S CLUSTER FORMATION IN CHLOROPLASTS

The efficiency of formation of "S-labeled holo-Fd was afunction of chloroplast concentration as shown in Figure 2.This increase in the "S-labeled holo-Fd was saturated at aconcentration of about 1.2 mg Chl/mL. For most experi-ments, 100 to 120 ,g of Chl/100 ,uL reaction mixture wasused. Higher chloroplast concentrations were not necessarilyadvantageous because incomplete lysis of the chloroplastsfrequently resulted in poor yields.

Figure 3 shows the effect of incubation time on the forma-tion of "S-labeled holo-Fd. After a short lag period, theradioactive holo-Fd increased up to about 90 min. When thereaction was carried out in the absence of externally addedATP, less than 10% of the 3"S-labeled holo-Fd was foundwhen compared to that in the presence of ATP. When thereaction was carried out in the dark in either the presence orabsence of ATP, no detectable amount of labeled holo-Fdwas observed.

Identification of 35S-Labeled Fe-S Cluster Assembled intoHolo-Fd

The "S-labeled spinach holo-Fd was prepared from a large-scale reaction mixture and purified by preparative PAGEunder nondenaturing conditions. To confirm that the 35S-labeled protein was holo-Fd, its ability to bind FNR wasexamined using FNR-linked Sepharose 4B (19). There is asubstantial conformational difference between holo- and apo-Fd (12), and only the native holo-Fd can interact with the Fd-linking enzyme, FNR. Figure 4, lane 2, shows that most ofthe radioactive protein that comigrated with holo-Fd wasadsorbed onto the FNR-Sepharose, indicating that this radio-active protein had the same conformation as the native holo-Fd. Further characterization ofthe 3"S-moiety in holo-Fd wascarried out by incubating the purified "S-labeled holo-Fdwith either 1% 2-mercaptoethanol or 10% TCA. As expected,the 3"S-moiety in holo-Fd was removed by the TCA treatment

lysed chloroplasts

(mg chl./ml) 0 0.2 0.4 0.6 0.8 1.0 1.2 1.5

holo-Fd -

Figure 2. 35S-Labeled holo-Fd at different chloroplast concentrations.The concentration of lysed chloroplasts in the reaction mixture,expressed on the basis of Chl concentration, is indicated in the figure.Reactions were carried out in the light with 5 mM ATP under condi-tions similar to those described in the legend to Figure 1, and thelabeled holo-Fd was analyzed by nondenaturing PAGE andautoradiography.

L+ATPo6.0E

xEc4.0/U0I~0

S 2.0

co/uC /

L0 .D+ATP0 30 60 90 120

Incubation Time (min)

Figure 3. Time course of the formation of 35S-labeled holo-Fd inlysed chloroplasts. Reactions were performed in the light in thepresence (0) or absence (A) of added ATP (5 mM), or in the dark (-)with ATP. Other experimental conditions were similar to those de-scribed in the legend to Figure 1. After the nondenaturing PAGE andautoradiography, gel slices containing holo-Fd were solubilized andtheir radioactivities were determined as described in "Materials andMethods."

but not by the 2-mercaptoethanol treatment (Fig. 4, lanes 4and 5). These results confirmed that the radioactivity in holo-Fd was derived from the "S-labeled Fe-S cluster, and notfrom mere binding of [35S]cysteine via a disulfide bond.

In order to evaluate whether the observed reaction is in-volved in the conversion of apo-Fd to holo-Fd during Fdbiosynthesis, it is necessary to determine whether the acceptorFd ofthe "S-labeled Fe-S cluster is apo- or holo-Fd. Therefore,the following experiments were carried out utilizing the dif-ference in electrophoretic mobility between spinach and peaFds. Figure 5A shows the electrophoretic mobilities of holo-and apo-Fds on PAGE under nondenaturing conditions. PeaII holo-Fd, one of the two iso-Fds prepared from pea leaves(4), migrated in the gel slightly more slowly than spinachholo-Fd. Therefore, we examined the destiny of added pea IIapo-Fd distinguishable from the endogenous spinach holo-and apo-Fds contained in the lysed chloroplast system. FigureSB shows that the externally added pea II apo-Fd was con-verted to "S-labeled holo-Fd when incubated in the spinachlysed chloroplast system (lane 2). Spinach holo-Fd also ac-cepted [35S]sulfide (lane 3) probably by substitution of eitherthe Fe-S cluster moiety or the sulfide molecule. When the peaII apo-Fd and spinach holo-Fd were incubated together, con-version ofapo-Fd to radioactive holo-Fd was stimulated abouttwofold, whereas conversion of holo-Fd to radioactive holo-Fd was rather suppressed (lane 4), showing that apo-Fd waspreferentially utilized as an acceptor of the "S-labeled Fe-Scluster in the lysed chloroplast system. These results clearlydemonstrated that apo-Fd was converted to radioactive holo-

99




reat r -re Q''>*1lz- ..,

LL(-IC)

C)

C

z

Figure 4. Identification of 35S-labeled Fe-S cluster assembled intoholo-Fd. The reaction was carried out under the conditions describedin Figure 1 except for scaling up of the total volume to 1 mL, and35S-labeled holo-Fd was purified by preparative PAGE under nonde-naturing conditions. An aliquot was incubated with FNR-linked Seph-arose 4B and eluted with 0.5 M NaCI as described previously (19).Lanes 2 and 3, FNR-bound and unbound fractions, respectively.Another aliquot of the labeled holo-Fd was incubated with 1% 2-mercaptoethanol at 0°C for 1 h and subjected to PAGE (lane 4).Another aliquot was incubated with 10% TCA, and the TCA-insolublefraction was analyzed by PAGE (lane 5). An autoradiogram of thedried gel is shown.

Fd through correct integration of the "S-labeled Fe-S clusterand that, therefore, the activity for biological Fe-S clusterformation can be represented as the relative amount of radio-active holo-Fd labeled in the lysed chloroplast system.A number of proteins other than holo-Fd were also labeled

from [35S]cysteine, and some of them showed the same elec-trophoretic mobility as apo-Fd (Fig. 1). However, the radio-activity in apo-Fd was not reproducible and it is not clearwhether the labeled apo-Fd is an intermediate form to buildup Fe-S cluster or a [35S]cysteine-binding form via a nonspe-cific disulfide bond.

Dependence of Fe-S Cluster Formation on ATP

Figure 6 shows that Fe-S cluster formation depends on

externally added ATP. The amount of "S-labeled Fe-S cluster

ofholo-Fd increased with increasing ATP concentration, withan apparent Km (ATP) of about 2 mm and a maximum rateat about 5 mm. The activity of Fe-S cluster formation variedin proportion to ATP concentration over a physiological rangein chloroplasts, in which ATP concentration depends on abalance between its synthesis by photophosphorylation andits consumption by the carbon cycle. These results indicatethat the observed in vitro activity represents in vivo events,since Fe-S cluster formation in both intact chloroplasts (19)and lysed chloroplasts has a common requirement for ATP.By contrast, ATP-independent Fe-S cluster formation wasalso observed when the lysed chloroplast system was supple-mented with >10 mM 2-mercaptoethanol (data not shown).The 2-mercaptoethanol-dependent Fe-S cluster formationproceeded in the dark and was suppressed by light. This isunlikely to be a physiological reaction, because reconstitutionof holo-Fd from apo-Fd has been demonstrated in a chemicalreaction involving sulfide and ferric or ferrous salts in thepresence of 2-mercaptoethanol (10, 13).When the specificity of adenine nucleotides (ATP, ADP,

and AMP) was examined (Table I), ATP and ADP signifi-cantly stimulated Fe-S cluster formation but AMP did not.In order to clarify whether both ADP and ATP were involvedin Fe-S cluster formation, the following experiments wereconducted using ATP-consuming systems. Three reactionmixtures were prepared; the first was the control with noATP-consuming system, the second contained apyrase, andthe third contained hexokinase and D-Glc. Potato apyrasehydrolyzes ATP to ADP and Pi and ADP to AMP and Pi.nexokinase transfers Pi from ATP to D-hexose to yield ADPand D-hexose 6-phosphate. Table I shows that both apyraseand hexokinase significantly inhibited ATP- and ADP-stim-ulated Fe-S cluster formation, suggesting that the effectiveform of adenine nucleotide is ATP.To confirm the result that ATP is required for Fe-S cluster

formation, and to rule out the possibility that any contami-nant in the hexokinase or D-Glc preparation abolished thecluster formation, we attempted to inhibit Fe-S cluster for-mation by hexokinase with or without D-Glc. Figure 7 showsthat an increase in the amount ofadded hexokinase decreasedthe Fe-S cluster formation, and that this hexokinase activityinhibiting the cluster formation was significantly enhanced bythe addition of D-Glc. D-Glc was not a significant inhibitorby itself, because addition of D-Glc without hexokinase wasineffective. The inhibitory effect of hexokinase in the absenceof added D-Glc was explained by the endogenous D-hexosepresent in the chloroplast preparations. In conclusion, theadenine nucleotide active in Fe-S cluster formation in chlo-roplasts is ATP. Observed stimulation by ADP was probablydue to the synthesis of ATP from ADP by photosyntheticphosphorylation or by the action of adenylate kinase. Thisconclusion was further confirmed using an ATP-regenerationsystem composed of phosphocreatine and phosphocreatinekinase (Table I). When the reaction was carried out in thepresence of an externally added ATP-regeneration systeminstead of ATP, Fe-S cluster formation was stimulated signif-icantly, whereas either of the individual components, phos-phocreatine or phosphocreatine kinase, did not stimulate thereaction when added independently.When the reaction was supplemented with the nonhydro-

100 TAKAHASHI ET AL.

I....

-11-I %,




0z.j

A B

pea 11 holo-Fd _

spinach holo-Fd '

.0

N

.. -

0

0)

0-

Figure 5. Conversion from apo-Fd to 35S-la-beled holo-Fd. A, Electrophoretic mobilities ofspinach holo-Fd (lane 1), apo-Fd (lane 2), pea 11holo-Fd (lane 3), and apo-Fd (lane 4). Holo-Fd (5jig) and apo-Fd (20 gg) were subjected to PAGEunder nondenaturing conditions and the gel wasstained with Coomassie blue R. B, 35S-LabeledFe-S cluster formation. Reactions were carriedout as described in Figure 1 except for thenonaddition of Fd (lane 1), addition of Pea II apo-Fd (lane 2), addition of spinach holo-Fd (lane 3),or addition of pea 11 apo-Fd plus spinach holo-Fd (lane 4). 35S-Labeled holo-Fd was analyzedby nondenaturing PAGE and autoradiography.

1 2 3 4

staining

1 2 3 4

- autoradiography -

lyzable ATP analog AMP-PNP, no significant Fe-S clusterformation was detected (Table I). dATP also had no effect onthe reaction. These results strongly suggest that hydrolysis ofATP is essential for Fe-S cluster formation in chloroplasts.

ATP(mM) 0 0.6 1.3 2.5 5.0

holo-Fd

10

Iww

UJ

:' *

.. 9cn

CUc

0

CZ

Figure 6. ATP requirement for the formation of the Fe-S cluster ofFd. The experimental conditions were similar to those described in

Figure 1, except that the reactions were carried out in the light with

various concentrations of ATP as indicated in the figure. An autoia-

diogram of the dried gel is shown.

ATP versus Other Nucleotides

Several other nucleotides listed in Table I were also testedfor their effectiveness in Fe-S cluster formation. GTP was ableto substitute for ATP, whereas CTP and UTP were able tostimulate about 50% of the ATP. This apparent lack ofnucleotide specificity suggests two possibilities for the involve-nient of nucleotide in Fe-S cluster formation. One is that theapparatus for Fe-S cluster formation contains NTPase activityand directly utilizes any nucleoside triphosphate. The other isthat Fe-S cluster formation is specific for ATP, which can bederived from NTP by the action of nucleoside diphosphatekinase. This enzyme catalyzes the reaction NTP + ADP --

NDP + ATP. Addition of apyrase or hexokinase showedinhibitory effects on GTP-, CTP-, or UTP-stimulated Fe-Scluster formation, but significant activity still remained whencompared to the ATP-stimulated reaction (Table I). On thebasis of this partial resistance to apyrase treatment, it seemslikely that NTP is directly utilized without its conversion toATP. Because the formation of ATP from NTP involves theexchange of terminal phosphate among nucleotide triphos-phates and a contaminating pool ofADP, and because apyrasetreatment completely diminished the internal ADP and ATP,apyrase could possibly inhibit the formation of ATP fromNTP completely. Therefore, direct hydrolysis of NTP mightbe involved in Fe-S cluster formation in a lysed chloroplastsystem.By contrast to these observations obtained in a lysed chlo-

roplast system, Fe-S cluster formation in intact chloroplastsshowed a somewhat different nucleotide specificity. We havereported previously that Fe-S cluster formation in intact chlo-roplasts was dependent on light and inhibited by DCMU and

101111111111 III0.1

101

-r

-"vIllpl ", "'' ...I

ll. .i -,MEw




Table I. Nucleotide Specificity of Fe-S Cluster FormationAssays were performed as described in Figure 6, and the values

represent the average of at least two experiments. All nucleotideswere tested at the same concentration (5 mM). Where indicated, thereactions were carried out in the presence of ATP-consuming sys-tems containing potato apyrase (5 units/mL) or yeast hexokinase (5units/mL) with 10 mM D-GIc and 5 mm MgCI2. The ATP-regeneratingsystem was composed of 20 ig/mL phosphocreatine kinase, 10 mMphosphocreatine, and 2 mM MgCI2.

% ATP ControlAdditions

None Apyrase Hexokinase

None 9 nda ndATP 100 nd 7ADP 99 nd 4AMP 6dATP 3AMPPNP 8GTP 109 12 36GDP 9CTP 50 8 4UTP 48 4 15Creatine kinase + 89 nd 3

phosphocreatineCreatine kinase 3Phosphocreatine 3

a Not detected.

the uncouplers, atebrin and gramicidin (19). These inhibitoryeffects were reversed by externally added ATP but not byGTP, CTP, or UTP. One possible reason for the difference innucleotide specificity between intact and lysed chloroplastsmay be the different permeabilities of these nucleotidesthrough the chloroplast membranes. Externally added ATP isincorporated into intact chloroplasts via the ATP translocator(6), but other nucleotides might be transported into chloro-plasts more slowly than ATP. Alternatively, the level ofGTP,CTP, and UTP in chloroplasts might remain constant atconcentrations lower than ATP. In view ofthe energy require-ment for Fe-S cluster formation in vivo, we concluded thatthe physiologically dominant energy source is ATP, and thatATP hydrolysis is essential for Fe-S cluster formation inchloroplasts.

Role of Light in Fe-S Cluster Formation

The formation of "S-labeled Fe-S cluster in lysed chloro-plasts was dependent on both added ATP and illumination(Figs. 1 and 3), but reaction in the dark could not be restoredeven in the presence of added ATP and an ATP-regenerationsystem (not shown). These results suggest that the light stim-ulation might be due to the production of some functionalcompounds in addition to the production of ATP. Severalspecific inhibitors were tested to determine the process re-sponsible for light stimulation (Fig. 8). NH4', an uncoupler,stimulated the Fe-S cluster formation about twofold (lane 3).DCMU inhibits transfer of electrons away from PSII andmethyl viologen is an artificial acceptor of electrons from PSI.These inhibitors suppressed the light stimulated formation of

the Fe-S cluster (lanes 4 and 5), and therefore a noncyclicelectron transport system is obviously involved in the light-stimulated reactions. In addition, NADPH substantially stim-ulated Fe-S cluster formation in the dark (lane 7). Sinceneither NADP+ nor NADH showed this stimulation (notshown), an NADPH-linked enzyme might be involved in thereaction. ATP was also required for the Fe-S cluster formationeven in the presence of NADPH (lanes 6-9). Thus, Fe-Scluster formation in lysed chloroplasts depends on both ATPand reducing compounds such as NADPH. Although a num-ber of photosynthetic reactions are regulated by thioredoxinsystem, further analysis in that line was unsuccessful becausethe addition of sulfhydryls, such as DTT, caused ATP-inde-pendent reactions as described above.The obvious requirement for ATP and NADPH in a lysed

chloroplast system might be due to dilution of these solublecomponents upon lysis of the chloroplasts, and this reconsti-tution system allowed us to confirm the light stimulation ofFe-S cluster formation observed previously in intact chloro-plasts (19). We also demonstrated that it was necessary forATP to be hydrolyzed, and that NADPH could not be sub-stituted by NADH. The requirement of ATP and NADPHgives rise to some speculation on the possible mechanism ofFe-S cluster formation in chloroplasts, which is distinct fromthe in vitro chemical reconstitutions reported previously (10,1 1, 13). It would be possible to consider that ATP hydrolysisoccurs through coupling of energy to formation of the Fe-Scluster. Alternatively, ATP might be required for the phos-phorylation of a certain protein involved in the reaction.NADPH might be related to the maintenance of a reductiveenvironment or to the reduction of sulfur, iron, or unknowncompounds. In order to understand the mechanistic back-ground of Fe-S cluster formation, it will be necessary toseparate the process into its various stages and to analyze each

100

75~0%

LA.0I0

~0a)co-JC'I

50 .

25 .

O L0 2 4 6

Hexokinase (units/ml)

Figure 7. Inhibition of Fe-S cluster formation by hexokinase. Fe-Scluster formation assay was done in the presence of various concen-trations of hexokinase with (@) or without (0) the addition of 10 mMD-GIc. The results for Fe-S cluster formation were quantitativelyevaluated by densitometry of the exposed film.

102 TAKAHASHI ET AL.




-- NADPH-

ATP + + +

D L L L L L D L D

w

0lL

C_

c

c

0

z

Figure 8. NADPH requirement for Fe-S clusterformation. 35S-Labeled Fe-S cluster formationwas examined either in the light (L) or in the dark(D), and in the presence (+) or absence (-) ofextemally added ATP (5 mM). Where indicated,10 mM NH4CI (lane 3), 10 uM DCMU (lane 4),0.16 mm methylviologen (MV, lane 5), or 5 mMNADPH (lanes 6-9) was supplied to the reactionmixture.

hcAo-Fd-

1 2 3 4 5 6 7 8 9

step separately. The system using osmotically lysed chloro-plasts described here should provide a means of elucidatingthe role of ATP and NADPH in cluster formation. Someinvestigations along these lines are presented in a companionstudy (20).

ACKNOWLEDGMENTS

The authors express their sincere appreciation to Dr. T. Hase(Nagoya University) for his interest and critical comments during thecourse of the present study.

LITERATURE CITED

1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Poly-phenoloxidase in Beta vulgris. Plant Physiol 24: 1-15

2. Buchanan BB, Arnon DI (1970) Ferredoxins: chemistry andfunction in photosynthesis, nitrogen fixation, and fermentativemechanism. Adv Enzymol 33: 119-176

3. Choules GL, Zimm BH (1965) An acrylamide gel soluble inscintillation fluid. Anal Biochem 13: 336-344

4. Dutton IE, Rogers LJ, Haslett BG, Takruri IAH, Gleaves JT,Boulter D (1980) Comparative studies on the properties of twoferredoxins from Pisum sativum L. J Exp Bot 31: 379-391

5. Fukuyama K, Hase T, Matsumoto S, Tsukihara T, Katsube Y,Tanaka N, Kakudo M, Wada K, Matsubara H (1980) StructureofS. platensis [2Fe-2S] ferredoxin and evolution ofchloroplast-type ferredoxins. Nature 286: 522-524

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