Plant Science, 90 (1993) 21-29 21 Elsevier Scientific Publishers Ireland Ltd.

The in vitro effects of ATP and protein phosphorylation on the activity of ferredoxin:NADP ÷ oxidoreductase from spinach

chloroplasts

Michael Hodges and Myroslawa Miginiac-Maslow

Laboratoire de Physiologic V~g~tale Mol~culaire, Bat. 430, CNRS ( UA 1128), Universit~ de Paris Sud, 91405 Orsay Cedex (France)

(Received August 31st, 1992; revision received December 24th, 1992; accepted January 26th, 1993)

When ferredoxin:NADP + oxidoreductase (FNR) was preincubated with [-t-a2p]ATP-Mg and separated by native PAGE it was found to be radioactive. This was not seen on SDS-PAGE, unless FNR was preincubated with a crude protein kinase extract with FNR kinase activity under phosphorylating conditions. These observations suggest that FNR contains an ATP-binding domain. It was found that ATP caused an inhibition of FNR diaphorase activity, which when analysed by Lineweaver-Burk plots indicated that ATP was a non-competitive inhibitor with respect to NADPH. This shows that the ATP site is distinct from the NADP(H) active site. The in vitro phosphorylation of FNR on a serine residue(s) by the crude FNR kinase extract led to a modification of ferredoxin (Fd)-dependent FNR activity. An analysis of the data showed that after phosphorylation the apparent K m for Fd and Vma x both in- creased. This observation suggests that the Fd-FNR interaction is modified after FNR phosphorylation in vitro. Limited proteolysis of phosphorylated FNR followed by SDS-PAGE infers that the phosphorylated amino acid(s) is located near the N-terminus.

Key words: ferredoxin; ferredoxin:NADP + oxidoreductase; protein kinase; protein phosphorylation; thylakoid membrane (spinach)

Introduction

Ferredoxin:NADP ÷ oxidoreductase (FNR) (EC 1.18.1.2) is located in the chloroplasts of higher plants and is the terminal enzyme of the non-cyclic photosynthetic electron transfer chain; it has also been implicated in cyclic electron flow around photosystem I (PSI) [1,2]. FNR is believed to be bound to the thylakoid membrane via an elec- trostatic mechanism [3]. Two pools of FNR ap- pear to exist in vivo; a loosely-bound pool which is easily removed from the membrane by a low-salt wash and a more tightly-bound pool (30-60% of

Correspondence to: Michael Hodges, Laboratoire de Physiologic Vrgrtale Mol~eulaire, Brit. 430, CNRS (UA 1128), Universit6 de Paris Sud, 91405 Orsay Cedex, France. Abbreviations: DPIP, 2,6-dichlorophenol indophenol; Fd, ferredoxin; FNR, ferredoxin:NADP + oxidoreductase; PS, photosystem; CHAPS, 3-((3-Cholamidopropyl)-dimethyl- ammonio)-l-propanesulfonate; Chl, chlorophyll.

0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Printed and Published in Ireland

the total enzyme) which requires several extensive low salt/EDTA washes and/or the addition of detergents (e.g. CHAPS) for its removal [4]. It is mainly located within the non-appressed stromal thylakoid membranes, which contain most of the PSI, where it can interact with ferredoxin (Fd) which donates electrons for NADP + reduction. However, 20% of the total FNR is located in the appressed, granal lamellae [5] which are enriched in PSII. Therefore, FNR may play a key role in the regulation of cyclic/non-cyclic electron flow and hence modify the NADPH/ATP ratio in chloro- plasts. Furthermore, the need to control the parti- tioning of photosynthetic electrons after Fd, between a number of different competing reactions [6] suggests that FNR could be subject to regula- tory processes.

This key enzyme appears to be activated by light in a process requiring a pH-gradient [7] and as- sociated with a conformational change in the FNR

Ltd.

22

protein [8]. We have previously shown that FNR can be phosphorylated either in the dark or by a light-dependent reaction in vitro, with the possibility that such a post-translational modifica- tion may alter its functioning [9]. Moreover, it has been reported that FNR can bind azido-ATP [10] and that ATP can protect FNR from inactivation by phenylglyoxal which occurs upon illumination [8]. As this latter compound reacts with arginine residues, this observation suggests that there is a light-induced conformational change in FNR that unmasks an arginine located near or at the active site. Indeed, arginine residues have been proposed to play a role in both NADP ÷ binding and the in- teraction of FNR with Fd [11].

In this work we have tried to distinguish be- tween the effect of FNR phosphorylation on enzyme functioning in vitro, and a possible direct effect of ATP on FNR activity. Our results are discussed in terms of two types of ATP induced changes in FNR functioning; a phosphorylation- dependent modification of the FNR-Fd interac- tion and a direct inhibition by ATP which appears to affect the active site.

Materials and Methods

Ferredoxin-Sepharose affinity chromatography, as described in Droux et al. [12], was used for the purification of spinach FNR (peak gradient elu- tion at 140 mM NaCI), followed by 2 ' ,5 ' -ADP- Sepharose affinity chromatography where FNR was released by a step elution with 400 mM NaCI.

A crude protein kinase preparation with FNR kinase activity was prepared from market spinach leaves. Thylakoids were isolated and washed twice in the presence of 1 mM EDTA [131. The final pel- let was resuspended in 30 mM Tris (pH 7.9), 10 mM KC1 and 5 mM MgC12 to give a final chlorophyll (Chl) concentration of 1.5 mg Chl/ml. CHAPS was slowly added to the washed thyla- koids to a final concentration of 10 mM, and the membranes were left at 4°C for 30 min. The mix- ture was centrifuged at 40 000 x g for 1.5 h and the resulting supernatant fluid was used for am- monium sulphate fractionation. After the addition of 40% saturation ammonium sulphate and subse- quent centrifugation for 15 min at 25 000 x g, the

supernatant fluid was taken and ammonium sul- phate added to give 60% saturation. After cen- trifugation at 25 000 × g for 30 min, the resulting pellet was resuspended in 30 mM Tris (pH 7.9), I0 mM CHAPS and dialysed overnight against 2 liters of 30 mM Tris (pH 7.9) to remove the deter- gent and ammonium sulphate before phase parti- tioning with Triton X-114 (see Ref. 14). The dialyzed sample was diluted 1:1 (v/v) with 4% (w/v) Triton X-114, 40 mM Tris (pH 7.9), 300 mM NaCI, and 10-ml aliquots of this mixture were loaded onto 35-ml cushions containing 20 mM Tris (pH 7.9), 6% (w/v) sucrose, 150 mM NaC1. The tubes were incubated at 30°C until the Triton X-114 phase became cloudy (approx. 15 min) and then centrifuged at 6000 x g for 10 rain at 30°C. The resulting oily, Triton-enriched pellets were gathered and pooled. This fraction was dialysed against 2 liters of 30 mM Tris (pH 7.9) overnight before being loaded onto a 10-ml histone- Sepharose column equilibrated with 30 mM Tris (pH 7.9) and subsequently eluted with a 0.1 M NaC1 linear gradient. The peak of FNR kinase ac- tivity eluted at the end of the gradient (1 M NaC1). This fraction was dialysed and concentrated on a Diaflow system to give the crude FNR kinase ex- tract. It was interesting to note that two other pro- tein peaks containing histone kinase activity but no FNR kinase activity were eluted at lower salt concentrations (150 mM and 500 mM NaCI).

FNR phosphorylation was carried out using the crude kinase extract as follows; affinity-purified FNR (3 #g) was resuspended in 30 mM Tris (pH 8), 10 mM NaF, 10 mM MgC12 and 20 #1 of crude FNR kinase extract to give a final volume of 100 #1. Phosphorylation was carried out for 30 min at 30°C in the presence of 100 #M ATP (sometimes containing [7-32p]ATP-Mg at a specific radioac- tivity of 200 Ci/mol to verify that FNR was, in- deed, phosphorylated). Control samples were incubated in the absence of the FNR kinase ex- tract or in the absence of ATP. To investigate the effect of FNR phosphorylation on enzymatic ac- tivity, 30-#1 aliquots of the reaction mixture were taken for Fd-dependent cytochrome c reductase and diaphorase activity measurements (see below).

The effect of ATP on FNR function was assayed by the direct addition of ATP to the reaction

cuvette at concentrations ranging between 0 and 10 mM before measuring FNR activity. The stock ATP solution contained Mg 2÷ cations so as to have MgATP.

Diaphorase activity was measured at 30°C by the absorbance decrease at 600 nm in a routine reaction medium (1 ml final volume) containing 100 mM Tris (pH 7.9), 100 #M DPIP, and 25 #M NADPH in the presence of 2.4/zg FNR.

Fd-dependent FNR activity was measured at 30°C by the absorbance increase at 550 nm in a routine reaction medium (1 ml final volume) con- taining 30 mM Tris (pH 7.9), 40 #M cytochrome c, 25 #M NADPH and various concentrations of Fd (0.5-7 /~M) in the presence of 30 /~1 of the above phosphorylation reaction medium contain- ing FNR (giving a final concentration of carry- over ATP in the reaction medium of 3 #M when ATP was present, a concentration too low to give rise to a direct ATP effect).

SDS-PAGE (12.5% acrylamide) and non- denaturing PAGE (7.5% acrylamide in the absence of SDS, 4°C) were carried out as in Laemmli [15] before protein-staining with Coomassie brilliant blue. The gels were dried and, when appropriate, the phosphorylated proteins were detected by autoradiography using Kodak XAR-5 X-ray film (1-day exposure at -60°C)

The stoichiometry of [32p]phosphate incorpora- tion into FNR was estimated by either liquid scin- tillation counting of repurified, phosphorylated FNR or by Cerenkov counting of the phosphory- lated FNR bands excised from SDS-PAGE gels. Occasionally these methods were used in conjunc- tion with densitometric scans of the autoradio- grams.

To identify the amino-acid type phosphorylated by the crude kinase extract, [32p]phosphorylated FNR was repurified by Fd-Sepharose affinity chromatography and subjected to partial acid hydrolysis in 6 N HC1 followed by separation by thin layer chromatography and electrophoresis on cellulose plates as described in Hodges et al. [9].

Trypsin, chymotrypsin and S. aureus V8 pro- tease were used to locate the phosphorylation site by limited proteolysis of phosphorylated FNR. FNR (75 ttg) was phosphorylated in the presence of [3,-32p]ATP-Mg and the crude kinase extract as

23

described above and after 30 min at 30°C the reac- tion was stopped by the addition of 25 mM EDTA. Protease (7.5 #g) was then added to the phosphorylation mixture and a time course of pro- teolysis was carried out at 25°C. Aliquots of 10/zl were taken at various incubation times and im- mediately heated at 100°C for 5 min to stop further proteolysis. The samples were then prepared for SDS-PAGE as described above.

R ~

Fig. 1 shows a non-denaturing electrophoresis gel of affinity-purified spinach FNR, which gives rise to two major protein bands that are well separated (lane 1) compared to two very closely migrating bands when the purified FNR is run on SDS-PAGE (see Fig. 2, lane 1). Recently it has been shown that the lower Mr form found on SDS-PAGE arises from degradation of the larger Mr form at the N-terminus [16]. By comparing FNR preparations containing different propor- tions of the degradation product we concluded that the lower band on the native gel corresponded to the non-proteolysed form of FNR (data not shown). When FNR was incubated in the presence of ['r-32p]ATP-Mg alone and subsequently allow- ed to migrate on a non-denaturing gel, the two protein bands were labeled (Fig. 1, lane 2). If the [32p]ATP was chased with unlabeled ATP prior to electrophoresis, the labeling of the protein bands disappeared (Fig. 1, lane 3), while a chase with either ADP (Fig. 1, lane 4) or NADPH (Fig. 1, lane 5) was much less effective. When FNR was incubated with [3,32p]ATP-Mg and the crude FNR kinase extract under phosphorylating condi- tions, its labeling could not be removed by chasing with unlabeled ATP (Fig. 1, lane 6). A notable ob- servation is that neither the incorporation of phos- phate by phosphorylation nor the non-covalent binding of ATP alters the migration of FNR on a native gel. When FNR is incubated with [3'- 32p]ATP-Mg and then separated by SDS-PAGE (after heating) instead of native PAGE, no labeling occurs (data not shown) unless FNR is also in- cubated in the presence of the FNR kinase extract (Fig. 2, lane 2). These results indicate that ATP can bind non-covalently to FNR besides the al-

2 4

:il ̧. i~i

i~ ~c @i ~

:(i((((iii

2 3 4 5 6 Fig. 1. Labeling of purified spinach leaf FNR with [~/'~2PIATP-Mg (100/~M ATP containing [~,. 32plAT P at a spe- cific radioactivity of 200 Ci/mol) as described in the Materials and Methods and the effect of different unlabeled nucleotide chase treatments. FNR was incubated with radiolabeled ATP for 5 rnin at 30°C; then unlabeled nucleotide was added for a further 5 min before analyzing the samples by non-denaturing electrophoresis at 4°C. The different lanes correspond to: 1, Coomassie blue-stained FNR; 2, autoradiogram of FNR; 3, autoradiogram of FNR chased with 250 #M unlabeled ATP; 4, autoradiogram of FNR chased with 250 #M unlabeled ADP; 5, autoradiogram of FNR chased with 250 /zM NADPH; 6, autoradiogram of FNR phosphorylated in the presence of the crude kinase extract followed by a chase with 250 ~M unlabeled ATP. The arrows correspond to the three FNR bands found on the native gel. The • (lane 6) corresponds to a phosphoprotein which is found in the crude kinase extract. The+and-s igns correspond to the anode and cathode, respectively.

ready described covalent transfer of its gamma- phosphate during protein phosphorylation [9].

In order to investigate a possible effect of the non-covalent binding of ATP (Fig. 1) on FNR ac- tivity the kinetic properties of the enzyme were measured in the presence of various ATP (0-10 mM) and NADPH (12-250 #M) concentrations. DPIP reduction was followed as a test for diaphorase activity and an inhibition was observed in the presence of ATP. This might have arisen from competitive interaction between the two dif- ferent nucleotides at the active site. However, a Lineweaver-Burk plot of FNR diaphorase activity (Fig. 3) showed that the inhibition by ATP was non-competitive with respect to NADPH. A non- competitive inhibition by ATP was also seen for

, i i* Fig. 2. Phosphorylation of purified spinach leaf FNR by ['Y'32PlATP-Mg and crude FNR kinase extract, The FNR was analyzed by SDS-PAGE. The different lanes correspond to: 1, Coomassie blue-stained FNR: 2, autoradiogram of FNR phos- phorylated in the presence of kinase extract. The arrows corre- spond to the two FNR bands found on an SDS-PAGE gel, The + and - signs correspond to the anode and cathode, respectively,

4 2 , ,

ii O - 0 0 5 0.00

0 i- _ - 0 05

/

0 0 5 0 10

/ / / /

_ L _ - - . . . . . .

r iO0 0 05 f,~

/ /NAOPH (,~zM- ')

Fig. 3. Inhibition of FNR diaphorase activity (NADPH to DPIP) by the addition of different concentrations of A T P to the assay medium. The ATP concentrations (in mM) were: O, 0; O, 3; V, 6; v, 10. The Lineweaver-Burk plot shows that ATP is a non-competitive inhibitor with respect to NADPH. I/(DPIP reduction) is based on the decrease in absorbance at 600 nm/min. The inset shows a double-reciprocal plot of FNR diaphorase activity versus NADPH concentration (~M-I) in the absence of adenylates (O) or in the presence of 10 mM ADP (V) or 10 mM ATP (0).

25

the Fd-dependent FNR activity (NADPH to cytochrome c) (data not shown). In the latter case, it was also found that the presence of ATP did not alter the Km for Fd. The inset of Fig. 3 shows a Lineweaver-Burk plot for diaphorase activity in the presence of 10 mM ADP compared to 10 mM ATP and no additions. It can be seen that ADP is somewhat less effective than ATP in bringing about an inhibition of FNR activity. This confirm- ed the chasing experiments where ADP was less ef- fective than ATP in removing the non-covalent labeling of FNR (Fig. 1, lane 4). These data sug- gest the presence of an ATP-binding site that is distinct from the NADP(H) active site. A Dixon plot of a set of ATP inhibition data showed that the apparent K i for ATP is high ( -2 .5 mM).

We have previously shown that FNR can be phosphorylated in vitro and that perhaps this covalent modification can influence FNR- thylakoid membrane interactions and hence change PSI activity and possibly FNR function [9]. Fig. 4 shows the effect of FNR phosphoryla- tion in vitro, using the crude FNR kinase extract from spinach, on Fd-dependent FNR activity. FNR was phosphorylated and aliquots were taken to measure activity as a function of Fd concen-

1.2

1.o- 0.8"

0.6"

0.4"

0.2"

0.0 0

I i I I i I I

2 4 6 8 10 12 Ferredoxin j.LM

Fig. 4. The effect of phosphorylation by the crude F NR kin- ase extract on Fd-dependent FNR activity (NADPH to cytochrome c) normalized to the calculated Vma x as a function of Fd concentration, e , - A T P ; O, + A T E The calculated Vma x values were 1.0 and 1.4 nmol cyt. c reduced s -] for - A T P and +ATP, respectively. The data of five similar experiments were analysed by a single Michaelis-Menten reaction using the curve fit Maths function of Sigmaplot v. 4.02. and the average kinetic values are shown in Table I.

tration in the presence of a saturating concentra- tion of NADPH and cytochrome c. The non- phosphorylated protein was subjected to the same treatment but no ATP was included during the preincubation period with the crude kinase ex- tract. The 30-min phosphorylation treatment led to the incorporation of 0.25 mol phosphate per mol FNR as judged by the techniques described in the Materials and Methods. The reason(s) for the incomplete phosphorylation of FNR is not known. Nevertheless, it can be seen from Table I that phosphorylation led to small, but statistically significant changes in both the Vma ~ and apparent Km for Fd. The data of five different experiments, similar to those shown in Fig. 4, were fit to a single Michaelis-Menten reaction using the Marquardt- Lavenberg algorithm incorporated in the Maths function of Sigmaplot v.4.02. The calculated Vma x

and apparent Km (Fd) values are given in Table I. Such changes suggest that phosphorylation of FNR modifies the Fd-FNR interaction, as postulated in Hodges et al. [9].

It has previously been shown that FNR is phos- phorylated in vitro on serine (in a light- independent reaction) and threonine (light- dependent) residues in the presence of thylakoid membranes [9]. Partial acid hydrolysis of phos- phorylated FNR followed by two-dimensional separation of the phosphoamino acids showed that the crude kinase extract only phosphorylated serine residues (data not shown). The phosphory- lation site(s) giving rise to the observed changes in Fd-dependent FNR activity brought about by the crude kinase extract (Fig. 4, Table I) was indirectly

Table I. The effect of in vitro phosphorylation of FNR on its kinetic constants. The values (+S.E.) were calculated from 5 different experimental data sets using the curve fit Maths func- tion of Sigmaplot v. 4.02 based on a single Michaelis-Menten type reaction.

- A T P +ATP

K m Fd (#M) 1.3 ± 0.1 5.4 + 0.4 Vmax 0.86 + 0.15 1.44 + 0.15 (nmol cyt. c reduced s - l)

26

M 1 2 3 4 5 6 7

;; i~!i! ̧!~̧

ili̧ ~ v ~:'~i:;~ ~;~%~ ~ !~i~;~!i ¸ ~iii~ ~ ~ '~i~i'~i!:

Fig. 5. Time course of limited proteolysis of phosphorylated FNR by trypsin as analysed by SDS-PAGE. The samples were run on a 12.5% gel. Lanes 1-7: 0-, 1-, 5-, 10-, 20-, 40- and 60-min incubation with protease. (a) The protein bands stained with Coomassie blue and (b) the corresponding autoradiogram. Lane M contains the molecular weight markers of 116, 84, 58, 48.5, 36.5 and 26.6 kDa (from top to bottom, respectively).

analysed by limited proteolysis of phosphorylated FNR. The fragments arising from proteolysis of FNR by trypsin, chymotrypsin or S. aureus V8 protease have already been documented by Gadda et al. [17]. Fig. 5 shows the time course for limited tryptic proteolysis of FNR (and the crude kinase extract) by separating the peptides on SDS-PAGE. The corresponding autoradiogram of the Coomassie-stained gel (Fig. 5a) is given in Fig. 5b. During proteolysis the region(s) of FNR contain- ing the 32p-labeled amino acid(s) disappears from the gel, suggesting that the phosphoamino acid is associated with a small proteolytic fragment. It has been shown in Ref. 17 that trypsin initially removes the N-terminal domain of FNR leaving Thr 36 as the first amino acid (31 kDa fragment). Fig. 5 suggests that the phosphorylation site is located in the N-terminal region since the large

tryptic fragments were no longer labeled after 20 min of incubation. Similar experiments using either chymotrypsin or SV8 protease confirmed this view. The lower molecular weight bands generated by chymotrypsin and SV8 correspon- ding to fragments between amino acids 33 and 314 (chymotrypsin) and 23/24 and 314 (SV8) I17], were no longer 32p-labeled. The small 32p-labeled phosphopeptide obtained after proteolysis by SV8 was purified by HPLC; however, attempts to se- quence it by Edman degradation have failed. This result seems to confirm that the phosphorylation site may be close to the N-terminus since spinach FNR is N-blocked [18].

Conclusions

The data presented herein clearly indicate that FNR activity can be modified in vitro by ATP both directly and indirectly via a posttranslational phosphorylation mechanism. The results of Fig. 1 show that ATP can bind to purified FNR in vitro. This finding confirms the observations of Matthijs et al. [4] that FNR can be labeled by azido-ATP. This non-covalent binding brings about an inhibi- tion of FNR activity (both Fd-dependent and- independent) (Fig. 3). Although a high Ki value for ATP of 2.5 mM is obtained, this ATP concen- tration can be found in illuminated intact chloro- plasts [19]. Moreover, such a value was calculated for the whole chloroplast, and local ATP concen- trations well above this level might be expected to occur around FNR, which is mainly located in the stromal lamellae within close proximity to the ATP synthase complex. The question arises whether this type of inhibition could play a physiological role where NADPH production is controlled by high ATP (or adenylate) concentra- tions. An interesting observation is that the inhibi- tion does not alter the Km for NADPH or Fd. This non-competitive type of inhibition suggests that the ATP binding site is distinct from the NADP(H) site which has been assigned to the C- terminal end of FNR, between amino acids 169-314 of the spinach FNR sequence [18].

27

When the spinach FNR primary sequence of Karplus et al. [18] is examined for possible ATP- binding domain motifs (e.g., GXXXXGK(T)XX- XXXXIN, see Ref. 20), a possible candidate can be found between amino acids 71 and 97. This gives the following sequence where the numbers correspond to the numbering of the primary se- quence of spinach FNR [18] and asterisk indicates an amino acid proposed to be conserved in at least one type of ATP-binding domain [20].

REGQSVGVIPDGEDKNGKPHKLRLYSI 71 88 97

This putative ATP-binding site is not close to the NADP(H) site in the primary structure. Thus, the inhibition could be due to an ATP-induced con- formational change which affects the transfer of electrons between NADP(H) and Fd but which does not affect either Fd or NADP(H) binding.

A second effect on the activity of FNR which is indirectly linked to ATP is due to a posttransla- tional phosphorylation requiring a yet to be deter- mined protein kinase. It has previously been shown that the phosphorylation of FNR in vitro can modify FNR-membrane interactions as well as inhibit PSI-dependent NADP ÷ reduction in isola- ted thylakoids [9]. The data presented above show that after phosphorylation on a serine residue(s), Fd-dependent FNR activity is modified. It has al- ready been shown that FNR phosphorylation does not alter FNR diaphorase activity (NADPH to DPIP) [9]. It is seen from Fig. 4 and Table I that alter phosphorylation by the crude FNR kinase extract, the apparent Km for Fd as well as the Vma x are both increased. The covalent incorpora- tion of phosphate into FNR alters the Fd-FNR in- teraction such that the K m (Fd) is increased by a factor of approx. 4, while Vma x is increased by about 50%. These changes appear to be small, but they are statistically significant and it should be noted that the well-documented phosphorylation of C4 phosphoenolpyruvate carboxylase only doubles the apparent Ki for L-malate [21] but this

posttranslational modification seems to be very important in the regulation of C4-metabolism. It has been proposed that the Fd-FNR interaction involves an electrostatic mechanism exploiting two negatively charged amino acids on Fd and two positively charged amino acids (Arg and Lys) on

• FNR [22]. Therefore, it can be envisaged that the introduction of a negatively charged phosphate group(s) could possibly perturb this electrostatic interaction.

The results from the limited proteolysis of phos- phorylated FNR by trypsin (Fig. 5) and other pro- teases suggest that the crude kinase extract phosphorylates FNR close to the N-terminus, be- tween amino acids 1 and 23/24. This region of FNR contains two serine residues. When the N- terminal sequence of spinach FNR is examined for possible phosphorylation site motifs (e.g., K/R x(x) S/T, see Ref. 23) then the following is found:

10 25 QIASDVEAPPPAPAKVEKHSKKMEEGIT

where the numbers correspond to the numbering of the primary sequence of spinach FNR [18] and asterisk indicates a possible phosphorylation-site motif. Indeed, trypsination of FNR near its N- terminus would give rise to a phosphorylation site being located in a fragment too small to be seen by standard SDS-PAGE. Similarly, it has been reported that Fd binding to FNR involves the N- terminus of the reductase [17]. Therefore, the changes in the Fd-dependent kinetic properties of phosphorylated FNR are consistent with this ob- servation and with the putative localisation of the phosphorylation site close to the N-terminus.

The physiological role for this phosphorylation is not apparent. It might be that FNR phosphory- lation, by modifying the Fd-FNR interaction, favors cyclic electron flow around PSI or perhaps changes the partitioning of photosynthetic elec- trons between NADP + reduction and other Fd- dependent reactions, e.g. Fd-thioredoxin reduc- tase, nitrite reductase. Nonetheless, it appears that

28

FNR activity can be modified in vitro directly by ATP, which can bind to the enzyme at a site distinct from the NADP(H) site, and also indirect- ly via protein phosphorylation. In the first case, ATP in some way interferes with electron transfer between oxidant and reductant, while in the sec- ond case covalent phosphorylation weakens the Fd-FNR interaction.

Acknowledgements

This work was funded by the Centre National de la Recherche Scientifique and a grant (No. 89C0665) from the Ministre de la Recherche et Technologie. We would like to thank Margot Weinbaum for her photographic skills, Pierre LeMar6chal for the phosphoamino acid analyses, Paulette Decottignies for the separation of FNR proteolytic peptides by HPLC and Jean-Marie Schmitter (Ecole Polytechnique, Palaiseau, France) for the sequencing of the radiolabeled peptides.

References

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13 M. Droux, M. Miginiac-Maslow, J-P. Jacquot, P. Gadal, N.A. Crawford, N.S. Kosower and B.B. Buchanan, Ferredoxin-thioredoxin reductase: A catalytically active dithiol group links photoreduced ferredoxin to thioredox- in functional in photosynthetic enzyme regulation. Arch. Biochem. Biophys., 256 (1987) 372-380.

14 C. Bordier, Phase separation of integral membrane pro- teins in Triton X114 solution. J. Biol. Chem., 256 (1981) 1604-1607.

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