Eur. J. Biochem. 171,95-100 (1988) 0 FEBS 1988

Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex I1 in chloroplast photosynthetic membranes John BENNETT, Elizabeth K. SHAW and Hanspeter MICHEL Biology Department, Brookhaven National Laboratory, Upton, New York

(Received September 28, 1987) - EJB 870968

The light-harvesting chlorophyll a/b complex (LHC 11) and four photosystem I1 (PS 11) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS I1 proteins, LHC I1 is phosphorylated only when the cytochrome b6 f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6- isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC I1 phosphorylation in spinach thylakoids. (2 ) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6 f complex, phosphorylates the four PS I1 proteins but fails to phosphorylate LHC I1 in vivo or in vitro.

The chloroplast photosynthetic membranes, or thyla- koids, contain more than ten phosphoproteins, of which the two most conspicuous are the 25 -27-kDa LHC I1 [l] and the 8.3-kDa psbH gene product [2]. The herbicide diuron blocks electron transport between PS I1 and PQ and concomitantly inhibits phosphorylation of thylakoid proteins [3]. This suggests that a component of the electron transport chain after PS I1 must be reduced before the thylakoid-bound pro- tein kinase is activated. Several studies have provided evidence that PQ itself is the key redox component. (a) A series of single-turnover flashes progressively reduced the PQ pool and in parallel activated LHC I1 phosphorylation [4]. (b) Electron donors which fed into the PQ pool activated LHC I1 phosphorylation in the dark [3 - 51. (c) Redox titrations in the dark at pH 7.5-7.8 indicated that kinase activation is controlled by a two-electron carrier with an Em of 0 mV [6], + 55 mV [7] or + 80 mV [S]. These results are consistent with the involvement of PQ, which is a two-electron carrier with an Em of +50 mV at pH 7.5 [9]. (d) No electron transport component after the PQ pool appeared to be involved in kinase activation, because the PQ antagonist DBMIB (1 pM) can block electron flow from PQHz to the cytochrome b6f complex without inhibiting LHC I1 phosphorylation [4]. The fact that higher concentrations of DBMIB (> 3 pM) inhibited LHC I1 phosphorylation was attributed [4] to a secondary site of action of DBMIB within PS TI, at which DBMIB diverted electrons away from PQ to 0 2 [lo].

Recently we re-investigated the secondary site of DBMIB action within PS I1 [ll]. We found that addition of ascorbate

Correspondence to J. Bennett, Biology Department, Brookhaven National Laboratory, Upton, New York, USA-1 1973

Abbreviations. Chl, chlorophyll; DBMIB, 2,5-dibromo-3-methyl- 6-isopropyl-p-benzoquinone; DNP-INT, the 2,4-dinitrophenyl ether of iodonitrothymol; DQHZ, duroquinol; HQNO, 2-(n-heptyl)-4- hydroxyquinoline N-oxide; LHC, light-harvesting chlorophyll a/b complex; PQ, plastoquinone; PS, photosystem.

to DBMIB-poisoned thylakoids restored PS I1 protein phosphorylation, even in the presence of diuron. Presumably, reduced DBMIB was acting as an electron donor to the PQ pool. However, phosphorylation of LHC I1 and two other proteins (12 and 54 kDa) remained substantially inhibited by DBMIB/ascorbate. These results are difficult to understand in terms of a single PQ-controlled protein kinase but they are entirely consistent with the growing body of evidence which suggests that LHC I1 and the psbH protein are probably phosphorylated by different kinases. In addition to being more sensitive than PS I1 phosphorylation to DBMIB [ll, 121, LHC I1 phosphorylation is more sensitive to inhibition by fluorosulfonylbenzoyladenosine [12], hydrophobic thiol reagents such as N-phenylmaleimide [8] and exposure to photoinhibitory conditions [I 31. The difference in sensitivity to DBMIB is also displayed when thylakoids phosphorylate synthetic peptide analogs of the phosphorylation sites of LHC I1 and thepsbH protein [14].

Various explanations can be put forward for the sensitivity of LHC I1 phosphorylation to DBMIB. First, DBMIB may itself be acting as a hydrophobic thiol reagent, by virtue of the ability of its bromines to be displaced by protein-bound thlols [15]. As such it would be expected to inhibit LHC I1 phosphorylation preferentially. Second, LHC I1 phosphoryla- tion may be sensitive to inhibition by peroxides that can be generated by the autooxidation of DBMIBHz [lo, 161. Third, activation of LHC I1 phosphorylation may require the pres- ence of the cytochrome b6f complex and not simply the re- duction of the PQ pool. DBMIB is known to bind to the complex at the PQH2 oxidase site, in the vicinity of the Rieske FeS protein and the low-potential cytochrome b6, and prevent binding of PQHz [17, 181. Here we present evidence strongly favoring this third hypothesis, in agreement with the obser- vation that a mutant of Chlamydomonas reinhardtii lacking the cytochrome b6 f complex is unable to phosphorylate two out of four apo-proteins of LHC I1 [19].

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MATERIALS AND METHODS

Chemicals

DBMIB and DNP-INT were generously supplied by Prof. A. Trebst ; 2-(n-heptyl)-4-hydroxyquinoline N-oxide (HQNO) and duroquinone were obtained from Sigma. Duroquinone was reduced to duroquinol (DQH,) as described [5].

Plants

Spinach (Spinacia oleracea L. var. hybrid 424) was grown as described [20]. The corn (Zea mays) used in this study was the hcf6 mutant [21], which is propagated as the heterozygote. Seeds were generously provided by Dr D. Miles, University of Missouri, and were sown in pots containing a mixture of potting fiber and soil. The pots were placed in cardboard cylinders covered with neutral density filters so that the pho- ton flux density provided by continuous cool white fluorescent tubes was 25-30 pmol m-'s-l at plant height.

Identification of mutant seedlings

After 7 days each corn seedling was ringed with a label, and the tip (1 cm) of the primary leaf was removed for identifi- cation of mutants. Each segment was ground with 2 ml ice- cold buffer (50 mM Tris, pH 8.0, 5 mM NaKHP04, 5 mM MgC12, 5 mM NaF) in a cold mortar and pestle, the homog- enate was filtered once through damp Miracloth (Calbio- chem), the filtrate was centrifuged at 15 000 x g for 5 min at 4 "C, and the membrane pellet was thoroughly dispersed in 50 pl 0.1 M Na2C03, 0.1 M dithiothreitol and dissolved in 50 pl 4% (w/v) sodium dodecyl sulfate, 15% (v/v) glycerol and 0.05% bromophenol blue without heating. Homozygous mutants were identified as those which failed to give a detect- able cytochrome f band after sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and heme staining [22]. Wild-type and heterozygous seedlings gave a blue band at M, = 38000.

Spinach protein phosphorylation

Thylakoids were prepared from isolated, intact spinach chloroplasts as described [ll, 201. They were incubated at 50 pg chlorophyll/ml with [p3'P]ATP (20 pM, 2000 Ci/mol) in 100 pl buffer A (50 mM Tricine/NaOH, pH 8.0, 10 mM MgC12, 10 mM NaF, 200 pM phenylmethylsulfonyl fluoride, 1 mM 6-aminohexanoic acid, 1 mM benzamidine). Electron transport inhibitors were supplied in a total volume of less than 5 p1 methanol. Reactions were at 23°C for 4 min in darkness or illuminated at 50 pmol photon m-2 s-'. Reac- tions were terminated by addition of 1 ml 10% (w/v) trichloro- acetic acid. Pellets were washed with 5% trichloroacetic acid, resuspended in 50 pl NazC03/dithiothreito1 and 50 pl sodium dodecyl sulfate/glycerol/bromophenol blue and analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography.

Corn protein phosphorylation

Individual mutant and wild-type corn leaves were supplied with [32P]orthophosphate as described previously for barley leaves [20]. Thylakoids were isolated from labeled leaves and analyzed for phosphoproteins [20]. Protein phosphorylation in vitro was analyzed in thylakoids which had been isolated from 4- 8 mutant or wild-type seedlings (10 days old). Leaves

PC DNP-INT

Fig. 1. Schematic diagram of electron transfer in the cytochrome b6f complex. High-affinity sites of inhibition by DBMIB and DNP-INT are located at the PQH, oxidase site, whereas the high-affinity site of inhibition by HQNO is located at the PQ reductase site. PC, plastocyanin

were homogenized in buffer B (0.3 M sucrose, 25 mM Hepes/ NaOH, pH 7.6,2 mM EDTA, 5 mM MgClZ, 5 mM ascorbate) in a chilled mortar and pestle, the homogenate was filtered once through damp Miracloth and centrifuged at 5000 x g for 2 min. The crude chloroplast pellet was washed once in 10 ml buffer B (without ascorbate but containing 200 pM phenyl- methylsulfonyl fluoride, 1 mM 6-aminohexanoic acid, 1 mM benzamidine) and was resuspended in 2 ml buffer A and incu- bated at 250 pg chlorophyll/ml under phosphorylation con- ditions as described above for spinach thylakoids.

RESULTS

Inhibitor studies implicate the cytochrome b6f complex in LHC IIphosphorylation by spinach thylakoids

The ability of DBMIB to inhibit LHC I1 phosphorylation without inhibiting phosphorylation of PS I1 proteins such as the 8.3-kDa protein was investigated by comparing the effects of DBMIB with those of two other PQ antagonists, DNP- INT and HQNO (Fig. 1). DNP-INT resembles DBMIB in being a potent inhibitor of PQH2 oxidation by the cytochrome b6fcomplex [17, 18, 231 but differs from DBMIB in lacking potentially reactive bromine atoms and in lacking redox prop- erties. DNP-INT is therefore unable to act as a thiol reagent, or to divert electrons from PS I1 to oxygen with the generation of potentially toxic H20z. HQNO binds to the PQ reductase site of the cytochrome complex and inhibits a key step in the proposed Q-cycle, viz. the oxidation of cytochrome b6 by PQ

Fig. 2 shows the effects of DBMIB, DNP-INT and HQNO on phosphorylation of LHC I1 and the 8.3-kDa protein in illuminated spinach thylakoids. PS I1 was source of reductant for kinase activation, and ascorbate (5 mM) was added to maintain DBMIB in the reduced state and prevent inhibition of protein phosphorylation at a site in PS I1 [lo, 1 I]. LHC I1 phosphorylation was at least 50 times more sensitive than phosphorylation of the 8.3-kDa protein to inhibition by DBMIB and DNP-INT, while HQNO failed to inhibit phosphorylation of either protein. The 150 values for inhi- bition of LHC I1 phosphorylation by DBMIB and DNP- INT were 0.2 pM and 0.6 pM, respectively, at a thylakoid concentration of 50 pg chlorophyll/ml. Care was taken to ensure that incorporation into LHC I1 was approximately linear over the assay period ; non-linear kinetics resulted in over-estimation of Zs0.

[24 - 261.

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I A

0-

[inhibitor], pM

Fig. 2. Sensitivity of spinach thylakoidprotein phosphorylation to inhi- bition by DBMIB, DNP-INT and HQNO in the light. A thylakoid suspension containing 50 mM Tricine (pH KO), 10 mM MgCI2, 10 mM NaF and 5 mM ascorbate was treated with the indicated concentrations of inhibitors and then illuminated (50 pmol photon m - 2 - 1 s ) at 23°C for 4 min in the presence of 20 pM [Y-~~PIATP. Phosphorylation of LHC I1 (0 ) and the 8.3-kDa PS I1 protein (0) was determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by autoradiography and liquid scintillation counting of gel slices

We also examined the effects of DBMIB, DNP-INT and HQNO on LHC I1 phosphorylation in the dark in the pres- ence of 10 pM diuron and 0.5 mM duroquinol (DQH2). Under these circumstances, DQH2 reduces the PQ pool di- rectly and thylakoid protein phosphorylation proceeds with- out the involvement of PS I1 [5]. Nevertheless, as Fig. 3A shows, LHC I1 phosphorylation was again very sensitive to inhibition by DBMIB (Zso = 0.5 pM) and DNP-INT (Zs0 = 0.3 pM) but insensitive to HQNO. Repetition of these exper- iments indicates that the slight differences in Zso for each inhibitor between Fig. 2 and Fig. 3A are not significant.

The above results argue that phosphorylation of LHC I1 requires an active cytochrome b6 f complex. Furthermore, inhibitors of the PQH2 oxidase site (DBMIB and DNP-INT) abolish LHC I1 phosphorylation but an inhibitor of the PQ reductase site (HQNO) does not. In contrast, phosphorylation of PS I1 proteins does not appear to require an active cytochrome b6 f complex.

Of the two electrons which enter the cytochrome b6 f com- plex from each molecule of PQH2 at the PQH2 oxidase site, one is transferred initially to the Rieske FeS protein and thence to cytochromef, plastocyanin and PS I (Fig. 1). The other electron is transferred to the low-potential form of cytochrome b6 (cyt bL), then to the high-potential cytochrome b6 (cyt bH) and to PQ at the PQ reductase site, as envisioned by the Q-cycle hypothesis [27]. It could be argued that the role of the cytochrome complex in activating the LHC I1 kinase is

linhibitor I , pM

Fig. 3. Effects of electron transport inhibitors on LHC II phosphoryla- tion. (A) Sensitivity to DBMIB, DNP-INT and HQNO in the dark in the presence of duroquinol. A spinach thylakoid suspension con- taining 50 mM Tricine (pH 8.0), 10 mM MgCIZ, 10 mM NaF, 10 pM diuron, 0.5 mM DQH2 and the indicated concentrations of DBMIB (O) , DNP-INT (0) or HQNO ( A ) was assayed in darkness with 20 pM [y-32P]ATP for LHC I1 phosphorylation. (B, C) Sensitivity of LHC I1 phosphorylation to HQNO and DNP-INT in the presence and absence of a terminal electron acceptor. Thylakoids in 50 mM Tricine (pH KO), 10 mM MgC12, 10 mM NaF were treated with the indicated concentrations of HQNO (B) or DNP-INT (C) and then incubated in the light with 20 pM [Y-~'P]ATP in the absence (0 ) or presence (0) of 5 mM NADP and 5 pM ferredoxin. LHC I1 phosphorylation was assayed by sodium dodecyl sulfate/polyacryl- amide gel electrophoresis, followed by autoradiography and liquid scintillation counting

merely to catalyze the redox equilibration of the PQ pool via the Q-cycle. Alternatively, the LHC I1 kinase might be responsive to reduction of the cytochrome complex itself. Support for the second alternative is provided by a compari- son of the effects of DNP-INT and HQNO on LHC 11 phosphorylation in the absence and presence of the terminal electron acceptors, NADP and ferredoxin (Fig. 3 B, C). Ter- minal electron acceptors oxidize the intersystem electron car- riers and inactivate LHC I1 phosphorylation [4]. If a complete Q-cycle is required for activation of LHC I1 phosphorylation, addition of HQNO should merely reinforce the inhibitory effect of NADP and ferredoxin. However, as Fig. 3B shows, LHC I1 phosphorylation was completely restored by addition of 5 pM HQNO, a concentration which is known to block electron transfer from cytochrome b6 to PQ [24 - 261. In con- trast, DNP-INT, which prevents reduction of the cytochrome complex, has no such effect (Fig. 3 C). Higher concentrations of HQNO begin to inhibit phosphorylation, presumably be- cause of their ability to bind weakly at the PQH2 oxidase site

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Fig. 4. Phosphorylation of thylakoidproteins in detached maize leaves supplied with [ 3’P]orthophosphate for 150 rnin. Autoradiogram of sodium dodecyl sulfate/pol yacrylamide gel electrophoretic analysis of membrane proteins recovered from wild-type leaves (WT) and leaves of hcf6 mutant (hcf6). Lanes 1 and 2 are for two different wild-type or mutant seedlings. Numbers on the side are molecular mass in kDa

Fig. 5. Protein phosphorylation in isolated maize thylakoids. Auto- radiogram of sodium dodecyl sulfate/polyacrylamide gel electropho- retic analysis of thylakoids which were isolated from wild-type leaves (WT) and leaves of hcf6 mutant (hcf6) and then incubated with 50 mm Tricine (pH 8.0), 10 mM MgCI2, 10 mM NaF, 20 pM [Y-~~PIATP in the light in the absence (1) and presence (2) of 10 mM sodium dithionite. Numbers on the side are molecular mass in kDa

[25]. These results are again consistent with the idea that the cytochrome b6 f complex is involved in the activation of LHC I1 phosphorylation, and strongly suggest that the involvement is not merely structural but actually requires reduction of part or all of the cytochrome complex.

A corn mutant lacking the cytochrome b6f complex fails to phosphorylate LHC II

As mentioned in the introduction, a mutant of C. rein- hardtii which lacks the cytochrome b6f complex fails to phosphorylate two out of four LHC I1 apo-proteins in vivo [19]. To confirm the dependence of LHC I1 phosphorylation on the presence of the cytochrome b6 f complex, we examined a maize mutant (hcfq which lacks the complex but retains PS 11 and PQ [23]. Proteins were phosphorylated in vivo by supplying leaves with [32P]orthophosphate for 150 min and were then analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography (Fig. 4). Wild-type leaves incorporated 32P into LHC I1 and the four PS I1 phosphoproteins (8.3, 32, 34 and 44 kDa) but mutant leaves labeled the PS II proteins without labeling LHC 11. Labeling of two minor phosphoproteins (15 and 54 kDa) was also greatly inhibited in the mutant. The two genotypes contained equal amounts of LHC I1 as judged by staining of the gel (see also [23]).

The most obvious interpretation of this result is that LHC I1 phosphorylation is abolished in the absence of the cytochrome complex. However, we reported previously that dephosphorylation of thylakoid proteins is accelerated in a chlorophyll-b-less mutant [28]. Since the absence of 32P from LHC I1 apo-proteins in vivo in the two cytochrome b&f-de- ficient mutants [19] (Fig. 4) could in principle also be due to accelerated phosphatase activity, we examined thylakoid protein phosphorylation by mutant and wild-type corn plants in vitro. Thylakoids prepared from isolated intact chloroplasts

were incubated with [Y-~’P]ATP (Fig. 5), and NaF was in- cluded to prevent dephosphorylation. Again, LHC I1 and the 15-kDa and 54-kDa proteins were phosphorylated only in wild-type membranes. The 15-kDa and 54-kDa phosphopro- teins presumably correspond to the 12-kDa and 54-kDa spin- ach proteins whose phosphorylation, like that of LHC 11, is sensitive to DBMIB [ll]. In contrast, PS I1 proteins such as the 8.3 kDa protein were phosphorylated approximately equally in wild-type and mutant. Addition of dithionite (Fig. 5) or DQH2 (not shown) failed to activate LHC I1 phosphorylation in the mutant, even though these reductants are able to replace PS I1 in the activation of LHC I1 phosphorylation in normal membranes [4, 51.

The results obtained with maize wild-type and mutant thylakoids, like those obtained with spinach thylakoids, indi- cate an absolute requirement for the cytochrome b6 f complex during phosphorylation of LHC I1 but not during phosphor- ylation of PS I1 proteins.

DISCUSSION

Our results, together with data from the mutant of C. reinhardtii [19], establish that two distinct protein phosphor- ylation reactions occur in thylakoids. One phosphorylates LHC I1 and two other proteins and requires the operation of the cytochrome b6 f complex, while the other phosphorylates PS I1 core proteins and is independent of the cytochrome complex. It has been thought for some time that LHC I1 phosphorylation is responsive to changes in the redox state of the PQ pool [4-81. The present results are consistent with PQ control of PS I1 protein phosphorylation but suggest that control of LHC I1 phosphorylation involves the interaction of PQH2 with the cytochrome complex. Three levels of inter- action seem possible: (a) PQHz could activate LHC I1 phosphorylation simply by binding to the cytochrome com- plex (no electron transfer required); (b) if LHC I1 kinase is

99

activated by PQ-, the cytochrome complex could be the site at which the semiquinone is generated (transfer of one elec- tron); and (c) the kinase could be activated by reduction of cytochrome b6 or another component of the complex (transfer of both electrons from PQH2). Whether the kinase senses the reduction of the cytochrome complex or some form of localized transmembrane potential is not clear.

We cannot distinguish rigorously among these possibilities at present. It seems clear that the Rieske FeS center, cytochrome f or plastocyanin cannot be the crucial redox regulator of the kinase, because they are one-electron carriers and their mid-point potentials (> + 290 mV) [29] are much higher than the 0 to + 80 mV values determined for the redox regulator of the kinase [6 - 81. These carriers are also located on the luminal side of the thylakoid [30], whereas phosphorylation occurs on the stromal side [31]. Cytochromes bH and bL might also be excluded, because they are one- electron carriers with very low mid-point potentials. However, there is some controversy [29] concerning the redox behavior of cytochrome b6 in thylakoids. Bohme and Cramer [32] rec- orded an Em = + 5 mV and n = 2 for cytochrome b6 in freshly prepared thylakoids, values which changed to Em = -20 mV and n = 1 after aging of the membranes. On the other hand, a re-investigation of this phenomenon [33] yielded only low redox potentials and n = 1 for all thylakoid preparations. In isolated cytochrome b6 f complexes, cyt b, and cyt bH exhibited Em values of - 150 mV and - 50 mV, respectively, with n = 1 in both cases, but addition of HQNO changed these values to - 50 mV and + 50 mV, respectively, with n = 2 in both cases [34]. Given this variability in its redox behavior, it may be premature to eliminate cytochrome b6 (and especially cyt bH, which is located near the stromal surface of the thylakoids [30]) from the list of possible kinase regulators.

It has been postulated that the cytochrome b6 f complex contains a bound quinone (U) analogous to Qz of the bacterial cytochrome bcl complex [29]. From studies on thylakoids, Bouges-Bocquet [35] estimated that U has an Em about 40 mV higher than that of the PQ pool (i.e. about f 6 0 mV at pH 8). Isolated cytochrome b6 f complexes retain tightly bound quinone molecules (PQA and/or PQ2) which display an Em of +40 mV at pH 8.0. It may be that DBMIB and DNP-INT inhibit LHC 11 phosphorylation by preventing communi- cation between the PQ pool and the bound quinone(s). The involvement of a bound quinone may explain why some authors [36] have obtained data which are not completely consistent with control of LHC I1 phosphorylation by the entire PQ pool.

Does the involvement of the cytochrome b6 f complex re- quire a change of views about the role of LHC I1 phosphoryla- tion? Two major roles have been attributed to LHC I1 phosphorylation: balancing of energy distribution between the two photosystems [4,37 - 391 and regulation of the relative rates of cyclic and linear photophosphorylation to optimize the relative rates of ATP synthesis and NADP reduction [7, 381. Energy distribution would appear to be controllable quite adequately through either the PQ pool or the cytochrome complex, but cyclic and linear photophosphorylation would be much more convincingly regulated through the cytochrome complex than through the PQ pool. This follows from the fact that electron flow through the cytochrome complex is inhibited by a high transmembrane ApH [40], which is in turn often an indication that there is little demand for ATP and hence little requirement for cyclic flow. Conversely, a demand for additional ATP would be signaled by a decline in the ApH, which would enhance electron flow through the cytochrome

complex and lead (our data suggest) to increased LHC I1 phosphorylation. Detachment of phosphorylated LHC I1 from PS I1 cores would follow [37] and would favor cyclic ATP synthesis over linear ATP synthesis and NADP reduction by (a) diminishing the effective cross-section of PS I1 [37], thereby reducing electron pressure from PS I1 and improving the ‘poising’ of the cyclic pathway [38], and (b) by increasing the effective cross-section of PS I [37, 391. Thus, the involve- ment of the cytochrome b6 f complex would allow the redox control over LHC I1 phosphorylation to be modulated by changes in the ApH. We suggest that the discovery by Fernyhough et al. [41] that LHC I1 phosphorylation in maize protoplasts is stimulated by relaxation of the dpH across the thylakoids is most readily explained in terms of the involve- ment of the cytochrome complex in kinase activation.

Our data are consistent with the existence of two distinct redox-controlled protein kinases in thylakoids, one respon- sible for phosphorylating LHC I1 and two other proteins, and a second responsible for phosphorylating PS I1 proteins. In addition to differing in substrate specificity and in sensitivity to N-phenylmaleimide, 5‘-fluorosulfonylbenzoyladenosine and photoinhibition, the kinases also differ in their require- ment for the cytochrome b6 f complex. However, the finding [42] that antibodies raised against a 64-kDa thylakoid protein kinase block phosphorylation of all thylakoid proteins suggests that the LHC I1 kinase and PS I1 kinase are immunologically related. Alternatively, there may be a single kinase which changes many of its properties, including redox control and substrate specificity, when associated with the cytochrome complex.

The loss of LHC I1 phosphorylation during photoinhibi- tion [13] could be due to damage sustained by either the kinase or the cytochrome complex. It cannot be due to damage suffered by PS 11, because recovery of DQH2-driven LHC I1 phosphorylation occurs in the presence of chloramphenicol, while recovery of PS I1 integrity does not [13]. It would be interesting to know whether loss of function in the cytochrome b6 f complex is another feature of photoinhibition.

We thank Dr. D. Miles for the gift of corn seedlings, Prof. A. Trebst for the gift of DBMIB and DNP-INT, and Drs. P. Falkowski, T. Owens and A. Sutton for helpful discussions. The research was supported by the Office of Basic Energy Sciences, US Department of Energy, and by the Office of Competitive Grants, US Department of Agriculture.

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