Light-dependent Inhibition of Photophosphorylation by N-EthyImaleimide*

SUMMARY The treatment of chloroplasts with 1 mM N-ethylmaleimide (NEM) in the light, but not in the dark, resulted in a partial, permanent inhibition of photophosphorylation. Noncyclic electron flow coupled to phosphorylation was also inhibited on incubation of chloroplasts with NEM in the light, but this inhibition was reversed by ammonium chloride. The uncouplers, carbonyl cyanide m-chlorophenylhydra-zone and ammonium chloride, largely prevented the inhibition. Adenosine triphosphate and adenosine diphosphate partially protected phosphorylation from inhibition by light and NEM. Light-induced hydrogen ion uptake was unaffected by NEM except that the uptake stimulated by adenosine triphosphate was inhibited. The calcium-dependent adenosine triphosphatase of coupling was also inhibited in chloroplasts treated with NEM in the light prior to extraction of the coupling It is conformational the of of


END \'OKO TSUCHIYA
From the Section of Biochemistry and Xolecular Biology, Cornell University, Ithaca, Sew York I.4850 SUMMARY The treatment of chloroplasts with 1 mM N-ethylmaleimide (NEM) in the light, but not in the dark, resulted in a partial, permanent inhibition of photophosphorylation. Noncyclic electron flow coupled to phosphorylation was also inhibited on incubation of chloroplasts with NEM in the light, but this inhibition was reversed by ammonium chloride. The uncouplers, carbonyl cyanide m-chlorophenylhydrazone and ammonium chloride, largely prevented the inhibition. Adenosine triphosphate and adenosine diphosphate partially protected phosphorylation from inhibition by light and NEM.
Light-induced hydrogen ion uptake was unaffected by NEM except that the uptake stimulated by adenosine triphosphate was inhibited.
The calcium-dependent adenosine triphosphatase of coupling factor 1 was also inhibited in chloroplasts treated with NEM in the light prior to extraction of the coupling factor.
It is suggested that light causes a conformational change in the membrane-bound coupling factor, allowing the reaction of a group (or groups) with NEM.
The sensitivity of the inhibition to adenine nucleotides suggests that these nucleotides modify the conformation of coupling factor 1 in the light.
We recently reported (1) that low concentrations of ATP enhance the estent of light-dependent H+ uptake (2) in spinach chloroplasts.
Because this stimulation was sensitive to an an& serum to the chloroplast coupling factor 1, we postulated that ATP modifies the conformation of CFQ on the membrane, \&ich in turn reduces the permeability of chloroplast membranes to H+. Ryrie and Jagendorf (3)  Only about 2 of the S to 10 -Sll groul)s iI1 CF1 are accessible to sulfhydryl reagent,s such as iodoncet,amide and dithiobisnitrobenzoic acid (4). Furthermore, the heat-actir-a.ted, CtiLf-dependent AYYPase of CF1 is resistant to iodoacetamide, but is slowly inhibited by AT-ethylmaleimide. A light-induced con-formations1 change in CF1 might cause the esposure of a sulfhydryl group which in the dark is not accessible to NEAL If the exposed group were required for CF, activity, its react,ion with NE;11 would be expected to inhibit phosphorylstion.
In this paper, we report that light ma.rkedly enhances the inhibition of phosphorylation by NE?\I. The characteristics of this inhibition are consistent with the concept that light causes a conformational change in CF1 which is influenced by adenine nucleotides.
The s:~mplrs were illuminated at room temperature for 2 min with 2 x 10" ergs per cm2 per s of white light. Phospliorylatioi~ coul1led to methyl viologen reduction was assayed in the same renctioll nrirture (1.75 nil), except that pyocyanine was replaced with 0.1 mnr methyl viologen, and 1 nl h% NaN3 was present.
Oxygen up take dependent on methyl viologen reduction with water as the electron donor was followed polarographically with a Clark-type electrode.
The Cat+-dependent ATPase of CFI was acti\vat,ed with either trypsin (7) or with DTT (8). The procedure of Vambutas and Racker (7) was used to assay ATPase activity, except that 1'; was determined by the method of Taussky and Shorr (9).
In most experiments, chloroplasts were incubated at 20" \vith NE&I at a chlorophyll concentration of 0.33 mg per ml in a iZfter a timed period in the light (2 x lo6 ergs per cm2 per s) or the dark, an amount of DTT calculated on the basis of its -SH content to be about 10% in excess of the amount of NEM present was added. Aliquots of the mixtures were either assayed directly for photophosphorylation or were further treated prior to assay as described in the legends to the figures and tables.

Inhibition of Phosphorylation by NEX-Although
the illumination of chloroplasts in the absence of NEXL for 1 min did not inhibit phosphorylation, illumination in the presence of 5 nlhf NEM severely inhibited it (Table I). Much less inhibition was observed in chloroplasts treated with NEAT in the dark, and this inhibition, but not that observed in the light, was abolished by the addition of DTT to remove the NEXI prior to the assay of photophosphorylation.
Thus, the inhibition due to NlGM treatment in the dark was probably caused by the presence of NEM during the phosphorylation assay. Further, since DTT did not reverse the light-dependent NE11 inhibition, it is appa.rent that the inhibition is irreversible. This was confirmed by washing the chloroplasts.
DTT, added to the incubation mixtures before the chloroplasts, prevented the inhibition.
The inhibition of phosphorylation by NEXI in the light was partial, ranging from 35 to 70%. As seen in Fig. 1, increasing the NEM concentration above 1 mM did not markedly augment the inhibition.
Furthermore, the onset in the light of the NEM inhibition of phosphorylation was fairly rapid, nearly reaching completion in about 60 s. Phosphorylation coupled to the reduction of methyl viologen with water as the electron donor was also inhibited by the treatment with NEM in the light, but not in the dark (Table II). Light and NEM also attenuated methyl viologen reduction assayed in the presence of XDP and Pi. This inhibition was reversed by NH&l, suggesting the inhibition by NEM and light is of the energy transfer type. However, electron flow assayed in the absence of ADP was stimulated by the incubation of the incubation of the chloroplasts with NEM in the light. For example, whereas phosphorylating electron flow was inhibited 20% by the light and NEM treatment, electron flow in the absence of ADP was stimulated by 33%. Thus, the NEM inhibition is distinct from that of the energy transfer inhibitors Dio-9 (10) and phlorizin (II), which have little effect on electron transport in the absence of ADP.
Uncoupling concentrations of NH&l and CCP protected chloroplasts from the action of NEM in the light (Table III) Chloroplasts were treated with the indicated concentrat.ions of NEM as described in Table II  Chloroplasts equivalent to 1 mg of chlorophyll were exposed to 1 mM NEM for 90 s in 3 ml of the standard incubation mixture, which also contained the indicated concentrations of NH&l or CCP. At the end of the incubation, 0.6 mM DTT was added and the mixtures centrifuged at 10,000 X g for 10 min. The pellets were resuspended in 5 ml of a solution which contained 0.4 M sucrose, 0.02 SC tris(hydroxymethyl)methylglycine-NaOH (pH 8), and 0.01 M N&l.
After centrifugation as above, the pellets \vere resuspended in a small volume of the buffered sucrose solution and pyocyanine-dependent phosphorylation determined.
Phosphorylation is given as /Imoles of Pi esterified per hour per mg of chlorophyll. , NEhI inhibition is related to the generation of the high energy state.

Protection of Phosphorylation from NEJI Inhibition by Adenine iliucleofides--Y1'1'
and ADP reduced the inhibition of phosphorylation elicited by NEM and light, whereas their presence during the incubation of chloroplasts with NE11 in the dark had little effect on phosphorylation (Table IV). Under phosphorylating conditions, the inhibition by NEM was minimal. Pi was shown in other experiments to have no effect on the inhibition.
As little as 5 PM ATP partially prevented the inhibition of phosphorylation by 1 mM NEM and light and saturation was achieved at about 20 pM.
For example, 5 pM ATP reduced the inhibition from 64 to 55% and 50 pM ATP reduced the inhibition to 31%. At similar concentrations, ATP stimulates Hf uptake (1) and inhibits electron How (12). UTP, GTP, CTP, and ITP (50 PCJZ) did not diminish the light-dependent phosphorylation inhibition by x;EM. Eflects of 1VEJl on IIf Uptake-Light-induced II+ uptake, assayed at pH 7.6, was little affected by the prior treatment of chloroplasts with NISI in the light. H+ uptake in chloroplasts illuminated for 90 s in the presence of 1 mnz NEM prior to assay was found in one experiment to be 0.18 peq per mg of chlorophyll, whereas that in chloroplasts incubated with 1 mM NE1\2 in the dark was 0.16. In contrast, II+ uptake assayed in the presence of 20 pcll"~ ATP was partially sensitive to the light and NEM treatmellt.
The stimulation by ATP of II+ uptake in chloroplasts t,reated with NEM in the light was only 54oj,, lyhereas that in chloroplasts incubated in the dark with NE1\1 \YtlS 1315°C. It would appear, t,herefore, that the ATP-stimulated II+ upt,ake is preferentially inhibited by incubation of chloroplnsts wit,h NEM in the light.

Evidence jar Interaction of NEX and Chloroplast Coupling
Facfor l-The sensitivity of the NEM inhibition to adenine nucleotides suggested that CF1 may be the site of the NEM inhibition.
To test this idea, chloroplasts were treated with NEM in the usual way and the Ca2+-dependent ATPase ac- To the s:~nplcs which contained NEM, DTT (0.6 mnl) \vas added at the end of the 2-min incubation in the light or the dark. The mixtures were centrifuged at 10,000 X g for 10 min, the precipitate resuspended in 10 ml of 10 rnM NaCl, and the chloroplasts collected by centrifllgation at 10,000 X g for 10 min.
The pellets were resuspended in 40 ml of 0.75 mM EDTA (pH 7) at room temperature, and, after 10 min, the mixture was centrifuged at 35,000 X g for 30 min.  (Table V, Experiment  II). DISCUSSION The inhibition of photophosphorylation by NEM may be traced to an irreversible reaction of the NEM with the CFI on the thylakoid membranes. This conclusion is supported by several liues of evidence, including the observation that the t,reatment of chloroplasts with NEbI in the light causes an inhibition of t,he Ca*+-ATPase of CFI. The light requirement for the inhibition may be understood if it is assumed that CFI 011 the thylakoid membranes undergoes a conformational chauge on illumination.
This conformational change could render a group(s) (possibly -SH) accessible to NEM, and, if this group is required for CF1 activity, its reaction with NEM would inhibit phosphorylation.
The light requirement for the inhibition is apparently related iu some manner to energy conservation, since the inhibition was abolished by uucouplers and by ADP and Pi. Ryrie and Jagendorf (3) showed that the light-induced conformational change in CFI, as detected by incorporation of 3H20, was diminished by uncouplers.
If it is accepted that the light potentiation of the NEM inhibition of phosphorylation is caused by light-dependent changes in CF1 conformation, then the fact that XTP aud ADP reduce the esteut of inhibitiou may be taken as evidence that these nucleotides modify the structure of CFI in the light.
Such a role for ATP was postulated to explain its stimulation of H+ uptake in chloroplasts (1). Similar low concentrations of ATP were required to protect from NEM inhibition and stimulate H+ upt,ake. ;\loreoyer, other nucleoside triphosphates were ineffect,ive in either blocking the NEI\I inhibition or stimulating H+ uptake (1).
Other -SII reagents have long been known to inhibit photophosphorylation (13,14), but a light dependence for their illhibitory activity has not been previously reported. Izawa and Good (15) established that p-chloromercuribenzoate, added directly to the reaction mixtures, is an energy transfer inhibitor. Hg*+ also i&ibit.ed phosphorylat.iou and coupled electron flow, but was found to stimulate electron flow under nonphosphorylating conditions. This stimulation was abolished by low conceutrationr of 1TI' .
. Similar results were obtained in chloroplasts treated with NEi\I in the light.
The inhibition of phosphorylation by treatment with NEM aud light, p-chloromercuribenzoate (15), or light plus ADP and sulfate (16) leveled off at, about 50%. It is clear that all three treatments affect in some way t.he activity of bound CF,. Extract.ion of about 50% of the CFI from chloroplast membranes cau inhibit phosphorylation completely (5). Yet phosphoryla-tion may be partially restored to these depleted particles by dicyclohexylcarbodiimide, indicating that the residual CF1 in the particles may participate in phosphorylation.
It is possible, therefore, that there is an excess of CF, in chloroplasts and that only about half of the CF1 in a populat.ion of chloroplasts uudergoes conformational changes and participates in photophosphorylation.
Thus, since NEM may react only with CF1 which has altered its conformation in the light, a 50 y0 inhibition lvould be expected.
Murakami (17) calculated that there may be as many as 1 CFI molecule for every 100 chlorophyll molecules, a value in excess of most of the components of the electrou transport chain.
Preliminary experiments with [%]NEhI have iudicuted &at more NEM is incorporated into a protein, ertract~able wit,11 EDTA, but not with 10 I~&I NaCl, in chloroplasts illuniinat.ed in the presence of NE:h/I thau in those kept iu the dark. Since CFI is certainly the major protein in ISDTX extra& of chloroplasts, it is probable that the [3H]NEM is associated wit.1~ CF,.
If it is established that there is a light-depeudent and uncouplersensitive reaction of NEM with CF, it will be of int#ereat t,o identify the functional group which reacts with NE11 and, further, to localize this group within the subunit struct,urc of CF,.