Sep 19, 1975 - the Mnor water oxidation site in photosystem II (salicylaldoxime, dithizone ... The action of OP as a chelator of nonheme iron proteins in bacterial systems ..... salicylaldoxime inhibition between Y, and Y2 near the man-.
Plant Physiol. (1976) 57, 450-453
Organization of Electron Transport in Photosystem II of Spinach Chloroplasts According to Chelator Inhibition Sites1 Received for publication September 19, 1975 and in revised form November 21, 1975
RITA BARR AND FREDERICK L. CRANE Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 ABSTRACT The organization of electron transport in photosystem II of spinach
(Spinacia oleracea) chloroplasts was studied by means of various chelators and uncouplers. The partial reactions used induded H20-+methyl viologen, H20--silicomolybdic acid H20--ferricyanide, and H20--dimethylbenzoquinone. Three types of chelator inhibition were found (a) inhibition common to al pathways and presumably affecting the Mn or water oxidation site in photosystem II (salicylaldoxime, dithizone, acridine, 4,4,4-trifluoro-1-(2-thienyl)-1,1-butanedione, 4,4,4trifluoro-O-(2-furyl)-1,3-butanedione; (b) strong inhibition of the H20--silicomolybdic acid pathway in presence of 3(3,4-dichlorophenyl)1,1-dimethylurea by lipophilic chelators (bathocuproine, tertoctylcatechol) but stimulation by orthophenanthroline; and (c) 2,5-dibromo-3methyl-6-isopropyl-p-benzoquinone-insensitive dimethylbenzoquinone reduction inhibited by all phenanthrolines while ferricyanide reduction was remarkably stimulated by bathophenanthroline but inhibited by orthophenanthroline and bathocuproine. The action of lipophilic chelators on silicomolybdic acid reduction presumes the presence of a metallo protein in photosystem II. The differential action of bathophenanthroline on dimethylbenzoquinone and ferricyanide reduction indicated the possible existence of a metafloprotein in this pathway which is different from the site of orthophenanthroline inhibition.
Salicylaldoxime, a copper chelator, was usel in chloroplasts by Trebst (24) and Katoh and San Pietro (14). It proved to be ineffective as a Cu chelator of plastocyanin in photosystem I but inhibited a site in photosystem 11 (22). Chelator inhibition of ATPase activity has recently been investigated by Phelps and Crane (21) in mitochondria, Crane et al. (7) in Escherichia coli, and Bering et al. (6) in spinach chloroplasts. A preliminary study by Barr and Crane (4) using metal chelators on several partial PS I and PS II reactions tentatively found three chelator inhibition sites in PS II. This is a continuation of PS II chelator inhibition studies using a more diverse group of chelators and uncouplers than before. Again, we find three groups of chelators with different sites of action: (a) those which affect every partial PS II reaction and are presumably acting on the Mn site before P680; (b) those which show stronger inhibition in the water-1*silicomolybdate reaction before the DCMU block and which, therefore, act before or at Q, the primary electron acceptor for PS II; and (c) those which act after the DCMU block but before the DBMIB inhibition site and presumably inhibit at the PS II phosphorylation site. Since group b consisted of many copper chelators, the implication is that there is a Cu protein in PS II, located in the water--silicomolybdate pathway. MATERIALS AND METHODS
The effect of various chelators on photosynthetic electron transport goes back to Warburg and Luttgens (19) in 1946 and to Macdowall (17) who in 1949 used orthophenanthroline and 8hydroxyquinoline to inhibit the Hill reaction. Satoh (23) compared the inhibition pattern of many OP2 derivatives, including BP. Oettmeier and Grewe (19) found that the incorporation of a single nitrogen atom into an aromatic azaphenanthrene ring system led to inhibition of electron transport activity whereas an aromatic two ring system proved to be inactive. The effectiveness of 1,10-diazaphenanthrene as an inhibitor of electron transport in photosystem II was due to its chemical properties, as well as to its rigidity and planarity of the aromatic system. The action of OP as a chelator of nonheme iron proteins in bacterial systems has been studied by Jackson et al. (11) and in mitochondria by Harmon and Crane (9).
Chloroplasts were isolated from market spinach (Spinacia oleracea) in sucrose-NaCl medium (0.4 M sucrose with 0.05 M NaCl) by the method of Jagendorf and Avron (12). Chlorophyll was determined according to Arnon (2). Oxygen evolution by PS II of spinach chloroplasts was monitored with a Clark-type electrode in saturating white light at 25 C. Individual reaction mixtures are given in figure legends. The water cobaltinitrite reaction was done as described by Barr et al. (5). Lipophilic chelators, such as BP or BC, were dissolved in hot ethanol and added to reaction mixtures not to exceed 20 ,l of ethanol/1.5 ml. Corresponding amounts of ethanol were added to all controls.
RESULTS
Eight different types of chelators were tested on the well known PS I and II reaction H2O0-methyl viologen to determine the overall effects of these chelators on electron transport in spinach chloroplasts. As Figure 1 shows, this reaction was inhibited by these chelators to varying degrees. The most severe inhibition was shown by OP presumably located at or close to the DCMU inhibition site in PS II. BP and BC also inhibited this pathway at low concentrations. Acridine, TTFA, and FTFA required higher concentrations for 50% or more inhibition, while dithizone and salicylaldoxime, primarily known as copper
' Research was supported by National Science Foundation Grant 7419689. 2 Abbreviations: OP: orthophenanthroline; ACR: acridine; BC: bathocuproine; BP: bathophenanthroline; DBMIB: 2,4-dibromo-3-methyl-
6-isopropyl-p-benzoquinone; DITH: dithizone; DMBQ: 2,4-dimethylp-benzoquinone; DPIP: 2,6-dichlorophenolindophenol; FTFA: 4,4,4trifluoro-0-(2-furyl)-1,3-butanedione; MV: methyl viologen; PS I: photosystem I; PS II: photosystem II; SA: salicylaldoxime; TTFA: 4,4,4-
trifluoro-1-(2-thienyl)-1,3-butanedione. 450
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CHELATOR INHIBITION OF PHOTOSYSTEM II
and FTFA in moderately high concentrations like all the other PS II partial reactions. Likewise, it was hardly affected by salicylaldoxime or dithizone. DISCUSSION
The rationale behind using chelators as tools in investigations of photosynthetic electron transport lies in finding inhibition sites which reflect the presence of some metal, such as iron, copper, or manganese, presumably in conjunction with a protein. Thus in mitochondria, it is well established that several nonheme iron sites are a part of the electron transport chain (10) and that they can be correlated to chelator inhibition sites (9). The same is true for electron transport in photosynthetic bacteria (I 1) and E. coli (7). In bacterial systems, OP blocks electron transport from primary to secondary acceptors because it causes changes in the midpoint potential of the primary acceptor (11). Chelator inhibition of spinach chloroplasts is not so well understood, although a nonheme iron ESR signal has been recently reported by Malkin and Aparicio (18) in PS I between plastoquinone and Cyt f. In PS II, Oettmeier and Grewe (19), who studied azaphenanthrene inhibition, are convinced that at the DCMU inhi198 330 462 594 Chelotor Added (PM)
726
FIG. 1. Effect of chelators and uncouplers on reduction of methyl viologen by spinach chloroplasts. Oxygen uptake by MV was measured with a Clark-type electrode. Reaction mixture contained: chloroplasts (50 ug chl), 37.5 mm Trizma-MES, pH 7, with 20 AM NH4Cl and MgCl2, and 0.05 mg of methyl viologen. Chelators and uncouplers were added in ethanol in concentrations indicated. Control rate: 360 MLeq/mg Chl-hr.
0
SA
A
chelators, did not have much effect on the H20- methyl viologen reaction. For testing chelator inhibition near the water oxidation step of photosynthesis, the water-*silicomolybdate reaction which accepts electrons from Q before the DCMU block, proved to be valuable. It was found (Fig. 2) that this reaction in presence of DCMU was particularly sensitive to BC but not to OP or BP. TTFA, acridine, and FTFA were also good inhibitors but they required higher concentrations. Dithizone and salicylaldoxime, being more hydrophilic, proved less effective. Complete inhibition of the water silicomolybdate pathway was given by the lipophilic chelator, tertoctylcatechol, with concentrations double or higher than those indicated in Table I. It is known that without DCMU silicomolybdic acid accepts electrons further down the electron transport chain. If the water-*silicomolybdate reaction is run in presence of DBMIB to stop PS I reactions, a different chelator inhibition pattern is noted (Fig. 3). OP, BP, and BC in low concentrations stimulate this reaction. Best inhibitors of this reaction were acridine, TTFA, and FTFA. Salicylaldoxime or dithizone showed only slight inhibition as before. Since the reduction of ferricyanide by PS II is completely sensitive to DCMU, ferricyanide must accept electrons after the DCMU block. This portion of the electron transport chain was found to be inhibited by low concentrations of OP and BC. The most distinguished feature of this reaction was its stimulation by BP in freshly made chloroplasts (Fig. 4). A 2-fold increase of the rate was commonly encountered during the 1st hr. Thereafter the stimulation by BP decreased. The effect of other chelators on ferricyanide reduction was similar to those observed in the silicomolybdate pathway. Another PS II acceptor which accepts electrons just before DBMIB, the presumed plastoquinone antagonist, is dimethylbenzoquinone. The reduction of this quinone was most strongly inhibited by OP, BC, and BP (Fig. 5). This portion of the electron transport chain was again inhibited by acridine, TTFA,
DITH 25
TTFA
c
*FTFA
0
C
ACR
0
~~~A
0IR
BP
C~~~~~~~~*S OP
C
75
66132 Chelotor
196
264
3-30
Added (riM)
FIG. 2. Effect of chelators and uncouplers on silicomolybdate reduction in spinach chloroplasts. Oxygen evolution in presence of DCMU was measured with a Clark-type electrode. Reaction mixture as in Fig. 1 except 0.2 mg of silicomolybdic acid added in place of MV and 0.2 Mm DCMU was present. Control rate: 271 Aeq silicomolybdate reduced/mg Chl-hr.
Table I. Inhibition of Silicomolybdic Acid Reduction by Lipophilic Chelators in Photosystem II of Spinach Chloroplasts Rate of Silicomolybdic Acid Reduction
peq/mg Chl-hr 203 197 214 192 214
Concn of Chelator Added AddCeao Chelator
Bathocuproine Tertoctylcatechol 8-Quinolinol Dithizone Salicylaldoxime
Inhibition
PM
%
3 40 200 400 1000
56 51 64 64 48
452
BARR'AND CRANE.
Plant Physiol. Vol. 57, 1976
bition site OP does not act as a chelator. Their conclusion is based on structural studies of various mono- and diazaphenanthrene derivatives. Satoh (23), who found that 4,7-dimethyl1,10-phenanthroline was the most potent inhibitor of DCIP photoreduction and fluorescence induction at room temperature, is of the opinion that if a metal ion is involved in the inhibition it is not present as a hydrated ion in the chloroplast
SA
DITH TTFA FTFA ACR BP BC
0
OP
0
z
c
Chelotor Added (1,uM)
FIG. 5. Effect of chelators and uncouplers on dimethylbenzoquinone reduction by PS II of spinach chloroplasts. Oxygen evolution was measured with a Clark-type electrode in presence of DBMIB. Reaction mixture as in Fig. 1, except MV was replaced by 1 iLmole of DMBQ. Control rate: 328 Aeq dimethyobenzoquinone reduced/mg Chl-hr. Chelotor Added FIG.
3.
Effect
of chelators
and
(AM)
uncouplers
on
silicomolybdic
acid
reduction by PS II of spinach chloroplasts. Oxygen evolution in presence of DBMIB was measured with a Clark-type electrode. Reaction mixture as in Fig. 2; DCMU replaced by 0.2 ,UM DBMIB. Control rate: 231 leq silicomolybdate reduced/mg Chl-hr.
+100[ A
o +50
SA
*
DITH TTFA
*
FTFA
o
ACR
*
A& BP
C-)
0
BC
o
OP
0 r-
Eo
Io
4-
c
0
._ c
50
'
132
264 396 528 660
Chelotor Added
(JMM)
FIG. 4. Effect of chelators and uncouplers on ferricyanide reduction by PS II of spinach chloroplasts. Oxygen evolution in presence of DBMIB was measured with a Clark-type electrode. Reaction mixture as in Fig. 1, except MV replaced by 2.5 Mmoles of ferricyanide. Control rate: 293 ,ueq ferricyanide reduced/mg Chl-hr.
membranes or is attached by some coordinate bonds to protein with a low stability constant because of studies of reversal of chelator inhibition by metal ions. BP, if it reacts with a metal ion, would cause the coordination of 2 molecules of ligand at a reactive site, whereas OP would favor monomolecular coordination but still cause an inhibition. According to Oettmeier and Grewe (19) OP, despite its 2 spacially separated nitrogen atoms, is monobasic and forms a monohydrochloride and a mono-Noxide. Thus, although in this study OP strongly inhibited the H20-I+methyl viologen pathway (Fig. 1), the H20-.dimethylbenzoquinone pathway (Fig. 5), and ferricyanide reduction by PS II (Fig. 4), it does not necessarily implicate the presence of a nonheme iron protein at its inhibition site. On the other hand, the strong BP inhibition noted in the water-+methyl viologen (Fig. 1) and in the water dimethylbenzoquinone pathway (Fig. 5) may reflect the presence of nonheme iron proteins at phosphorylation sites in PS I and II. Evidence for the presence of a nonheme iron protein between plastoquinone and Cyt f by ESR studies has recently been provided by Malkin and Aparicio (18). The most interesting and unexpected chelator inhibition site in PS II has been provided by studies of the water-+silicomolybdate pathway which is mostly DCMU-insensitive suggesting that silicomolybdic acid is able to accept electrons from Q, the primary electron acceptor in PS II. Silicomolybdate reduction was found to be sensitive to chelators, especially those of lipophilic nature, such as BC (Fig. 2) and tertoctylcatechol (Table I). Salicylaldoxime, found by Katoh and San Pietro (14) to give complete inhibition of the Hill reaction with DPIP, was not a very effective chelator in this study. Postulations about its site of action in PS II vary. Renger et al. (22) came to the conclusion that salicylaldoxime inhibited a plastocyanin-independent step in the complete electron transport chain or in isolated PS II particles. Katoh (13), Kimimura and Katoh (16), and Katoh et al. (15) place salicylaldoxime inhibition between Y, and Y2 near the manand the water oxidation sites. This is the same site where data from this study would allow us to locate salicylaldoxime, dithizone, TTFA, acridine, and FTFA inhibition. At least, this
ganese
Plant Physiol. Vol. 57, 1976
CHELATOR INHIBITION OF PHOTOSYSTEM II
type of inhibition which varies from strong with acridine to weak with dithizone and salicylaldoxime is found in all of the partial electron transport reactions tested in this study. This means that these chelators and uncouplers are able to disturb the Mn or the water oxidation site in PS II. The presence of a Cu2+ ion in the silicomolybdic acid pathway in chloroplasts would not be inconsistent with the data by previous workers. Park and Pon (20) found 3 Cu atoms per Mn in the nonlipid portion of their lamellar unit. Anderson et al. (1) found nearly equal amounts of Cu in PS I and PS II particles isolated by digitonin fractionation of chloroplasts. Renger et al. (22) were forced to conclude from studies of salicylaldoxime inhibition in whole chloroplasts compared to an isolated PS II cycle that the action of this compound was the same in either case. This meant that salicylaldoxime inhibited a plastocyaninindependent step in PS II. Katoh and San Pietro (14) also obtained complete inhibition of the Hill reaction with DPIP using 10 mm salicylaldoxime. In this study we found that tertoctylcatechol and BC gave better inhibition of the proposed Cu2+ site in PS II than salicylaldoxime (Table I). Extreme sensitivity of the water->silicomolybdate pathway to inhibition by ethanol and other alcohols has been found in this study. This sensitivity may have to do with the generation of an ethanol cation radical under conditions not well understood at present, as was found by Harbour and Tollin (8) at low temperatures when Chl sensitized the production of a quinone anion radical and an ethanol cation radical in a one-electron transfer from water. LITERATURE CITED 1. ANDERSON, J. M., N. K. BOARDMAN, AND D. J. DAVID. 1964. Trace metal composition of fractions obtained by digitonin fragmentation of spinach chloroplasts. Biochem. Biophys. Res. Commun. 17: 685-689. 2. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenol-oxidase in Beta vulgaris. Plant Physiol. 24: 1-15. 3. BANASZAK, J., R. BARR, AND F. L. CRANE. 1975. Evidence for multiple sites of ferricyanide reduction in chloroplasts. J. Bioenerget. In press. 4. BARR, R. AND F. L. CRANE. 1974. Chelator-sensitive sites in chloroplast electron transport. Biochem. Biophys. Res. Commun. 60: 748-755.
453
5. BARR, R., D. RosEN, AND F. L. CRANE. 1975. New ionic redox agents for the study of photosynthesis. Proc. Indiana Acad. Sci. 84: 147-159. 6. BERING, C. L., JR., R. A. DILLEY, AND F. L. CRANE. 1975. Inhibition of the membrane-bound Mg++-ATPase of chloroplasts by lipophilic chelators. Biochem. Biophys. Res. Commun. 63: 736-741. 7. CRANE, R. T., 1. L. SUN, AND F. L. CRANE. 1975. Lipophilic chelator inhibition of electron transport in Escherichia coli. J. Bacteriol. 122: 686-690. 8. HARBOUR, J. R. AND G. TOLLIN. 1974. ESR evidence for chlorophyll-photosensitized oneelectron oxidation of water by benzoquinone. Photochem. Photobiol. 20: 271-277. 9. HARMON, H. J. AND F. L. CRANE. 1974. Inhibition of cytochrome c oxidase by hydrophobic metal chelators. Biochim. Biophys. Acta 268: 125-129. 10. HATEFI, Y. 1963. The pyridine nucleotide-cytochrome c reductases. In: P. Boyer, H. Lardy and K. Myrback, eds., The Enzymes, Vol. 7. Academic Press, New York. pp. 495-515. 1 1. JACKSON, J. B., R. J. COGDELL, AND A. R. CRoFrs. 1973. Some effects of o-phenanthroline on electron transport in chromatophores from photosynthetic bacteria. Biochim. Biophys. Acta 292: 218-225. 12. JAGENDORF, A. T. AND M. AVRON. 1958. Cofactors and rate of photosynthetic phosphorylation by spinach chloroplasts. J. Biol. Chem. 231: 277-290. 13. KATOH, S. 1972. Inhibitors of electron transport associated with photosystem II in chloroplasts. Plant Cell Physiol. 13: 273-286. 14. KATOH, S. AND A. SAN PIETRO. 1966. Inhibitory effect of salicylaldoxime on chloroplast photooxidation-reduction reactions. Biochem. Biophys. Res. Commun. 24: 903-908. 15. KATOH, S., K. SATOH, I. IKEGAMI, M. KIMIMuRA, AND A. TAKAMIYA. 1971. Electron transport system associated with oxygen evolution in chloroplasts. In: G. Forti, M. Avron, and A. Melandri, eds., Reaction sites of inhibitors and electron donors. Proc. 2nd Int. Cong. on Photosynthesis., Vol. I. Stresa, Italy. pp. 525-537. 16. KIMIMURA, M. AND S. KATOH. 1972. On the functional site of manganese in photosynthetic electron transport system. Plant Cell Physiol. 13: 287-296. 17. MACDOWALL, F. D. H. 1949. The effects of some inhibitors of photosynthesis upon the photochemical reduction of a dye by isolated chloroplasts. Plant Physiol. 24: 462-480. 18. MALKIN, R. AND P. J. APARICCO. 1975. Identification of a g = 1.90 high-potential iron sulfur protein in chloroplasts. Biochem. Biophys. Res. Commun. 63: 1157-1160. 19. OEIrMEIER, W. AND R. GREWE. 1974. Inhibition of photosynthetic electron transport by azaphenanthrenes. Z. Naturforsch. 29c: 545-551. 20. PARK, R. B. AND N. G. PON. 1963. Chemical composition and the substructure of lamellae isolated from Spinacea oleracea chloroplasts. J. Mol. Biol. 6: 105-114. 21. PHELPS, D. C. AND F. L. CRANE. 1974. Lipophilic chelator inhibition of mitochondrial membrane-bound ATPase activity and prevention of inhibition by uncouplers. Biochem. Biophys. Res. Commun. 61: 671-676. 22. RENGER, G., J. VATER, AND H. T. WrI-r. 1967. Effect of salicylaldoxime on the complete electron transport system of photosynthesis and on the isolated reaction cycle II. Biochem. Biophys. Res. Commun. 26: 477-480. 23. SATOH, K. 1974. Action of some derivatives of 1,10-phenanthroline on electron transport in chloroplasts. Biochim. Biophys. Acta 333: 127-135. 24. TREBST, A. 1963. Zur Hemmung photosynthetischer Reaktionen in isolierten Chloroplasten durch Salicylaldoxim. Z. Naturforsch. 18b: 817-821.
