Electron Flow Through Plastoquinone and Cytochromes B6 and F

Home , Plastoquinone

Proc. Nati. Acad. Sci. USA Vol. 76, No. 6, pp. 2765-2769, June 1979 Botany Electron flow through plastoquinone and cytochromes b6 and f in chloroplasts (chemosmotic hypothesis/photophosphorylation/quinone-cytochrome b-c oxidoreductase/proton translocation/plastocyanin) B. R. VELTHUYS Martin Marietta Laboratories, 1450 South Rolling Road, Baltimore, Maryland 21227 Communicated by Bessel Kok, March 26, 1979

ABSTRACT With dark-adapted chloroplasts in which the MATERIALS AND METHODS plastoquinone was oxidized, a partial reduction of cytochrome Chloroplasts were prepared according to ref. 7. They were be was obtained upon illumination with a pair of short satu- of rating flashes. The second flash of the pair was much more ef- stored as a concentrated suspension (5 mg chlorophyll per fective than the first, and the reduction was inhibited by the ml) in the dark at 0C. They were permeable to ferricyanide, system II inhibitor diuron. When the plastoquinone pool was as was checked by fluorescence induction measurements. For reduced, both the reduction and the oxidation of cytochrome some experiments (Fig. 2) the chloroplasts were osmotically be were accelerated. The cytochrome be oxidation appeared to shocked by suspension in distilled water (at 0C) followed by proceed in association with the reduction of cytochrome f, al- the addition of concentrated isolation medium. This treatment though these cytochromes are not simply connected in series. caused no qualitative changes in the behavior of the chloroplasts From these observations it is inferred that electron flow to the in experiments, but decreased scattering and accelerated secondary donors of system I alternately causes the reduction the and the oxidation of cytochrome be. An interpretation is offered the oxidation of system I donors by ferricyanide. that also accounts for the transmembrane proton translocation Flash-induced absorbance changes were measured as in ref. that is associated with the oxidation of plastohydroquinone. 1. For Figs. 3 and 4, a Fabritec 1052 signal averager was used. The reaction mixture used in the measurements contained 200 In a preceding paper (1) it was shown by spectroscopic analysis mM sucrose, 10 mM 3-(N-morpholino)propanesulfonic acid that the oxidation of plastohydroquinone by system I is associ- (Mops)/5 mM NaOH (pH -7.6), 5 mM NaCl, 2 MiM gramicidin ated with the generation of a transmembrane field and proton D, and 100 uM methylviologen or ferricyanide (50-500 MM). uptake. This and other results confirmed a previous observation Before each measurement, 5 ,ul of chloroplast suspension was in this laboratory, by means of pH measurements, that electron diluted in the dark to a final chlorophyll concentration of 100 flow through the chain connecting systems II and I is associated jig per ml. The optical pathlength was 2 mm. Spectral half- with the translocation across the thylakoid membrane of two bandwidth was 3.2 nm. protons per electron (2). The extents of cytochrome redox changes were calculated These results imply that the reaction chain between photo- by using the following differential extinction coefficients (ab- systems II and I is quite complicated both mechanistically and sorbance per mM per cm, from reduced-minus-oxidized - = structurally. It then seems no surprise that these reactions limit spectra): cytochromef, e (553 nm 540 nm) 18 (ref. 8); cy- - = the maximal rate of the photosynthetic process. tochrome b6, f (563 nm 570 nm) 14 (ref. 9). The literature shows a great deal of confusion concerning the RESULTS role of the various cytochromes in electron transport of chloroplasts (3, 4). A simple but strong argument that they do play Dependence of Cytochrome b6 Reduction on System II. a crucial role is that the capacity for high photosynthetic rates In the experiment of Fig. 1, chloroplasts were used that had as seen in sun vs. shade leaves correlates with a high content of been kept in the dark (at 00C) for several hours. This dark ad- cytochromes f and b (5). aptation causes oxidation of the plastoquinone pool, but not of This paper brings evidence that, as hinted at by Mitchell (6), electron donors closer to system I, such as cytochrome f and cytochromes and f are involved in electron flow towards plastocyanin (11). Illumination by a closely spaced flash pair b6 I system I in a kinetically peculiar interplay with plastoquinone. (interval ms) gave rise to the spectral changes shown in Fig. The results suggest that both the oxidation and the reduction 1A. The spectrum 15 ms after the illumination shows a con- are induced by the oxidation of plastohy- tribution of reduced-minus-oxidized cytochrome b6, charac- of cytochrome b6 mole- droquinone. The oxidation of cytochrome b6 appears to be as- terized by a maximum at 563 nm. Per 500 chlorophyll sociated with reduction of cytochrome f, whereas the oxidation cules, an estimated 0.4 molecule of cytochrome b6 is reduced. of plastohydroquinone by another system I donor (possibly This absorbance change due to cytochrome b6 did not develop extent I ms after the illumination. Evidently, plastocyanin) causes the cytochrome b6 reduction. To explain to any significant under the conditions of Fig. 1., the cytochrome b6 reduction these data, a tentative hypothesis is developed that, in addition, occurs rather slowly. accounts for the high proton-to-electron ratio mentioned above. II Mitchell's "Q- In Fig. 1B, a parallel experiment to Fig. IA, the system The proposed scheme differs crucially from inhibitor diuron was added before the illumination. In this case cycle" model (6). It assumes two parallel oxidizing pathways the the cytochrome b6 reduction was fully inhibited, and the dif- for the oxidation of plastohydroquinone and accommodates ference spectra at I ms and 15 ms after the illumination are reservoir ("pool") function of this electron/proton carrier. essentially identical. The short flash spacing used in these experiments excludes the possibility that diuron could have af- The publication costs of this article were defrayed in part by page fected electron flow through the primary reaction of system charge payment. This article must therefore be hereby marked "ad- vertisenment" in accordance with 18 U. S. C. §1734 solely to indicate I. Obviously, in the experiment of Fig. IA, the electrons that this fact. reduce cytochrome b6 come from system II. 2765 Downloaded by guest on September 27, 2021 2766 Botany: Velthuys Proc. Natl. Acad. Sci. USA 76 (1979) periment of Fig. 2B, the (first) half-time of cytochrome b6 reduction after the second flash was t10 ms; even 80 ms after the second flash the reduction was still progressing. Evidently, the reduced cytochrome b6 is not readily reoxidized by ferricyanide or endogeneous components such as cytochrome f or plastocyanin-components that remain largely oxidized after two flashes. Both reduction and reoxidation-as observed after a light flash-are considerably accelerated when (prior to the flashes) reducing equivalents are present in the plastoquinone pool. The x 0- latter condition was achieved by simply shortening the time the chloroplast samples were kept in the dark. Fig. 3A shows an experiment made with chloroplasts that had -2- been stored in the dark (at 0C) for 12 hr. The data show that long dark incubation produces a state similar to that achieved by incubation with ferricyanide (cf. Fig. 2): the reduction and -4 reoxidation of cytochrome b6 are slow, and the difference in the efficacies of the first and the second flash is large. The data shown in Fig. 3B were obtained with chloroplasts -6- that were briefly preilluminated so that the plastoquinone pool was about halfway reduced. The rates of both reduction and 550 560 570 550 560 570 Wavelength, nm oxidation were then much faster, so that the absorbance in- crease, which was also smaller, reverted between the flashes. FIG. 1. (A) Time-resolved spectra of the absorbance change in- Under these conditions of a partially reduced pool, there is no duced by a pair of flashes spaced 1 ms apart. I, light intensity. 0, immediate dependence upon electron donation by system II; Measurements made 1 ms after the second flash; *, measurements made 15 ms later. Each point gives the average result of two obser- there is no difference in yield for the first and the second flash; vations, which typically varied by 0.4 X 10-4. Chlorophyll concen- and diuron does not inhibit-at least not during the initial tration, 100 pg/ml; electron acceptor, 50 AM ferricyanide. In the flashes (not shown). standard reaction mixture (with gramicidin D and NaCl), the light- Fig. 4 A and B, with an expanded time axis, illustrates this induced electrical field, with its associated absorbance changes, de- acceleration in more detail. In the experiment of Fig. 4B, the cayed in the submillisecond time range. (B) Same experiment as for pool was about halfway reduced by a preillumination; for the A, except that 10 1AM diuron was added, in the dark, 30 s before the measurement. The additional bleaching (compared to A) around 550 experiment of Fig. 4A, the chloroplasts from the same batch nm reflects reduction of the system II acceptor Q (C550) (10). were used without preillumination. Whereas in Fig. 3A the change after the second flash was half maximal after t6 ms and the decay was very slow, in Fig. 4A the (first) half-times of rise and decay were 4 and 80 ms, respectively; in Fig. 4B they were Electron flow from photosystem II into the quinone pool is 1.5 and 30 ms. These latter half-times correspond to those re- known to involve a secondary acceptor, R, that acts as a charge ported earlier by Dolan and Hind (14). These authors used re- accumulator. It stores the single charge it obtains from the petitive flashes in the absence of a terminal electron acceptor, primary acceptor, Q, after one photoact. Only after it obtains and therefore presumably measured under conditions of a another charge after a second photoevent and is doubly reduced largely reduced pool. does it transfer its reducing equivalents to the plastoquinone Cytochrome f Reduction Correlates with Cytochrome b6 pool (12, 13). Oxidation. The spectra of Fig. 1 clearly show a negative change The experiment of Fig. 2 shows that, when the plastoquinone at 553 nm that is insensitive to diuron and characteristic for pool is empty (oxidized), electron transfer through R is reflected cytochrome f oxidation (8). This change is present at both 1 and in the reduction of cytochrome b6. For this experiment the 15 ms after the flashes. More detailed kinetic measurements chloroplasts were preincubated with ferricyanide to oxidize all showed that most of the cytochrome f oxidation induced by a intermediates in the intersystem chain, including the system flash occurs between 0.1 and 1 ms (see also ref. 15). While the I donors and R. Two flashes were given, spaced 90 ms apart so photooxidation is always rapid, the rate of the subsequent re- that the effect of the individual flashes was revealed. The results duction of cytochrome f depends upon the redox state of the are shown in the form of time-resolved difference spectra (Fig. plastoquinone pool. For instance, in the experiment of Fig. 3A, 2A) and in the kinetics at four wavelengths (Fig. 2B). the first flash caused a rapid oxidation of cytochromef (about The difference spectrum recorded 80 ms after the second 0.4 molecule per 500 molecules of chlorophyll) that was prac- flash of the pair shows a considerable reduction of cytochrome tically irreversible. Only after the subsequent flashes did some b. The maximum at 563 nm indicates that the reduced cyto- cytochrome f reduction occur. The oscillation in the production chrome is predominantly cytochrome b6. In contrast, the dif- of hydroquinone was reflected in a (weak) oscillation in the rate ference spectra in Fig. 2A that were recorded at 10 ms and 80 of cytochrome f reduction. ms after the first flash show hardly any b component. The ki- Under the conditions of the experiment of Fig. 4A, a partial netic traces in Fig. 2B also show that the reduction of cyto- rereduction of cytochrome f occurred even after the first flash chrome b6 is very small after the first flash, but predominant (not shown). With an increasing number of reducing equiva- after the second. This result is readily explained by the notion lents in the pool, dark reduction between the flashes became that cytochrome b6 is reduced by plastohydroquinone, which, more rapid and complete, as is illustrated by the experiments because of charge accumulation in R, is not formed until after of Fig. 4 C and D, made in parallel with those of Fig. 4 A and the second flash. B. It is evident that in the various experiments cytochrome b6 Reduction As Well As Oxidation of Cytochrome b6 Is reoxidation and cytochrome f rereduction (see also Fig. 3A) Rapid When the Plastoquinone Pool Is Reduced. In the ex- resembled one another. However, their first half-times were Downloaded by guest on September 27, 2021 Botany: Velthuys Proc. Natl. Acad. Sci. USA 76 (1979) 2767

la 0 x -I-

III. I 540 550 560 570 0 100 200 Wavelength, nm Time after first flash, ms FIG. 2. (A) Time-resolved spectra of the absorbance change induced by a pair of flashes, spaced 90 ms apart. Osmotically shocked chloroplasts were incubated with 0.5 mM ferricyanide for 1 min. 0 and 0, Measurements 10 ms and 80 ms, respectively, after the first flash; O. measurements at 170 ms-i.e., 80 ms after the second flash. Each point gives the (calculated) average of four observations. (B) The absorbance change measured in A as function of time, at four wavelengths. The time axis starts at the moment of the first flash. not simply equal; e.g., in Fig. 4 B and D they are approximately Prereduction of plastoquinone, as in the experiment of Fig. 30 ms and 10 ms, respectively. The fact that the two compo- 3B, causes the flash-induced reduction of cytochrome b6 to be nents are not directly connected in series (see above) might accelerated and diuron insensitive. This result persuasively preclude a simple kinetic relationship, but makes the existence substantiates the inference that the cytochrome b6 reduction of a correlation the more striking. occurs coupled with the reduction of an oxidized system I donor by plastohydroquinone. However, there seems to be a kinetic DISCUSSION problem. Electron flow from plastohydroquinone to P700 occurs with a half-time of >5 ms (24) (but see also ref. 25). In Fig. The cytochrome b6 redox changes reported in this paper oc- 4B and ref. 14 the reduction of cytochrome b6 occurred faster curred in the presence of an electron acceptor of system I-i.e., (nz1.5 ms first half-time). According to arguments described under conditions of linear electron transport. Apparently, this later in this paper, the discrepancy may only be apparent. cytochrome not only turns over in cyclic flow (16-18) but also It is uncertain whether a is cytochrome f. The reduction of is generally involved in electron flow, whether cyclic or non- cytochrome f is often considerably slower than that of cyto- cyclic, that goes through plastoquinone. Unmistakably, the chrome b6 (Fig. 3A, Fig. 4 A and C). Possibly, a rapid phase in results accentuate similarity with electron flow through ubi- the reduction of cytochrome f is masked by a simultaneous quinone in mitochondria (19) and photosynthetic bacteria reoxidation by its electron acceptor, plastocyanin. The relation (4). between cytochrome f and plastocyanin is not well understood Reduction of Cytochrome b6 Is Coupled with Reduction (3); their respective redox states correlate poorly (10, 13, 26, 27). of a System I Donor by Plastohydroquinone. The data of Figs. Alternatively, a might be a plastocyanin, or the "Rieske" 1, 2, and 3A show that, under conditions in which the plasto- iron-sulfur center (27). quinone pool is empty, a reduction of cytochrome b6 takes place Oxidation of Cytochrome b6, Also, Is Associated with upon electron flow from photosystem II. A similar observation Oxidation of Plastohydroquinone. The presented data show was made earlier by Rumberg (20). He concluded that electron that not only the reduction but also the oxidation of cytochrome flow from plastoquinone, a carrier of two reducing equivalents, b6 is accelerated with a more reduced pool. The kinetics of this branched into two one-equivalent acceptors (cytochrome b6 oxidation showed similarities with those of the reduction of and, supposedly, cytochrome f). This concept is also used in cytochromef. Such a parallelism has been noticed earlier, both Mitchell's Q-cycle and in variations of this model for mito- with chloroplasts (16) and mutatis mutandis with photosyn- chondria and bacteria (8, 21, 22). In chloroplasts, the process thetic bacteria (28). However, because reduced cytochrome b6 can be visualized to proceed as follows: The oxidation of plas- and oxidized cytochrome f can coexist for hundreds of milli- tohydroquinone (PQH2) by a high-potential one-electron ac- seconds (when plastoquinone is oxidized; see Fig. 2) the two ceptor (a, cytochrome f?) generates a strong reductant (plas- cytochromes are evidently not simply connected in series. tosemiquinone) (23), which in turn reduces cytochrome b6. A plausible explanation of the concerted disappearance of PQH2 + aox PQ- + ared + 2H+ [la] reduced cytochrome b and oxidized cytochrome c in chroma- tophores of purple bacteria was given by Crofts et al. (21). PQ- + b6ox - PQ + b6rel [lb] Translated to chloroplasts, the suggestion is that the oxidation Downloaded by guest on September 27, 2021 2768 Botany: Velthuys Proc. Natl. Acad. Sci. USA 76 (1979)

A c

cyt f

cyt b6 tms 40 ms D B * > A/lI~~~2X 1-4

^r ~~~~~200ms

B I FIG. 4. (A) Cytochrome b6 redox changes after the second flash t t t t (first flash, 1 s earlier). Eight measurements at 570 nm subtracted from eight measurements at 563 nm. Chloroplasts were dark adapted for -4 hr (at 0WC). (B) Same measurements as for A, except for a cyt be preillumination of the chloroplasts in the absence of electron acceptor, 1 min before the measurement. (C) Cytochrome f redox changes (540 nm - 553 nm; reduction downward). Parallel experiment to that of A. (D) Cytochrome f redox changes measured parallel to the experi- FIG. 3. (A) Redox changes of cytochromes b6 and f in a series ment of B. of flashes after long dark adaptation (t12 hr at 0VC). Electron acceptor, 100 ,uM methylviologen. The cytochrome b6 trace is the result of subtracting four measurements at 570 nm from four measurements currently with its reduction, its net reduction ceases earlier than at 563 nm. The cytochrome f was measured similarly, using 553 nm the oxidation of plastohydroquinone. Therefore, its kinetics, minus 540 nm; it is shown with sign reversed (reduction downward). in terms of (first) half-time (though not in terms of molecules The first few milliseconds of the cytochrome f traces (also Fig. 4) per second), will be faster than that of electron flow to P700. contain interference from C550 (10). (B) Cytochrome b6 redox changes This may explain why the rate of the cytochrome b6 reduction observed with chloroplasts that had been preilluminated, in the ab- apparently exceeds that of the flow to P700 (see above). For sence of electron acceptor, for 5 s, 1 min before the measurement. similar reasons, the net oxidation of cytochrome b6, in terms of half-time, will be slower than the reduction of cytochrome f, of cytochrome b6 (Eq. 2b) is caused by a plastosemiquinone that but the kinetic correlation between these two processes should is generated in the oxidation of plastohydroquinone by cyto- be loose anyway. Cytochrome b6 reoxidation by "pairing" chrome f (Eq. 2a) (cf. Eq. la): (involving the reversal of reaction 1, followed by the transfer of a positive charge, via plastocyanin, to another chain, and PQH2 + fox PQ- + fred + 2H+ [2a] completed by the occurrence of reaction 2 in this chain) may + b6red + 2H+ - + b6ox. proceed after the reduction of cytochrome f is virtually com- PQ- PQH2 [2b] plete. Rudimentary Scheme; Transfer of Oxidizing Equivalents Scheme to Qualitatively Account for Both Cytochrome b6 between Chains. It will be assumed that the interpretations Turnover and the Associated Proton Translocation. In elec- represented by Eqs. 1 and 2 are both basically correct. It follows tron flow from system II to system I, two protons are translo- that the turnover of cytochrome b6 is associated with the flow cated per electron passage (1, 2). No doubt this phenomenon of two electrons to system I. We may initially pretend that there is related to the behavior of cytochrome b6; both phenomena is only a single plastosemiquinone-generating site-i.e., that should be accounted for in a single scheme. To achieve this, the a is cytochrome f. However, the following discussion of the scheme discussed in the previous section was extended. In the cytochrome b6 kinetics is equally pertinent if reactions la and model represented by Fig. 5, plastoquinone reacts with other 2a occur in fact at separate sites-i.e., with a not being cyto- electron carriers at three different sites. A fourth site, at system chrome f. II, is not shown. Between these sites, it can diffuse only in the A most noteworthy aspect of the experiment of Fig. 3B is that fully reduced and fully oxidized states (cf. ref. 6). the cytochrome b6 reduction almost completely returns be- Two, rather than one, plastosemiquinone-generating sites tween flashes. This result implies that oxidizing equivalents are are located at the inner side of the thylakoid membrane, one distributed in such a way that an even number of electrons (or at a and another one at cytochrome f. Reaction 1 proceeds as zero electrons) are transferred by each chain. It is evident what already described. However, the semiquinone formed in the the driving force for such "pairing" is: electrons are at a higher second plastohydroquinone oxidizing pathway (reaction 2a) potential in plastoquinone than in cytochrome b6. But for it to cannot react directly with the cytochrome b6 reduced in reac- occur, a transfer of oxidizing equivalents between chains must tion 1, but, instead, reduces an unidentified compound, des- be allowed. Presumably such a transfer, similar to that occurring ignated ?b. Because chloroplasts contain at least two molecules between the plastoquinone of different chains (29), takes place of b6 per cytochrome f (17, 31), ?b might be a second cyto- at the level of plastocyanin (5, 23, 25, 30). chrome b6. Its reduction would not be observed because a pair Because the oxidation of cytochrome b6 takes place con- of reduced cytochrome b6 molecules is reoxidized more rapidly Downloaded by guest on September 27, 2021 Botany: Velthuys Proc. Natl. Acad. Sci. USA 76 (1979) 2769

2H 1. Velthuys, B. R. (1978) Proc. Natl. Acad. Sca. USA 75, 6031- 1.11. I,,; f.. I.11 " t 4 NIYI., I 2. Fowler, C. F. & Kok, B. (1976) Biochim. Biophys. Acta 423, Out I I 510-523. 0 1 3. Cramer, W. A. & Whitmarsh, J. (1977) Annu. Rev. Plant Physiol. , _ #- - -_ 28, 133-172. 0 1% , 4. Crofts, A. R. & Wood, P. M. (1978) in Current Topics in Bioen- ergetics, eds. Sanadi, D. R. & Vernon, L. P. (Academic, New York), Vol. 7A, pp. 175-244. 5. Boardman, N. K., Bjorkman, O., Anderson, J. M., Goodchild, D. J. & Thorne, S. W. (1975) in Proceedings of the Third Interna- tional Congress on Photosynthesis, ed. M. Avron (Elsevier, Amsterdam), pp. 1809-1827. 6. Mitchell, P. (1975) FEBS Lett. 59, 137-139. 7. Schwartz, M. (1966) Biochim. Biophys. Acta 112,204-212. 8. Bendall, D. S., Davenport, H. S. & Hill, R. (1971) Methods En- ~~A zymol. 23A, 327-344. A. L. & Wasserman, A. R. (1973) Biochim. Biophys. Acta .I 9. Stuart, PQ -., a--- 314, 284-297. 10. Knaff, D. B. & Arnon, D. I. (1969) Proc. Natl. Acad. Sci. USA 63, i / PCy 963-969. .,@Cyt f.___ 11. Marsho, T. V. & Kok, B. (1970) Biochim. Biophys. Acta 223, 240-250.

In 12. Velthuys, B. R. & Amesz, J. (1974) Biochim. Biophys. Acta 333, 85-94. 13. Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250- 2H 2H+ 256. 14. Dolan, E. & Hind, G. (1974) Biochim. Biophys. Acta 357, FIG. 5. Tentative scheme of electron transfer between plasto- 380-385. quinone and plastocyanin and associated proton translocation. Solid 15. Bouges-Bocquet, B. (1977) Biochim. Biophys. Acta 462, 362- lines show H transfer. Dotted lines show transfer of protons; dashed 379. lines, transfer of electrons. PQ, plastoquinone pool; cyt b6, cytochrome 16. Slovacek, R. E. & Hind, G. (1978) Biochem. Biophys. Res. b6; ?b, hypothetical intermediate, possibly also a cytochrome b6; cyt Commun. 84,901-906. f, cytochrome f; a, unidentified system I donor; PCy, plastocyanin. 17. Heber, U., Boardman, N. K. & Anderson, J. M. (1976) Biochim. Note: the reduction of plastoquinone by system II (not shown) binds Biophys. Acta 423,275-292. protons from the outside. 18. Bohme, H. (1977) Eur. J. Biochem. 72,283-289. 19. Rieske, J. S. (1976) Biochim. Biophys. Acta 456,195-197. than it is formed. This reoxidation occurs by plastoquinone at 20. Rumberg, B. (1966) in Currents in Photosynthesis, eds. Thomas, a site that adjoins the outside medium. Thereby, protons are J. B. & Goedheer, J. C. (Donker, Rotterdam, The Netherlands), bound from the outside. pp. 375-382. with reactions 2' plus 3' replacing 21. Crofts, A. R., Crowther, D. & Tierney, G. V. (1975) in Electron The following equations, Phosphorylation, eds. Quagli- reaction 2 of the simpler scheme, serve to further clarify the Transfer Chains and Oxidative ariello, E., Papa, S., Palmieri, E., Slater, E. C. & Siliprandi, N. proposed hypothesis. (North Holland, Amsterdam), pp. 233-241. 22. Dutton, P. L. & Prince, R. C. (1978) FEBS Lett. 91, 15-20. PQH2 + b6ox + aox PQ + b6rEJ + are2H~1n+ [1] 23. Wood, P. M. & Bendall, D. S. (1976) Eur. J. Biochem. 61, 337-344. 24. Stiehl, H. H. & Witt, H. T. (1969) Z. Naturforsch. 246, 1588- PQH2 + ?box + fox PQ + ?bred + fred + 2H+in [2'] 1598. 25. Bouges-Bocquet, B. (1975) Biochim. Biophys. Acta 396, 382- 391. PQ + b6red + ?bred + - PQH2 + b6ox + ?box. [3'] 2H+o,0t 26. Haehnel, W. (1977) Biochim. Biophys. Acta 459,418-441. 27. White, C. C., Chain, R. K. & Malkin, R. (1978) Biochim. Biophys. The result of these reactions is that the proton translocation Acta 502, 127-137. associated with electron transfer through plastoquinone is 28. Prince, R. C. & Dutton, P. L. (1975) Biochim. Biophys. Acta 387, doubled. 609-613. 29. Siggel, U., Renger, G., Stiehl, H. H. & Rumberg, B. (1972) Bio- chim. Biophys. Acta 256,328-335. This work was supported in part by National Science Foundation 30. Haehnel, W. (1978) in Photosynthesis 77, eds. Hall, D. O., Grant PCM77-20526, by the Department of Energy, Contract EY- Coombs, J. & Goodwin, T. W. (The Biochemical Society, Lon- 76-C-02-3326, and by the Department of Agriculture, Contract don), pp. 777-786 5901-0410-8-0179-0. 31. B6hme, H. (1976) Z. Naturforsch. 31c, 68-77. Downloaded by guest on September 27, 2021