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 chlo- roplasts (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 ex- periments 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 re- duction 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 plasto- cyanin-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.