Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

Photoreactions of Cytochrome b-559 and cyclic electron flow in photosystem II of intact chloroplasts.

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO2 showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

La photooxydation du cytochrome b-559, en presence de carbonylcyanure-p-trifluorométhoxyphenylhydrazone et de 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone,ou de p-benzoquinone,chez trois mutants non-photosynthétiques de Chlamydomonas reinhardti

Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardtiStudies of absorbance changes related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563.In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 μmol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl 5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome.Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553.A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also.An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Simultaneous photoreduction and photooxidation of cytochrome b-559 in Photosystem II treated with carbonylcyanide-m-chlorophenylhydrazone

The possibility of a Photosystem II (PS II) cyclic electron flow via Cyt b-559 catalyzed by carbonylcyanide m-chlorophenylhydrazone (CCCP) was further examined by studying the effects of the PS II electron acceptor 2,6-dichloro-p-benzoquinone (DCBQ) on the light-induced changes of the redox states of Cyt b-559. Addition to barley thylakoids of micromolar concentrations of DCBQ completely inhibited the changes of the absorbance difference corresponding to the photoreduction of Cyt b-559 observed either in the presence of 10 M ferricyanide or after Cyt b-559 photooxidation in the presence of 2 M CCCP. In CCCP-treated thylakoids, the concentration of photooxidized Cyt b-559 decreased as the irradiance of actinic light increased from 2 to 80 W m^-2 but remained close to the maximal concentration (0.53 photooxidized Cyt b-559 per photoactive Photosystem II) in the presence of 50 M DCBQ. The stimulation of Cyt b-559 photooxidation in parallel with the inhibition of its photoreduction caused by DCBQ demonstrate that the extent of the light-induced changes of the redox state of Cyt b-559 in the presence of CCCP is determined by the difference between the rates of photooxidation and photoreduction of Cyt b-559 occuring simultaneously in a cyclic electron flow around PS II.We also observed that the Photosystem I electron acceptor methyl viologen (MV) at a concentration of 1 mM barely affected the rate and extent of the light-induced redox changes of Cyt b-559 in the presence of either FeCN or CCCP. Under similar experimental conditions, MV strongly quenched Chl-a fluorescence, suggesting that Cyt b-559 is reduced directly on the reducing side of Photosystem II.Abbreviations ADRY acceleration of the deactivation reactions of the water-splitting system Y - ANT-2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - CCCP carbonylcyanide-m-chlorophenylhydrazone - DCBQ 2,6-dichloro-p-benzoquinone - FeCN ferricyanide - MV methyl viologen - P₆₈₀ Photosystem II reaction center Chl-a dimer CIW-DPB publication No. 1118.     

Cytochrome b-563 redox changes in intact CO-fixing spinach chloroplasts and in developing pea chloroplasts.

Intact spinach chloroplasts, capable of high rates of photochemical oxygen evolution with CO2 as electron acceptor (120-350 mumol O2 mg chlorophyll-1 h-1) were examined for cytochrome redox changes. The response of the cytochromes in intact chloroplasts to oxidants and reductants appears to be governed by the permeability of the chloroplast envelope. The low potential cytochromes (b-559LP and b-563) were more slowly reduced at 25 degrees C by dithionite than is the case with broken chloroplasts. At 0 degrees C, the reduction of the low potential cytochromes in intactchloroplasts was extremely slow. The chloroplast envelope is impermeable to ferricyanide, slowly permeable to ascorbate and rapidly permeable to reduced dichlorophenolindophenol. Light-induced redox changes of cytochrome b-563 in intact chloroplasts were examined both at 0 degrees and 25 degrees C. A red/far-red antagonism on the redox changes of cytochrome b-563 was observed at 0 degrees C under anaerobic conditions. 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea (DCMU) inhibited the photoreduction of cytochrome b-563 in red light following far-red illumination. The photooxidation of cytochrome b-563 under anaerobic conditions was not influenced by DCMU or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). The photoreduction of cytochrome b-563 under aerobic conditions was much less efficient than its photooxidation under anaerobic conditions. Developing pea chloroplasts showed much greater light-induced redox changes of cytochrome b-563 than did intact spinach chloroplasts. Our data are consistent with the view that cytochrome b-563 functions on a cyclic pathway around Photosystem I, but it appears that cyclic flow is sensitive to the relative poising of the redox levels of cytochrome b-563 and the components of the non-cylic pathway.     

The relationship between the activity of chloroplast Photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with E_m7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, E_m7.8 = +383 mV, in addition to some of the low potential (E_m7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to ‘dark’ chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, E_m7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.     

Kinetics of the photoreduction of cytochrome b-559 by Photosystem II in chloroplasts

The kinetics of the photoreduction of cytochrome b-559 and plastoquinone were measured using well-coupled spinach chloroplasts. High potential (i.e. hydroquinone reducible) cytochrome b-559 was oxidized with low intensity far-red light in the presence of N-methyl phenazonium methosulfate or after preillumination with high intensity light. Using long flashes of red light, the half-reduction time of cytochrome b-559 was found to be 100±10 ms, compared to 6–10 ms for the photoreduction of the plastoquinone pool. Light saturation of the photoreduction of cytochrome b-559 occurred at a light intensity less than one-third of the intensity necessary for the saturation of ferricyanide reduction under identical illumination conditions. The photoreduction of cytochrome b-559 was accelerated in the presence of dibromothymoquinone with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 25–35}\text{ms}$. The addition of uncouplers, which caused a stimulatory effect on ferricyanide reduction under the same experimental conditions, resulted in a decrease in the rate of cytochrome b-559 reduction. The relatively slow photoreduction rate of cytochrome b-559 compared to the plastoquinone pool implies that electrons can be transferred efficiently from Photosystem II to plastoquinone without the involvement of cytochrome b-559 as an intermediate. These results indicate that it is unlikely that high potential cytochrome b-559 functions as an obligatory redox component in the main electron transport chain joining the two photosystems.     

Studies on cytochromes in photosynthetic electron transport system I. photoreduction and photo-oxidation of cytochrome b559 by photosystem II in spinach chloroplasts

Light-induced redox-reactions of cytochrome b₅₅₉ in spinachchloroplasts were investigated. Illumination of chloroplastsinduced photoreduction of cytochrorne b₅₅₉ Red light (650 nm)was more effective than far-red light (725 nm), indicating thatthe photoreduction is a photosystem II-mediated reaction. Onaddition of DCMU, the photoreduction was eliminated and a photooxidationof cytochrome b₅₅₉ was observed. The rate of this photooxidationwas faster with photosystem II light than with photo-systemI light. On addition of Mn⁺⁺ the photooxidation was partly suppressed;far-red light became as effective as red light in inducing photooxidationof cytochrome b₅₉₉, in the presence of DCMU and Mn⁺⁺. Ascorbate completely suppressed photooxidation of cytochromeb⁵⁵⁹ In the presence of ascorbate, however, photooxidation wasobserved in the presence of inhibitors or after inhibitory treatmentsof chloroplasts which affected the oxidizing side of systemII. These inhibitors and inhibitory treatments, but not DCMU,decreased the redoxpotential of cytochrome b₅₅₉. Reactivationof Hill reaction in Tris-washed chloroplasts by indophenol-ascorbatetreatment was not accompanied by an abolishment of photooxidationof cytochrome b₅₅₉. A possible mechanism is proposed to account for these reactionsof cytochrome b₅₅₉ in the photosynthetic electron transportin chloroplasts. (Received April 4, 1972; )     

Photooxidation of the cytochrome b-559 in the presence of various substituted 2-anilinothiophenes and of some other compounds,in Chlamydomonas reinhardtii

Five substituted 2-anilinothiophenes and two substituted carbonylcyanide-phenylhydrazones were comparatively studied with respect to their capacities for inducing photooxidation of the cytochrome b-559 in chloroplast fragments and in whole cells of Chlamydomonas reinhardtii (wild type and P-700-lacking mutant Fl 5). In addition, some other compounds: antimycin A, picric acid, tetraphenylboron and NH₄Cl were also tested.Cytochrome b-559 photooxidations were clearly observed in the presence of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), 2-(3,4,5-trichloro)anilino-3,5-dinitrothiophene (ANT 2s), 2-(4-chloro)anilino-3,5-dinitrothiophene and, with greater amplitudes, in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, both in whole cells and in chloroplast fragments. Picric acid, antimycin A and tetraphenylboron were also able to induce cytochrome b-559 photooxidation in chloroplast fragments, but not in whole cells. In the wild type, the highest photoinduced redox changes were 1.1 (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, carbonylcyanide-m-chlorophenylhydrazone) and 0.6 (ANT 2p, ANT 2s) μmol of oxidized cytochrome b-559/1 mmol of chlorophyll, corresponding to 40% and 23% of the redox changes which could be induced chemically. All these cytochrome b-559 photooxidations, the greater part of which was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and occurred in the mutant Fl 5, appeared to be mainly Photosystem II-dependent reactions. But 3-(3,4-dichlorophenyl)-1,1-dimethylureainsensitive Photosystem I-dependent photooxidations of cytochrome b-559 occurred also in the wild type. On the other hand, 2-(4-dimethylamine)-anilino-3,5-dinitrothiophene, 2-N-methyl-(3-chloro-4-trifluoromethyl)anilino3,5-dinitrothiophene and NH₄Cl did not induce any cytochrome b-559 photooxidation.These results were discussed taking in consideration the nature of the molecular substitutions of the various tested substances and their respective acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis capacities which had been defined elsewhere by Renger (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439) for spinach chloroplasts. Like the acceleration of the deactivation reactions of the water-splitting enzyme system Y effect, the capacity for inducing the cytochrome b-559 photooxidation appeared dependent on the acidity of the NH group and on the number of halogenous substituents in the aromatic ring of the molecule. The greatest action towards cytochrome b-559 photooxidation was obtained with the most active acceleration of the deactivation reactions of the water-splitting enzyme system Y agents: carbonylcyanide-p-trifluoromethoxyphenylhydrazone, ANT 2p and ANT 2s.     

Characterization of new strains of nonphotosynthetic mutants of Chlamydomonas reinhardtii II. Quinones and cytochromes b-559, b-563 and c-553 in twelve mutants having impaired Photosystem II function

Twelve new strains of nonphotosynthetic mutants of Chlamydomonasreinhardtii having impaired functioning of Photosystem II werestudied with respect to their quinone and chloroplastic cytochromecontents and to various photooxidation reactions of cytochromesb-559 and c-553. The quinones were analyzed by chromatography,cytochromes b-563 and c-553 were measured spectrophotometricallyafter solubilization by Triton X-100, and cytochrome b-559 wasstudied by means of low-temperature difference spectra. Noneof these mutants showed a great deficiency of plastoquinoneA, ubiquinone Q9, cytochrome b-563 or cytochrome c-553. Butall lacked an ascorbate-reducible pool of cytochrome b-559 photooxidizableat 77?K. In spite of this deficiency, five mutants (Fl 18, Fl29, Fl 47, Fl 50, Fl 59) showed an appreciable photooxidationof cytochrome b-559 in the presence of FCCP at room temperature.The other strains performed only weak cytochrome b-559 photooxidationin the presence of FCCP, DCMU and DBMIB or p-benzoquinone (Fl39, Fl 42, Fl 52, Fl 54, Fl 57, Fl 60); in the mutant Fl 33,no cytochrome photooxidation was observed. These results pointed out that the pool of ascorbate-reduciblecytochrome b-559 photooxidizable at 77?K is different from thepool photooxidizable in the presence of FCCP at room temperature. (Received February 8, 1979; )     

pH-dependent photoreactions of the high- and low-potential forms of cytochrome b559 in spinach PS II-enriched membranes

Cytochrome b559 (Cyt b559) is a well-known intrinsic component of Photosystem II (PS II) reaction center in all photosynthetic oxygen-evolving organisms, but its physiological role remains unclear. This work reports the response of the two redox forms of Cyt b559 (i.e. the high- (HP) and low-potential (LP) forms) to inhibition of the donor or acceptor side of PS II. The photooxidation of HP Cyt b559 induced by red light at room temperature was pH-dependent under conditions in which electron flow from water was diminished. This photooxidation was observed only at pH values higher than 7.5. However, in the presence of 1 M CCCP, a limited oxidation of HP Cyt b559 was observed at acidic pH, At pH 8.5 and in the presence of the protonophore, this photooxidation of the HP form was accompanied by its partial transformation into the LP form. On the other hand, a partial photoreduction of LP Cyt b559 was induced by red light under aerobic conditions when electron transfer through the primary quinone acceptor Q_A was impaired by strong irradiation in the presence of DCMU. This photoreduction was enhanced at acidic pH values. To the best of our knowledge, this is the first time that both photoreduction and photooxidation of Cyt b559 is described under inhibitory conditions using the same kind of membrane preparations. A model accommodating these findings is proposed.Abbreviations CCCP carbonylcyanide 3-chlorophenylhydrazone - Cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - DCMU dichlorophenyldimethylurea - E _m midpoint redox potential - HP and LP high- and low-potential forms of Cyt b559 - P680 primary donor - I_A acceptor side inhibition - I_D donor side inhibition - Pheo pheophytin - PS II photosystem II - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

A pathway for the reduction of cytochrome b-559 by Photosystem II in chloroplasts

Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 180}\text{ms}$), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.     

The relationship between Q, C-550 and cytochrome b 559 in photoreactions at −196° in chloroplasts

Absorbance changes and fluorescence yield changes induced by irradiating spinach chloroplasts with red light at −196° were measured as a function of the redox potential of the chloroplast suspension. Absorbance changes at 546 nm indicate the photoreduction of C-550 and changes at 556 nm indicate the photooxidation of cytochrome b 559. The changes of fluorescence yield indicate the photoreduction of Q, the fluorescence quencher of chlorophylla a in Photosystem II. The titration curves for all three changes were essentially the same and showed the same midpoint potential. In other experiments as well, it was found that when C-550 is in the reduced state the fluorescence yield of the chloroplasts is high and the low-temperature photooxidation of cytochrome b 559 is blocked. These data indicate that C-550 may be equivalent to Q and that cytochrome b 559 serves as the electron donor for the photoreduction of C-550 at low temperature.     

Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.