Electron-transfer reactions in manganese-depleted photosystem II

We have used flash-detection optical and electron paramagnetic resonance spectroscopy to measure the kinetics and yield per flash of the photooxidation of cytochrome b559 and the yield per flash of the photooxidation of the tyrosine residue YD in Mn-depleted photosystem II (PSII) membranes at room temperature. The initial charge separation forms YZ+ QA-. Following this, cytochrome b559 is oxidized on a time scale of the same order and with the same pH dependence as is observed for the decay of YZ+; under the conditions of our experiments, the decay of YZ+ is determined by the lifetime of YZ+ QA-. In order to explain this observation, we have constructed a model for electron donation in which YZ+ and P680+ are in redox equilibrium and cytochrome b559 and YD are oxidized via P680+. Using our results, together with data from earlier investigations of the kinetics of electron transfer from YZ to P680+ and charge recombination of YZ+ QA-, we have obtained the first global fit for electron donation in Mn-depleted PSII that accounts for the data over the pH range from 5 to 7.5. From these calculations, we have obtained the intrinsic rate constants of all the electron-donation reactions in Mn-depleted PSII. These rate constants allow us to calculate the free energy difference between YZ+ P680 and YZ P680+, which is found to increase by 47 +/- 4 mV/pH from pH 5 to 6 and is observed to increase more slowly per pH unit for pH greater than 6. An important conclusion of our experimental work is that the rates of photooxidation of cytochrome b559 and YD are determined by the lifetime of the oxidizing equivalent on YZ/P680. Extension of our model to oxygen-evolving PSII samples leads to the prediction that the kinetics and yields of electron donation from cytochrome b559 and YD to P680+ will depend on the S2- or S3-state lifetime.     

Water-splitting manganese complex controls light-induced redox changes of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>559</Subscript> in Photosystem II

The effect of water-splitting Mn complex on light-induced redox changes of cytochrome b ₅₅₉ (cyt b ₅₅₉) was studied in spinach photosystem II (PSII) membranes. Photoreduction of the heme iron in the intact PSII membranes was completely suppressed by DCMU, whereas photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were unaffected by DCMU. Interesingly, photoreduction and photooxidation of the heme iron in the Mn-depleted PSII membranes were completely diminished by exogenous superoxide dismutase (SOD), whereas no effect of SOD on photoreduction of the heme iron was observed in the intact PSII membranes. The current work shows that the light-induced redox changes of cyt b ₅₅₉ proceed via a different mechanism in the both types of PSII membranes. In the intact PSII membranes, photoreduction of the heme iron is mediated by plastoquinol. However, in the Mn-depleted PSII membranes, photoreduction and photooxidation of the heme iron are mediated by superoxide anion radical formed in PSII.     

Evidence that cytochrome b559 mediates the oxidation of reduced plastoquinone in the dark

The function of cytochrome b(559) in photosystem II (PSII) was investigated using a mutant created in tobacco in which the conserved phenylalanine at position 26 in the beta-subunit (PsbF) was changed to serine (Bock, R., K?ssel, H., and Maliga, P. (1994) EMBO J. 13, 4623-4628). The mutant grew photoautotrophically, but the amount of PSII was reduced and the ultrastructure of the chloroplast was dramatically altered. Very few grana stacks were formed in the mutant. Although isolated PSII-enriched membrane fragments showed low PSII activity, electron paramagnetic resonance indicated the presence of functional PSII. Difference absorption spectra showed that the cytochrome b(559) contained heme. The plastoquinone pool was largely reduced in dark-adapted leaves of the mutant, based on chlorophyll fluorescence and thermoluminescence measurements. We therefore propose that cytochrome b(559) plays an important role in PSII by keeping the plastoquinone pool and thereby the acceptor side of PSII oxidized in the dark. Structural alterations as induced by the single Phe --> Ser point mutation in the transmembrane domain of PsbF evidently inhibit this function.     

A theoretical study on effect of the initial redox state of cytochrome b559 on maximal chlorophyll fluorescence level (F(M)): implications for photoinhibition of photosystem II

In this work, we extended the reversible radical pair model which describes energy utilization and electron transfer up to the first quinone electron acceptor (Q(A)) in photosystem II (PSII), by redox reactions involving cytochrome (cyt) b559. In the model, cyt b559 accepts electrons from the reduced primary electron acceptor in PSII, pheophytin, and donates electrons to the oxidized primary electron donor in PSII (P680+). Theoretical simulations of chlorophyll fluorescence rise based on the model show that the maximal fluorescence, F(M), increases with an increasing amount of initially reduced cyt b559. In this work we applied, the first to our knowledge, metabolic control analysis (MCA) to a model of reactions in PSII. The MCA was used to determine to what extent the reactions occurring in the model control the F(M) level and how this control depends on the initial redox state of cyt b559. The simulations also revealed that increasing the amount of initially reduced cyt b559 could protect PSII against photoinhibition. Also experimental data, which might be used to validate our theory, are presented and discussed.     

Evidence that cytochrome b559 is involved in superoxide production in photosystem II: effect of synthetic short-chain plastoquinones in a cytochrome b559 tobacco mutant

Light-induced production of superoxide (O2*-) in spinach PSII (photosystem II) membrane particles was studied using EPR spin-trapping spectroscopy. The presence of exogenous PQs (plastoquinones) with a different side-chain length (PQ-n, n isoprenoid units in the side-chain) enhanced O2*- production in the following order: PQ-1>PQ-2>PQ-9. In PSII membrane particles isolated from the tobacco cyt (cytochrome) b559 mutant which carries a single-point mutation in the beta-subunit and also has a decreased amount of the alpha-subunit, the effect of PQ-1 was less than in the wild-type. The increase in LP (low-potential) cyt b559 content, induced by the incubation of spinach PSII membrane particles at low pH, resulted in a significant increase in O2*- formation in the presence of PQ-1, whereas it had little effect on O2*- production in the absence of PQ-1. The enhancement of O2*- formation induced by PQ-1 was not abolished by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea]. Under anaerobic conditions, dark oxidation of LP cyt b559 increased, as pH was decreased. The presence of molecular oxygen significantly enhanced dark oxidation of LP cyt b559. Based on these findings it is suggested that short-chain PQs stimulate O2*- production via a mechanism that involves electron transfer from Pheo- (pheophytin) to LP cyt b559 and subsequent auto-oxidation of LP cyt b559.     

Redox changes of cytochrome b(559) in the presence of plastoquinones

We have found that short chain plastoquinones effectively stimulated photoreduction of the low potential form of cytochrome b(559) and were also active in dark oxidation of this cytochrome under anaerobic conditions in Triton X-100-solubilized photosystem II (PSII) particles. It is also shown that molecular oxygen competes considerably with the prenylquinones in cytochrome b(559) oxidation under aerobic conditions, indicating that both molecular oxygen and plastoquinones could be electron acceptors from cytochrome b(559) in PSII preparations. alpha-Tocopherol quinone was not active in the stimulation of cytochrome photoreduction but efficiently oxidized it in the dark. Both the observed photoreduction and dark oxidation of the cytochrome were not sensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethylurea. It was concluded that both quinone-binding sites responsible for the redox changes of cytochrome b(559) are different from either the Q(A) or Q(B) site in PSII and represent new quinone-binding sites in PSII.     

Characterization of the multiple forms of cytochrome b559 in photosystem II

Cytochrome b559 is an essential component of the photosystem II (PSII) protein complex. Its function, which has long been an unsolved puzzle, is likely to be related to the unique ability of PSII to oxidize water. We have used EPR spectroscopy and spectrophotometric redox titrations to probe the structure of cytochrome b559 in PSII samples that have been treated to remove specific components of the complex. The results of these experiments indicate that the low-temperature photooxidation of cytochrome b559 does not require the presence of the 17-, 23-, or 33-kDa extrinsic polypeptides or the Mn complex (the active site in water oxidation). We observe a shift in the g value of the EPR signal of cytochrome b559 upon warming a low-temperature photooxidized sample, which presumably reflects a change in conformation to accommodate the oxidized state. At least three redox forms of cytochrome b559 are observed. Untreated PSII membranes contain one high-potential (375 mV) and one intermediate-potential (230 mV) cytochrome b559 per PSII. Thylakoid membranes also appear to contain one high-potential and one intermediate-potential cytochrome b559 per PSII, although this measurement is more difficult due to interference from other cytochromes. Removal of the 17- and 23-kDa extrinsic polypeptides from PSII membranes shifts the composition to one intermediate-potential (170 mV) and one low-potential (5 mV) cytochrome b559. This large decrease in potential is accompanied by a very small g-value change (0.04 at gz), indicating that it is the environment and not the ligand field of the heme which changes significantly upon the removal of the 17- and 23-kDa polypeptides.     

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.     

Cytochrome b-559 may function to protect photosystem II from photoinhibition

Although cytochrome b-559 is an integral component of the photosystem II complex (PSII), its function is unknown. Because cytochrome b-559 has been shown to be both photooxidized and photoreduced in PSII, one of several proposals is that it mediates cyclic electron transfer around PSII, possibly as a protective mechanism. We have used electron paramagnetic resonance spectroscopy to investigate the pathway of photooxidation of cytochrome b-559 in PSII and have shown that it proceeds via photooxidation of chlorophyll. We propose that this photooxidation of chlorophyll is the first step in the photoinhibition of PSII. The unique susceptibility of PSII to photoinhibition is probably due to the fact that it is the only reaction center in photosynthesis which generates an oxidant with a reduction potential high enough to oxidize chlorophyll. We propose that the function of cytochrome b-559 is to mediate cyclic electron transfer to rereduce photooxidized chlorophyll and protect PSII from photoinhibition. We also suggest that the chlorophyll(s) which are susceptible to photooxidation are analogous to the monomer chlorophylls found in the bacterial photosynthetic reaction center complex.     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

Effect of dehydration on light-induced reactions in photosystem II: photoreactions of cytochrome b559

The effect of dehydration on the reaction pattern of photosystem II (PS II) has been studied by measuring and analyzing spectral changes induced by continuous wavelength illumination in films of untreated and hydroxylamine-washed PS II membrane fragments dehydrated to different levels. The obtained data revealed (i) the extent of light-induced formation of about one Q(A)(-*)per 230 chlorophylls (Chl) remains virtually invariant to dehydration down to the lowest values of relative humidity (6-8% RH); (ii) a decrease of the RH to 30% leads to severe blockage of the electron transfer from Q(A)(-*) to Q(B) and the progressive replacement of water oxidation by photooxidation of high potential (HP) cytochrome (Cyt) b559 in untreated PS II samples or accessory Chl and carotenoid (Car) molecules in samples with preoxidized Cyt b559; (iii) photooxidation of Cyt b559 is followed by its photoreduction, concomitant with photooxidation of Chl and Car; (iv) in dry samples with preoxidized Cyt b559, not more than a half of total Cyt b559 can be photochemically reduced, independent of the extent of Cyt b559 in the HP form; (v) at low RH values, Cyt b559 photoreduction in samples with preoxidized heme groups and photoaccumulation of Q(A)(-*) take place with biphasic kinetics with similar rate constants for both processes; (vi) Cyt b559 photoreduction in dry samples is DCMU insensitive, while the dark rereduction of photooxidized Cyt b559 is inhibited by DCMU; (vii) fast and slow kinetic phases of Cyt b559 photoreduction dramatically differ in their dependencies on the intensity of CW illumination and are associated with electron donation to Cyt b559 from Q(A)(-*) and pheophytin(-*), respectively. The pathways of light-induced electron transfer in PS II involving Cyt b559 are discussed.     

Photoreduction of QA, QB, and cytochrome b-559 in an oxygen-evolving photosystem II preparation from the thermophilic cyanobacterium Synechococcus sp

Light-induced absorption changes in an oxygen-evolving photosystem II (PS II) preparation from the thermophilic cyanobacterium Synechococcus sp. were analyzed using continuous illumination which caused the reduction of both QA (first stable quinone electron acceptor) and QB (second quinone electron acceptor of photosystem II). In this photosystem II preparation in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) the amount of QA was estimated to be 1 per 42 chlorophylls. In the absence of DCMU, plastoquinone (1.68 per QA) was photoreduced to plastohydroquinone within a few seconds, indicating that QB is reduced and protonated during this period. An electrochromic band shift centered around 685 nm was observed with and without DCMU. The extent of this band shift caused by QB reduction per electron was about a third or half of that caused by QA reduction. A significant amount of cytochrome b-559 (0.86 per QA) was photoreduced. Only 60% of the photoreduction of cytochrome b-559 was inhibited by a DCMU concentration that inhibited electron transfer beyond QB, indicating that the site of the reduction of cytochrome b-559 is located before the QB site and possibly on the donor side of PS II.     

Light-adaptation of photosystem II is mediated by the plastoquinone pool

During the first few enzymatic turnovers after dark-adaptation of photosystem II (PSII), the relaxation rate of the EPR signals from the Mn cluster and Y(D)(*) are significantly enhanced. This light-adaptation process has been suggested to involve the appearance of a new paramagnet on the PSII donor side [Peterson, S., Ahrling, K., H?gblom, J., and Styring, S. (2003) Biochemistry 42, 2748-2758]. In the present study, a correlation is established between the observed relaxation enhancement and the redox state of the quinone pool. It is shown that the addition of quinol to dark-adapted PSII membrane fragments induces relaxation enhancement already after a single oxidation of the Mn, comparable to that observed after five oxidations in samples with quinones (PPBQ or DQ) added. The saturation behavior of Y(D)(*) revealed that with quinol added in the dark, a single flash was necessary for the relaxation enhancement to occur. The quinol-induced relaxation enhancement of PSII was also activated by illumination at 200 K. Whole thylakoids, with no artificial electron acceptor present but with an intact plastoquinone pool, displayed the same relaxation enhancement on the fifth flash as membrane fragments with exogenous quinones present. We conclude that (i) reduction of the quinone pool induces the relaxation enhancement of the PSII donor-side paramagnets, (ii) light is required for the quinol to effect the relaxation enhancement, and (iii) light-adaptation occurs in the intact thylakoid system, when the endogenous plastoquinone pool is gradually reduced by PSII turnover. It seems clear that a species on the PSII donor side is reduced by the quinol, to become a potent paramagnetic relaxer. On the basis of XANES reports, we suggest that this species may be the Mn ions not involved in the cyclic redox changes of the oxygen-evolving complex.     

Electron transfer in photosystem II at cryogenic temperatures

The photochemistry in photosystem II of spinach has been characterized by electron paramagnetic resonance (EPR) spectroscopy in the temperature range of 77-235 K, and the yields of the photooxidized species have been determined by integration of their EPR signals. In samples treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a single stable charge separation occurred throughout the temperature range studied as reflected by the constant yield of the Fe(II)-QA-EPR signal. Three distinct electron donation pathways were observed, however. Below 100 K, one molecule of cytochrome b559 was photooxidized per reaction center. Between 100 and 200 K, cytochrome b559 and the S1 state competed for electron donation to P680+. Photooxidation of the S1 state occurred via two intermediates: the g = 4.1 EPR signal species first reported by Casey and Sauer [Casey, J. L., & Sauer, K. (1984) Biochim. Biophys. Acta 767, 21-28] was photooxidized between 100 and 160 K, and upon being warmed to 200 K in the dark, this EPR signal yielded the multiline EPR signal associated with the S2-state. Only the S1 state donated electrons to P680+ at 200 K or above, giving rise to the light-induced S2-state multiline EPR signal. These results demonstrate that the maximum S2-state multiline EPR signal accounts for 100% of the reaction center concentration. In samples where electron donation from cytochrome b559 was prevented by chemical oxidation, illumination at 77 K produced a radical, probably a chlorophyll cation, which accounted for 95% of the reaction center concentration. This electron donor competed with the S1 state for electron donation to P680+ below 100 K.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between starch breakdown, acetate assimilation, and photosynthetic cyclic electron flow in Chlamydomonas reinhardtii

Spectroscopic studies on photosynthetic electron transfer generally are based upon the monitoring of dark to light changes in the electron transfer chain. These studies, which focus on the light reactions of photosynthesis, also indirectly provide information on the redox or metabolic state of the chloroplast in the dark. Here, using the unicellular microalga Chlamydomonas reinhardtii, we study the impact of heterotrophic/mixotrophic acetate feeding on chloroplast carbon metabolism by using the spectrophotometric detection of P700(+), the photooxidized primary electron donor of photosystem I. We show that, when photosynthetic linear and cyclic electron flows are blocked (DCMU inhibiting PSII and methylviologen accepting electrons from PSI), the post-illumination reduction kinetics of P700(+) directly reflect the dark metabolic production of reductants (mainly NAD(P)H) in the stroma of chloroplasts. Such results can be correlated to other metabolic studies: in the absence of acetate, for example, the P700(+) reduction rate matches the rate of starch breakdown reported previously, confirming the chloroplast localization of the upstream steps of the glycolytic pathway in Chlamydomonas. Furthermore, the question of the interplay between photosynthetic and non-photosynthetic carbon metabolism can be addressed. We show that cyclic electron flow around photosystem I is twice as fast in a starchless mutant fed with acetate than it is in the WT, and we relate how changes in the flux of electrons from carbohydrate metabolism modulate the redox poise of the plastoquinone pool in the dark through chlororespiration.     

The rapid component of electron paramagnetic resonance signal II: a candidate for the physiological donor to photosystem II in spinach chloroplasts.

Rapid light-induced transients in EPR Signal IIf (F-+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q minus is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E'Os.o equals to + 480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential. A model is proposed in which Q minus, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F-+). At high potentials D is oxidized in the dark, and the (Q-+F-+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Restoration of the high-potential form of cytochrome b559 of photosystem II occurs via a two-step mechanism under illumination in the presence of manganese ions

Spinach photosystem II membranes that had been depleted of the Mn cluster contained four forms of cytochrome (Cyt) b559, namely, high-potential (HP), HP', intermediate-potential (IP) and low-potential (LP) forms that exhibited the redox potentials of +400, +310, +170 and +35 mV, respectively, in potentiometric titration. When the membranes were illuminated with flashing light in the presence of 0.1 mM Mn2+, the IP form was converted to the HP' form by two flashes and then the HP' form was converted to the HP form by an additional flash. The quantum efficiency of the first conversion appeared to be quite high since the conversion was almost complete after two flashes. By contrast, the second conversion proceeded with low quantum efficiency and 40 flashes were required for completion. The effects of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the first conversion did not require electron transfer from QA to QB while the second conversion had an absolute requirement for it. It was also suggested that the first conversion involved the reduction of the heme of Cyt b559, probably by QA-, and we propose that direct reduction by QA- induces a shift in the redox potential of the heme. The second conversion was also accompanied by the reduction of heme but it appeared that this conversion did not necessarily involve the reduction. The effects of DCMU on the reduction of heme suggested that the heme became reducible by QB- after the first conversion had been completed. This observation implies that the efficiency of electron transfer from QA to QB increased upon the conversion of the IP form to the HP' form, and we propose that restoration of the high-potential forms of Cyt b559 itself acts to make the acceptor side of photosystem II functional.     

Temperature-induced greening of<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis>. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.