Ascorbate-mediated LHCII protein phosphorylation--LHCII kinase regulation in light and in darkness

A freeze-thaw cycle of isolated thylakoids in darkness in the presence of ascorbate was employed as a novel experimental system to activate the light-harvesting complex (LHC) II kinase. Under these conditions ascorbate reduces Q(A), the primary quinone electron acceptor of photosystem (PS) II, and the subsequent reduction of plastoquinone and the cytochrome (cyt) b(6)f complex results in the activation of the LHCII kinase. Using this activation system, several facets of regulation of LHCII protein phosphorylation were unravelled. (i) Myxothiazol inhibited the activation of LHCII protein phosphorylation, thus being a potent inhibitor of electron flow not only in cyt bc complexes but in darkness also in cyt b(6)f complexes. (ii) Oxygen, the only electron acceptor in darkness, was required for LHCII kinase activation demonstrating that after a full reduction of the cyt b(6)f complex, an additional plastoquinol oxidation cycle in the quinol oxidation (Qo) site is required for LHCII kinase activation. (iii) In the absence of electron flow, when the intersystem electron carriers are reduced, the activated LHCII kinase has a half-life of 40 min, whereas the fully activated LHCII kinase becomes deactivated in a time scale of seconds upon oxidation of the cyt b(6)f complex, indicating that the kinase constantly reads the redox poise of the cyt b(6)f complex. (iv) The LHCII kinase is more tightly bound to the thylakoid membrane than the PS II core protein kinase(s). It is concluded that oxidation of plastoquinol at the Qo site of the reduced cyt b(6)f complex is required for LHCII kinase activation, while rapid reoccupation of the Qo site with plastoquinol is crucial for sustenance of the active state of the LHCII kinase.     

Contrasted effects of inhibitors of cytochrome b6f complex on state transitions in Chlamydomonas reinhardtii: the role of Qo site occupancy in LHCII kinase activation

We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske protein.     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl(2), an inhibitor of cytochrome b(6)f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b(6)f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Dithiol oxidant and disulfide reductant dynamically regulate the phosphorylation of light-harvesting complex II proteins in thylakoid membranes

Light-induced phosphorylation of light-harvesting chlorophyll a/b complex II (LHCII) proteins in plant thylakoid membranes requires an activation of the LHCII kinase via binding of plastoquinol to cytochrome b(6)f complex. However, a gradual down-regulation of LHCII protein phosphorylation occurs in higher plant leaves in vivo with increasing light intensity. This inhibition is likely to be mediated by increasing concentration of thiol reductants in the chloroplast. Here, we have determined the components involved in thiol redox regulation of the LHCII kinase by studying the restoration of LHCII protein phosphorylation in thylakoid membranes isolated from high-light-illuminated leaves of pumpkin (Cucurbita pepo), spinach (Spinacia oleracea), and Arabidopsis. We demonstrate an experimental separation of two dynamic activities associated with isolated thylakoid membranes and involved in thiol regulation of the LHCII kinase. First, a thioredoxin-like compound, responsible for inhibition of the LHCII kinase, became tightly associated and/or activated within thylakoid membranes upon illumination of leaves at high light intensities. This reducing activity was completely missing from membranes isolated from leaves with active LHCII protein phosphorylation, such as dark-treated and low-light-illuminated leaves. Second, hydrogen peroxide was shown to serve as an oxidant that restored the catalytic activity of the LHCII kinase in thylakoids isolated from leaves with inhibited LHCII kinase. We propose a dynamic mechanism by which counteracting oxidizing and reducing activities exert a stimulatory and inhibitory effect, respectively, on the phosphorylation of LHCII proteins in vivo via a novel membrane-bound thiol component, which itself is controlled by the thiol redox potential in chloroplast stroma.     

Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b₆/f-less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light-dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III-S at comparable rates. However, the redox-controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b₆/f-less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b₆/f complex inhibited both the electron flow and duroquinol-activated LHCII phosphorylation, but had no effect on the duroquinol-dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox-activated protein kinase(s). A quinone-binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b₆/f complex.     

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.     

Environmental and metaboliccontrol of LHCII protein phosphorylation: revealing the mechanismsfor dual regulation of the LHCII kinase

Light‐harvesting complex II (LHCII) protein phosphorylation inplant chloroplasts is under complex regulation. Combination of the invivo monitoring of LHCII protein phosphorylation (by immunoblotting)with the in vitro[γ‐³²P]ATPphosphorylation assays revealed that the basic activation/deactivationmodel of the LHCII kinase, regulated by reversible occupation/releaseof plastoquinol at the plastoquinol oxidation (Q_o) siteof the cytochrome b₆f (cyt b₆f) complex, isconsistent with, but not sufficient to explain the data obtainedwith isolated chloroplasts, leaf discs or intact leaves. Not onlythe light conditions but also the metabolic state of the entireplant, particularly the sugar metabolism, exerted a control overLHCII protein phosphorylation. Feeding of leaves with glucose (alsowith glutathione) activated the LHCII kinase in darkness. On the otherhand, independently of the basic activation/deactivationmechanism of the kinase, a strong inhibition of LHCII protein phosphorylationoccurred in vivo at increasing irradiances and even at lowlight conditions, depending on the metabolic state of the plant.Both the experiments with intact chloroplasts and the reconstitutionexperiments with isolated thylakoids to mimic LHCII kinase inhibition,disclosed that the kinase in its activated state (plastoquinol at theQ_o site of cyt b₆f complex) is protected againstinhibition by thiol reductants. However, directly upon deactivationof the kinase (release of plastoquinol from the Q_o site) itbecomes a target for inhibition by thiol reductants. Thus the twointerdependent regulatory systems of the LHCII kinase, the constantlyoccurring activation and deactivation on the one hand and the inhibitionby thiol reductants on the other, are strongly dependent on theconcentration of reducing equivalents in the chloroplast stroma.A scheme demonstrating the interconversion of activated, deactivated andinhibited states of the LHCII kinase in the chloroplast environmentof intact leaves is presented.     

Redox-controlled thylakoid protein phosphorylation. News and views

Thylakoid protein phosphorylation regulates state transition and PSII protein turnover under light-dependent redox control via a signal transduction system. The redox-dependent activation/deactivation of the membrane-bound protein kinase(s), mostly localized in the grana partitions, differs for the various phosphoproteins. Reduction of the plastoquinone pool may be sufficient to activate phosphorylation of few of these proteins. Phosphorylation of LHCII, requires the presence of the cytochrome bf complex in an 'activating mode' characterized by the reduction of its high potential path components and ability to interact with a reduced plastoquinol without oxidizing it. Activation and maintenance of this kinase activity is considered to involve alternate interactions with a cytochrome bf in its activating mode and with the substrate PSII(LHCII). The segregation of the thylakoid components into grana and stroma partitions appears to be mandatory for the kinase activation process. The protein substrate specificity and kinetics differs for various kinases. The thylakoid redox-controlled kinase(s) have not yet been isolated. Preparations highly enriched in kinase activity capable to phosphorylate LHCII and PSII core proteins, contain two kinase active bands, resolved by denaturing electrophoresis and renaturation, and having apparent molecular masses of about 53 and 66 kDa. The roughly estimated abundance of these putative kinase(s) in the grana partitions may be compatible with a ratio of kinase(s): PSII(LHCII) dimers:cytochrome bf dimers in the range of 1:60:30 and a ratio of kinase:phosphorylation sites of about 1:2000. Only about 10–20% of these sites are phosphorylated during state transition. The low turnover rate of the LHCII kinase(s) (< 5) may be due to hindrance of the required random lateral migration within the grana domain rich in tightly packed PSII(LHCII) and cytochrome bf complexes.     

The effects of quinone analogues on cytochrome b6 reduction and oxidation in a reconstituted system

The reconstituted system containing Photosystem I, plastocyanin and the cytochrome b6-f complex is used to study the effects of various quinone analogues on the redox behavior of cytochrome b6. The effects of DBMIB, DNP-INT and HQNO are compared in an attempt to discern the modes of action of these quinone analogues. Both DBMIB and DNP-INT are potent inhibitors of the plastocyanin reductase activity of the isolated cytochrome complex. However, while DBMIB abolished the oxidant-induced reduction of cytochrome b6, DNP-INT only inhibited about 25% of the net reduction. On the other hand, HQNO does not show any significant inhibition of plastocyanin reductase activity of the isolated cytochrome complex at concentrations up to 20 microM. An enhancement of the net amount of cytochrome b6 reduced is observed in the presence of HQNO. Both DNP-INT and HQNO inhibited the dark oxidation rate of cytochrome b6. The possible identity of the oxidant for cytochrome b6 is discussed. Plastoquinone is concluded to be the most likely candidate. DNP-INT is concluded to have at least two sites of inhibition in the cytochrome complex. The implications of these findings on quinone functions in the cytochrome b6-f complex are discussed.     

Two pathways of CD11b/CD18-mediated neutrophil aggregation with different involvement of protein kinase C-dependent phosphorylation

The characteristics of homotypic neutrophil aggregation, mediated by the adhesion molecule CD11b/CD18, differ according to whether activation takes place via intracellular protein kinase C(PKC) inducers or chemoattractants. In response to diacylglycerol (DAG) analogues such as PMA and 1,2-dioctanoyl-sn-glycerol, a prolonged cellular aggregation occurs that is associated with intense phosphorylation of the CD18 beta-chain. In response to the chemoattractant FMLP, a more transient aggregation event results that is associated with minimal beta-chain phosphorylation. By using the PKC inhibitor staurosporine, we now show that these differences are likely to reflect two different pathways of activation. Both aggregation and phosphorylation induced by DAG analogues are completely abolished by staurosporine in a parallel dose-dependent manner. Conversely, FMLP-induced aggregation is enhanced and prolonged by staurosporine whereas the associated minimal phosphorylation event is further diminished by staurosporine. Accordingly, activation of neutrophil aggregation by DAG analogues is associated with and presumably due to phosphorylation of the CD18 beta-chain. This intense phosphorylation occurs via a staurosporine-sensitive kinase such as PKC. FMLP, on the other hand, appears to activate CD11b/CD18 by a distinct mechanism. This latter mechanism does not seem to be dependent on what may be a minor PKC-induced phosphorylation of the beta-chain, and indeed is enhanced by inhibition of PKC. Of note, staurosporine was also found to cause selective release of specific granules with concomitant increase in surface display of CD11b/CD18. These data further support previous observations that up-regulation of this adhesive molecule is not the primary event in the induction of cellular adhesiveness.     

植物光合机构的状态转换

植物光合机构的状态转换是一种通过光系统Ⅱ的捕光天线色素蛋白复合体（LHCⅡ）的可逆磷酸化调节激发能在两个光系统间的分配来适应环境中光质等短期变化的机制．一般植物光合机构的LHCⅡ磷酸化主要受电子递体质醌和细胞色素b6f复合体氧化还原状态的调节,从而影响其在两种光系统间的移动。植物光合机构的状态转换也可以通过两种光系统相互接近导致激发能满溢来平衡两个光系统的激发能分配。外界离子浓度骤变可以引起盐藻LHCⅡ磷酸化,其调节过程与电子递体的氧化还原状态无关。绿藻的状态转换可以调节细胞内的ATP供求关系。     

Salt-induced redox-independent phosphorylation of light harvesting chlorophyll a/b proteins in Dunaliella salina thylakoid membranes

This study investigated the regulation of the major light harvesting chlorophyll a/b protein (LHCII) phosphorylation in Dunaliella salina thylakoid membranes. We found that both light and NaCl could induce LHCII phosphorylation in D. salina thylakoid membranes. Treatments with oxidants (ferredoxin and NADP) or photosynthetic electron flow inhibitors (DCMU, DBMIB, and stigmatellin) inhibited LHCII phosphorylation induced by light but not that induced by NaCl. Furthermore, neither addition of CuCl₂, an inhibitor of cytochrome b₆f complex reduction, nor oxidizing treatment with ferricyanide inhibited light- or NaCl-induced LHCII phosphorylation, and both salts even induced LHCII phosphorylation in dark-adapted D. salina thylakoid membranes as other salts did. Together, these results indicate that the redox state of the cytochrome b₆f complex is likely involved in light- but not salt-induced LHCII phosphorylation in D. salina thylakoid membranes.     

Large-scale purification of active cytochrome b6/f complex from spinach chloroplasts

A preparation is described through which large quantities of pure, active cytochrome b6/f complex can be isolated from spinach chloroplasts. The resulting complex is at least 90% pure with respect to the maximum content of redox centers, consists of four polypeptides according to polyacrylamide gel electrophoresis, and lacks both ferredoxin: NADP+ oxidoreductase and the high molecular weight form of cytochrome f seen in some other preparations. The complex contains 2 mol b6 and 2 atoms of nonheme iron per mole of cytochrome f, and possesses a high plastoquinol-plastocyanin oxidoreductase activity (Cyt f turnover no. 20-35 s-1). The present preparation should be helpful in the effort to crystallize the cytochrome b6/f complex.     

Light affects the accessibility of the thylakoid light harvesting complex II (LHCII) phosphorylation site to the membrane protein kinase(s)

Redox-controlled, reversible phosphorylation of the thylakoid light harvesting complex II (LHCII) regulates its association with photosystems (PS) I or II and thus, energy distribution between the two photosystems (state transition). Illumination of solubilized LHCII enhances exposure of the phosphorylation site at its N-terminal domain to protein kinase(s) and tryptic cleavage in vitro [Zer et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 8277-8282]. Here we report that short illumination (5-10 min, 15-30 micromol m(-2) s(-1)) enhances the accessibility of LHCII phosphorylation site to kinase(s) activity also in isolated thylakoids. However, prolonged illumination or higher light intensities (30 min, 80-800 micromol m(-2) s(-1)) prevent phosphorylation of LHCII in the isolated membranes as well as in vivo, although redox-dependent protein kinase activity persists in the illuminated thylakoids toward exogenous solubilized LHCII. This phenomenon, ascribed to light-induced inaccessibility of the phosphorylation site to the protein kinase(s), affects in a similar way the accessibility of thylakoid LHCII N-terminal domain to tryptic cleavage. The illumination effect is not redox related, decreases linearly with temperature from 25 to 5 degrees C and may be ascribed to light-induced conformational changes in the complex causing lateral aggregation of dephosphorylated LHCII bound to and/or dissociated from PSII. The later state occurs under conditions allowing turnover of the phospho-LHCII phosphate. The light-induced inaccessibility of LHCII to the membrane-bound protein kinase reverses readily in darkness only if induced under LHCII-phosphate turnover conditions. Thus, phosphorylation prevents irreversible light-induced conformational changes in LHCII allowing lateral migration of the complex and the related state transition process.     

Consequences of the structure of the cytochrome b6f complex for its charge transfer pathways

At least two features of the crystal structures of the cytochrome b6f complex from the thermophilic cyanobacterium, Mastigocladus laminosus and a green alga, Chlamydomonas reinhardtii, have implications for the pathways and mechanism of charge (electron/proton) transfer in the complex: (i) The narrow 11 x 12 A portal between the p-side of the quinone exchange cavity and p-side plastoquinone/quinol binding niche, through which all Q/QH2 must pass, is smaller in the b6f than in the bc1 complex because of its partial occlusion by the phytyl chain of the one bound chlorophyll a molecule in the b6f complex. Thus, the pathway for trans-membrane passage of the lipophilic quinone is even more labyrinthine in the b6f than in the bc1 complex. (ii) A unique covalently bound heme, heme cn, in close proximity to the n-side b heme, is present in the b6f complex. The b6f structure implies that a Q cycle mechanism must be modified to include heme cn as an intermediate between heme bn and plastoquinone bound at a different site than in the bc1 complex. In addition, it is likely that the heme bn-cn couple participates in photosytem I-linked cyclic electron transport that requires ferredoxin and the ferredoxin: NADP+ reductase. This pathway through the n-side of the b6f complex could overlap with the n-side of the Q cycle pathway. Thus, either regulation is required at the level of the redox state of the hemes that would allow them to be shared by the two pathways, and/or the two different pathways are segregated in the membrane.     

Cytochrome b6f complex is required for phosphorylation of light-harvesting chlorophyll a/b complex II in chloroplast photosynthetic membranes

The light-harvesting chlorophyll a/b complex (LHC II) and four photosystem II (PS II) core proteins (8.3, 32, 34 and 44 kDa) become phosphorylated in response to reduction of the intersystem electron transport chain of green plant chloroplasts. Previous studies indicated that reduction of the plastoquinone (PQ) pool is the key event in kinase activation. However, we show here that, unlike PS II proteins, LHC II is phosphorylated only when the cytochrome b6f complex is active. Two lines of evidence support this conclusion. (1) 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and the 2,4-dinitrophenyl ether of iodonitrothymol (DNP-INT), which are known to block electron flow into the cytochrome complex, selectively inhibit LHC II phosphorylation in spinach thylakoids. (2) The hcf6 mutant of maize, which contains PQ but lacks the cytochrome b6f complex, phosphorylates the four PS II proteins but fails to phosphorylate LHC II in vivo or in vitro.     

Phosphorylation-dephosphorylation of light-harvesting complex II as a response to variation in irradiance is thiol sensitive and thylakoid sufficient: modulation of the sensitivity of the phenomenon by a peripheral component

Downregulation of phosphorylation of chlorophyll a/b-binding proteins (LHCII) of the photosystem II at high irradiance could only be demonstrated with leaf discs but not in isolated thylakoids. The present view suggests this phenomenon to be regulated by stromal thioredoxin. Here, we show that high-light inactivation of LHCII phosphorylation can be reproduced in isolated thylakoids and have explained the apparent absence of inactivation in vitro to be due to the derepressed activity of a peripheral kinase. We investigated this phenomenon with Arachis hypogea thylakoids prepared with (Th:A) or without (Th:B) tricine, where tricine is known for removing peripheral proteins from thylakoids. While LHCII remained phosphorylated at high irradiance in Th:B, the response of Th:A mimicked Arachis leaflets where LHCII was transiently phosphorylated with irradiance. LHCII phosphorylation in Th:A was sensitive to thiol reducing conditions, but in Th:B, the phenomenon became insensitive to thiol reduction following illumination. Washing Th:B with tricine made them resemble Th:A, and conversely, Th:A reconstituted with the Tricine extract resembled Th:B with respect to both irradiance response and thiol sensitivity. In vitro phosphorylation reactions indicated a thiol insensitive kinase activity to be present in the Tricine extract that was capable of phosphorylating histone H1 as well as purified LHCII. This peripherally associated kinase activity explained the sustenance of LHCII phosphorylation as well as its thiol insensitivity at high irradiance in Th:B thylakoids. Contrary to the current view, our results clearly show that irradiance dependent phosphorylation and dephosphorylation of LHCII is a thylakoid sufficient phenomenon, although it remained open to regulation by thiol redox state modulation.     

Movement of the Rieske iron-sulfur protein in the p-side bulk aqueous phase: effect of lumenal viscosity on redox reactions of the cytochrome b6f complex

Based on the atomic structures of the mitochondrial cytochrome bc(1) complex, it has been proposed that the soluble domain of the [2Fe-2S] Rieske iron-sulfur protein (ISP) must rotate by ca. 60 degrees and translate through an appreciable distance between two binding sites, proximal to cytochrome c(1) and to the lumen-side quinol binding site. Such motional freedom implies that the electron-transfer rate should be affected by the lumenal viscosity. The flash-induced oxidation of cytochrome f, the chloroplast analogue of cytochrome c(1), was found to be inhibited reversibly by increased lumenal viscosity, as was the subsequent reduction of both cytochrome b(6) and cytochrome f. The rates of these three redox reactions correlated inversely with lumenal viscosity over a viscosity range of 1-10 cP. Reduction of cytochrome b(6) and cytochrome f was not concerted. The rate of cytochrome f reduction was observed to be approximately half that of cytochrome b(6) regardless of the actual viscosity, implying that the path length traversed by the ISP in reduction of cytochrome f is twice that of cytochrome b(6). This suggests that upon initiation of electron transfer by a light flash, cytochrome b(6) reduction requires movement of reduced ISP from an initial position predominantly proximal to cytochrome f, apparently favored by the reduced ISP, to the quinol binding site at which the oxidant-induced reduction of cytochrome b(6) is initiated. Subsequent reduction of cytochrome f requires the additional movement of the ISP back to a site proximal to cytochromef. There is no discernible viscosity dependence for cytochrome b(6) reduction under oxidizing conditions, presumably because the oxidized ISP preferentially binds proximal to the quinone binding niche. The dependence of the cytochrome redox reaction on ambient viscosity implies that the tethered diffusional motion of the ISP is part of the rate limitation for charge transfer through the b(6)f complex.     

Isolation and characterization of photoautotrophic mutants of Chlamydomonas reinhardtii deficient in state transition.

In photosynthetic cells of higher plants and algae, the distribution of light energy between photosystem I and photosystem II is controlled by light quality through a process called state transition. It involves a reorganization of the light-harvesting complex of photosystem II (LHCII) within the thylakoid membrane whereby light energy captured preferentially by photosystem II is redirected toward photosystem I or vice versa. State transition is correlated with the reversible phosphorylation of several LHCII proteins and requires the presence of functional cytochrome b(6)f complex. Most factors controlling state transition are still not identified. Here we describe the isolation of photoautotrophic mutants of the unicellular alga Chlamydomonas reinhardtii, which are deficient in state transition. Mutant stt7 is unable to undergo state transition and remains blocked in state I as assayed by fluorescence and photoacoustic measurements. Immunocytochemical studies indicate that the distribution of LHCII and of the cytochrome b(6)f complex between appressed and nonappressed thylakoid membranes does not change significantly during state transition in stt7, in contrast to the wild type. This mutant displays the same deficiency in LHCII phosphorylation as observed for mutants deficient in cytochrome b(6)f complex that are known to be unable to undergo state transition. The stt7 mutant grows photoautotrophically, although at a slower rate than wild type, and does not appear to be more sensitive to photoinactivation than the wild-type strain. Mutant stt3-4b is partially deficient in state transition but is still able to phosphorylate LHCII. Potential factors affected in these mutant strains and the function of state transition in C. reinhardtii are discussed.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.