Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Implications of cytochromeb 6/f location for thylakoidal electron transport

The cytochromeb ₆/f complex of higher plant chloroplasts is uniformly distributed throughout both appressed and nonappressed thylakoids, in contrast to photosystem II and photosystem I, the other major membrane protein complexes involved in electron transport. We discuss how this distribution is likely to affect interactions of the cytochromeb ₆/f complex with other electron transport components because of the resulting local stoichiometries, and how these may affect the regulation of electron transport.     

Electron transport and photophosphorylation by Photosystem I in vivo in plants and cyanobacteria

Recently, a number of techniques, some of them relatively new and many often used in combination, have given a clearer picture of the dynamic role of electron transport in Photosystem I of photosynthesis and of coupled cyclic photophosphorylation. For example, the photoacoustic technique has detected cyclic electron transport in vivo in all the major algal groups and in leaves of higher plants. Spectroscopic measurements of the Photosystem I reaction center and of the changes in light scattering associated with thylakoid membrane energization also indicate that cyclic photophosphorylation occurs in living plants and cyanobacteria, particularly under stressful conditions.In cyanobacteria, the path of cyclic electron transport has recently been proposed to include an NAD(P)H dehydrogenase, a complex that may also participate in respiratory electron transport. Photosynthesis and respiration may share common electron carriers in eukaryotes also. Chlororespiration, the uptake of O₂ in the dark by chloroplasts, is inhibited by excitation of Photosystem I, which diverts electrons away from the chlororespiratory chain into the photosynthetic electron transport chain. Chlororespiration in N-starved Chlamydomonas increases ten fold over that of the control, perhaps because carbohydrates and NAD(P)H are oxidized and ATP produced by this process.The regulation of energy distribution to the photosystems and of cyclic and non-cyclic phosphorylation via state 1 to state 2 transitions may involve the cytochrome b ₆-f complex. An increased demand for ATP lowers the transthylakoid pH gradient, activates the b ₆-f complex, stimulates phosphorylation of the light-harvesting chlorophyll-protein complex of Photosystem II and decreases energy input to Photosystem II upon induction of state 2. The resulting increase in the absorption by Photosystem I favors cyclic electron flow and ATP production over linear electron flow to NADP and poises the system by slowing down the flow of electrons originating in Photosystem II.Cyclic electron transport may function to prevent photoinhibition to the photosynthetic apparatus as well as to provide ATP. Thus, under high light intensities where CO₂ can limit photosynthesis, especially when stomates are closed as a result of water stress, the proton gradient established by coupled cyclic electron transport can prevent over-reduction of the electron transport system by increasing thermal de-excitation in Photosystem II (Weis and Berry 1987). Increased cyclic photophosphorylation may also serve to drive ion uptake in nutrient-deprived cells or ion export in salt-stressed cells.There is evidence in some plants for a specialization of Photosystem I. For example, in the red alga Porphyra about one third of the total Photosystem I units are engaged in linear electron transfer from Photosystem II and the remaining two thirds of the Photosystem I units are specialized for cyclic electron flow. Other organisms show evidence of similar specialization.Improved understanding of the biological role of cyclic photophosphorylation will depend on experiments made on living cells and measurements of cyclic photophosphorylation in vivo.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FCCP carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone - LHC light harvesting chlorophyll - LHCP II light harvesting chlorophyll protein of Photosystem II - PQ plastoquinone - PS I, II Photosystem I, II - SHAM salicyl hydroxamic acid - TBT Tri-n-butyltin CIW/DPB Publication No. 1146     

Electron transfer from cytochromeb 5 to cytochromec

The reaction of cytochromeb ₅ with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.     

Electron transfer from cytochrome f to photosystem I in green algae

The time course of P700⁺ reduction and cytochrome f oxidation following a single-turnover flash excitation of photosystem I was measured under various conditions in different strains of green algae. P700⁺ was reduced with a half-time of 4 s. The rate of cytochrome f oxidation was found to depend widely on physiological factors. Reversible transitions are described from a slow-oxidation state (t _1/2=500 s) to a fast-oxidation state (t _1/2=80 s). The addition of ionophore strongly favours and stabilizes the fast-oxidation state. We suggest that these transitions reflect either reversible association between the cytochrome bf complex and the reaction center of photosystem I or changes in the mobility of oxidized plastocyanin. The transitions might be under the control of the membrane potential or the intracellular ATP content. The relation of these reversible transitions with the light state transitions, and their possible involvement in a switch from linear to cyclic electron transfer, are discussed.Abbreviations cyt cytochrome - DCHC dicyclohexyl-18-crown-6 - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT dinitrophenylether of iodonitrothymol - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - LHC light harvesting complex - PC plastocyanin - PS I photosystem I     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Electron transfer in the water-oxidizing complex of Photosystem II

An overview is presented of secondary electron transfer at the electron donor side of Photosystem II, at which ultimately two water molecules are oxidized to molecular oxygen, and the central role of manganese in catalyzing this process is discussed. A powerful technique for the analysis of manganese redox changes in the water-oxidizing mechanism is the measurement of ultraviolet absorbance changes, induced by single-turnover light flashes on dark-adapted PS II preparations. Various interpretations of these ultraviolet absorbance changes have been proposed. Here it is shown that these changes are due to a single spectral component, which presumably is caused by the oxidation of Mn(III) to Mn(IV), and which oscillates with a sequence +1, +1, +1, –3 during the so-called S₀ S₁ S₂ S₃ S₀ redox transitions of the oxygen-evolving complex. This interpretation seems to be consistent with the results obtained with other techniques, such as those on the multiline EPR signal, the intervalence Mn(III)-Mn(IV) transition in the infrared, and EXAFS studies. The dark distribution of the S states and its modification by high pH and by the addition of low concentrations of certain water analogues are discussed. Finally, the patterns of proton release and of electrochromic absorbance changes, possibly reflecting the change of charge in the oxygen-evolving system, are discussed. It is concluded that nonstoichiometric patterns must be considered, and that the net electrical charge of the system probably is the highest in state S₂ and the lowest in state S₁.     

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl--maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A molecular weight of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents.The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P₇₀₀. The full set of Fe-S clusters (F_A, F_B and F_X) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy.Abbreviations BA benzamidine - CAS 6-amino-n-caproic acid - C₈-G octyl--D-glucopyranoside - C₁₂-M lauryl--D-maltoside - C₁₀-M decyl--D-maltoside - C₈-TG octyl--D-thioglucoside - Chl a chlorophyll a - EPR electron paramagnetic resonance - F_A, F_B, F_X iron-sulfur centers - HPLC high perfomance liquid chromatography - kDa kilodalton(s) - LDAO lauryldimethylamine oxide - MES 2-(N-morpholino)ethanesulfonic acid - PSI Photosystem I - PS II Photosystem II - P₇₀₀ primary electron donor - SB12 sulfobetain 12 - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - Tris tris(hydroxymethyl)-aminomethane     

Supercomplexes of plant photosystem I with cytochrome b6f,light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b₆f (Cytb₆f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb₆f at the antenna side of PSI. The rectangular-shaped Cytb₆f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb₆f, may improve regulation of electron transport by the control of binding partners and distances in small domains.     

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH2 oxidation by cytb6/f complex

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.     

The chloroplast cytochrome b 6 f complex can exist in monomeric and dimeric states

The cytochrome b ₆ f complex isolated from spinach chloroplast membranes can be resolved into two forms, a monomeric and a dimeric form, by centrifugation on sucrose gradients. The conversion of the dimeric form of the complex into the monomeric form could be prevented by cross-linking with the homobifunctional reagent, dithiobis(succinimidylpropionate) but not by cross-linking with disuccinimidyltartrate or glutaraldehyde. SDS-PAGE analyses of the monomeric and dimeric forms of the cytochrome complex showed the presence of specific cross-linked products in each respective form of the complex. For example, the monomeric form contained a cross-linked product of cytochrome f, cytochrome b ₆ f and subunit IV while the dimeric form contained a cross-linked dimer of cytochrome b ₆ f. The presence of the former in the isolated cytochrome b ₆ f complex prepared by the method of Hurt and Hauska (Eur J Biochem 117: 591–599, 1981) indicates the presence of the monomer in his preparation.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSP dithiobis(succinimidylpropionate) - DST disuccinimidyltartrate     

Functional activities of monomeric and dimeric forms of the chloroplast cytochrome b6f complex

A monomeric form of the isolated cytochrome b6f complex from spinach chloroplast membranes has been isolated after treatment of the dimeric complex with varying concentrations of Triton X-100. The two forms of the complex are similar as regards electron transfer components and subunit composition. In contrast to a previous report (Huang et al. (1994) Biochemistry 33: 4401–4409) both the monomer and dimer are enzymatically active. However, after incorporation of the respective complexes into phospholipid vesicles, only the dimeric form of the cytochrome complex shows uncoupler sensitive electron transport, an indication of coupling of electron transport to proton translocation. The absence of this activity with the monomeric form of the cytochrome complex may be related to an inhibition by added lipids.Abbreviations CCCP- carbonyl cyanide m-chlorophenylhydrazone - mega-9- nonanoyl-N-methylglucamide     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.