Dimer to Monomer Conversion of the Cytochromeb6 f Complex

109

The cytochrome b 6 f complex of oxygenic photosynthesis carries out "dark reactions" of electron transfer that link the light-driven reactions of the reaction centers, and coupled proton transfer that generates part of the electrochemical potential utilized for ATP synthesis. In contrast to the bc 1 complex of the respiratory chain, with which there are many structural and functional homologies, the b 6 f complex contains bound pigment molecules. Along with the specifically bound chlorophyll a previously found to be bound stoichiometrically in the dimeric b 6 f complex, it was found in the present study that ␤-carotene is also present in the b 6 f complex at stoichiometric levels or nearly so. Chlorophyll and carotenoid pigments were quantitatively extracted from b 6 f complex purified from (i) the thermophilic cyanobacterium, Mastigocladus laminosus, (ii) spinach chloroplasts, and (iii) the green alga, Chlamydomonas reinhardtii. Visible and mass spectra showed the carotenoid to be a ␤-carotene of molecular weight ‫؍‬ 536, with a stoichiometry of 1.0:1 relative to cytochrome f in the highly active M. laminosus complex but somewhat lower stoichiometries, 0.77 and 0.55, in the b 6 f complex obtained from spinach chloroplasts and C. reinhardtii. A photoprotective function for the ␤-carotene was inferred from the findings that the rate of photobleaching of the chlorophyll a bound in the complex was found to vary inversely with ␤-carotene content and to decrease markedly in the presence of ambient N 2 instead of air. The presence of ␤-carotene in the b 6 f complex, and not in the related bc 1 complexes of the mitochondrial respiratory chain and photosynthetic bacteria, suggests that an additional function is to protect the protein complexes in oxygenic photosynthetic membranes against toxic effects of intramembrane singlet O 2 .

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Stoichiometrically Bound β-Carotene in the CytochromebfComplex of Oxygenic Photosynthesis Protects against Oxygen Damage

Zhang

Huang

Cramer

1999

109

“…3, where it appears that FCCP did not alter either the oligomeric state cytochrome b 6 f complex nor its composition. The treated complex migrated as a dimer (36), and all subunits comigrated, especially the Rieske protein. This is important evidence for the structural integrity of the complex, as it has been shown that in case of any perturbation of the complex, the Rieske protein is the first subunit to be lost (4,33,36).…”

Section: Effects Of Increasing Concentrations Of Fccp On the Cytochromentioning

confidence: 99%

Kinetic Effects of the Electrochemical Proton Gradient on Plastoquinone Reduction at the Qi Site of the Cytochrome b 6 f Complex

Barbagallo¹,

Breyton²,

Finazzi³

2000

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We have investigated the effects of the light-induced thylakoid transmembrane potential on the turnover of the b 6 f complex in cells of the unicellular green alga Chlamydomonas reinhardtii. The reduction of the potential by either decreasing the light intensity or by adding increasing concentrations of the ionophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) revealed a marked inhibition of the cytochrome b 6 oxidation rate (10-fold) without substantial modifications of cytochrome f oxidation kinetics. Partial recovery of this inhibition could be obtained in the presence of ionophores provided that the membrane potential was re-established by illumination with a train of actinic flashes fired at a frequency higher than its decay. Measurements of isotopic effects on the kinetics of cytochrome b 6 oxidation revealed a synergy between the effects of ionophores and the H 2 O-D 2 O exchange. We propose therefore, that protonation events influence the kinetics of cytochrome b 6 oxidation at the Qi site and that these reactions are strongly influenced by the lightdependent generation of a transmembrane potential.The cytochrome b 6 f complex is a central component of the photosynthetic chain, transferring electrons from the photosystem II (PSII) 1 to the photosystem I (PSI). It comprises four major subunits (1, 2): the Rieske protein, which binds a Fe 2 S 2 cluster, with an E m of ϩ290 mV (3), a c-type cytochrome, cytochrome f, with an E m of ϩ330 mV (2), a b-type cytochrome, cytochrome b 6 , which binds the high and low potential hemes, b h and b l , with E m values of Ϫ84 mV and Ϫ158 mV, respectively (2), and the subunit IV. Four additional small subunits PetG, PetL, PetM and PetN (2, 4 -5) have also been evidenced. Sequence comparisons have shown that cytochrome b 6 and subunit IV are, respectively, homologous to the N-and C-terminal parts of mitochondrial and bacterial cytochromes b (6).As a member of the bc-type proteins, cytochrome b 6 f complex couples proton translocation across the membrane to electron transfer from a lipophilic two-electron donor (plastoquinol) to a hydrophilic one-electron acceptor protein (plastocyanin or a c-type cytochrome). This electron transfer operates through a high potential chain (also called the linear path) formed by the Rieske protein and cytochrome f. The most widely accepted hypothesis to explain the function mechanism of bc complexes is the "Q cycle" hypothesis of Mitchell (7). It predicts that oxidation of quinols not only involves the linear path but also a cyclic path composed of the b hemes, b l and b h , and termed the low potential chain. This mechanism, as modified by Crofts et al. (8), postulates both an oxidation and a reduction of plastoquinol at two distinct sites of the protein, the Qo and Qi sites, on opposite sides of the membrane. The oxidation of plastoquinol at the Qo site is associated with the reduction of both cytochrome f and b l (7-8) and the release of two protons into the lumen.The recent elucidation of the structure of the respirator...

“…Briefly, specific solubilization of the thylakoid membranes by 6-O-(N-heptylcarbamoyl)-methyl-␣-d-glycopyranoside (Hecameg) 2 is followed by sucrose gradient and hydroxylapatite chromatography. The two last steps of purification must be performed in the presence of lipids (egg L-␣-phosphatidylcholine (PC)) and at the critical micellar concentration of the detergent to prevent dissociation of the complex (38).…”

Section: Methodsmentioning

confidence: 99%

Conformational Changes in the Cytochromeb 6 f Complex Induced by Inhibitor Binding

Breyton

2000

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Most energy-transducing membranes contain a bc-type complex such as the cytochrome bc 1 complex or the cytochrome b 6 f complex. Members of this family couple electron transfer and proton translocation across the membrane. They also share some structural homologies, having three redox-active subunits in common: the Rieske iron-sulfur protein, a b-type cytochrome (cytochrome b or b 6 ), and a c-type cytochrome (cytochrome c 1 or f). Moreover, there is sequence homology between cytochrome b 6 , subunit IV, and the Rieske protein of the b 6 f complex, and the N-and C-terminal parts of cytochrome b, and the Rieske protein of the bc 1 complex, respectively. Cytochrome c 1 and cytochrome f, on the other hand, do not share any sequence homology. Spectroscopic and EPR properties of the hemes and of the iron-sulfur cluster are similar, although not identical, and some inhibitors affect both complexes equally (reviewed in Refs. 4 -7).The b 6 f complex is found in the thylakoid membrane of higher plants and algae and in the membrane of some cyanobacteria (for reviews, see . Besides the four high molecular weight subunits mentioned above, the b 6 f complex comprises four smaller subunits, PetG, PetL, PetM, and , that have no direct counterpart in the bc 1 complex. The cytochrome b 6 f is the middle component of the photosynthetic chain, coupling the electron transfer from photosystem II (via plastoquinol) to photosystem I (via plastocyanin or a c-type cytochrome).The most widely accepted mechanism for the bc-type complexes is the so-called "Q-cycle" mechanism proposed by Mitchell (18) and modified by Crofts et al. (19). According to this model, two electron paths are distinguished within the complex as follows: a high potential chain, composed of the iron-sulfur cluster of the Rieske protein and the heme of the c-type heme, and a low potential chain, composed of the two hemes of the b-type cytochrome, the low potential b L and high potential b H hemes. Two quinol/quinone-binding sites, located on opposite sides of the membrane, are also predicted: the Q o site, on the intermembrane space/lumenal side of the membrane, and the Q i site, on the matrix/stromal side of the membrane. A turnover of the Q o site comprises the oxidation of a two-electron carrier quinol, one electron reducing the Rieske protein, which in turn reduces the c-type cytochrome and which ultimately reduces plastocyanin or cytochrome c, and the other electron reducing b L , which in turn is oxidized by b H . These events are associated with the release of two protons in the intermembrane space/ lumen. A second turnover reduces another plastocyanine molecule and places both hemes of the b-type cytochrome in a reduced state. The reduction of a quinone at the Q i site results in the oxidation of the two b hemes and an uptake of two protons from the matrix/stroma. Thus, these complexes contribute to generating the proton gradient that drives ATP synthesis in respiration and photosynthesis.Recently, the structure of several bc 1 complexes from mitochondria have ...