Identification of a Mr = 17,000 protein as the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts

An azidoquinone derivative, 3-azido-2-methyl-5-methoxy-6-(3,7-dimethyl[3H]octyl)-1,4-benzoquinone (azido-Q), was used to study the plastoquinone-protein interaction and to identify the plastoquinone-binding protein in the cytochrome b6-f complex from spinach chloroplasts. When the lipid- and plastoquinone-deficient cytochrome b6-f complex is incubated with varying concentrations of azido-Q and illuminated with long wavelength UV light for 7 min at 2 degrees C, the enzymatic activity, assayed after reconstitution with lipid, decreases as the concentration of azido-Q increases. Maximum inactivation (45%) is observed when 30 mol of azido-Q is used per mol of cytochrome f. The extent of the decrease in activity upon illumination correlates with the amount of azido-Q incorporated into the protein. The 50% inactivation is in good agreement with that expected based on the amount of plastoquinone deficiency of the isolated enzyme complex. When the photolyzed, [3H]azido-Q-treated sample is extracted with organic solvent and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, radioactivity is found primarily in the Mr = 17,000 subunit. When the enzyme is pretreated with the electron transfer inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, significantly less radioactive label is observed in the Mr = 17,000 protein, suggesting that the action sites of these inhibitors are the same or near the plastoquinone-binding site. When the deficient complex is reconstituted with glycolipid prior to the addition of azido-Q, less than 5% inactivation is observed upon photolysis, and the amount of radioactive label on the Mr = 17,000 protein decreases greatly, suggesting that the plastoquinone-binding site is easily masked by glycolipid when endogenous plastoquinone is absent. Plastoquinol-2 apparently competes with azido-Q for the plastoquinone-binding site since it decreases the radioactive label on the Mr = 17,000 protein.     

A ubiquinone derivative that inhibits mitochondrial cytochrome b-c1 complex but not chloroplast cytochrome b6-f complex activity

A ubiquinone derivative, 3-chloro-5-hydroxyl-2-methyl-6-decyl- 1,4-benzoquinone (3-CHMDB), which shows different effects on the mitochondrial cytochrome b-c1 complex and chloroplast cytochrome b6-f complex, has been synthesized and characterized. When the cytochrome b-c1 complex is treated with varying concentrations of 3-CHMDB and assayed at constant substrate (Q2H2) concentration, a 50% inhibition is observed when 2 mol of 3-CHMDB per mol of enzyme are used. The degree of inhibition is dependent on the substrate concentration. When ubiquinol-cytochrome c reductase is treated with 2 mol of 3-CHMDB per mol of enzyme, less inhibition is observed with a lower substrate concentration, suggesting the possible existence of two forms of reductases: one with a high affinity for ubiquinone and another with a low affinity. 2-Chloro-5-hydroxyl-3-methyl-6-decyl-1,4-benzoquinone (2-CHMDB), an isomer of 3-CHMDB, shows much less inhibition of the mitochondrial cytochrome b-c1 complex, suggesting that the quinone binding site in this complex is highly specific. In contrast to the inhibition observed with the cytochrome b-c1 complex, 3-CHMDB causes no inhibition of the plastoquinol-plastocyanin reductase activity of chloroplast cytochrome b6-f complex, regardless of whether plastoquinol-2 or ubiquinol-2 is used as substrate. 3-CHMDB restores the dibromothymoquinone-altered EPR spectra of iron-sulfur protein in both complexes. In the case of the cytochrome b6-f complex, 3-CHMDB also partially restores the dibromothymoquinone-inhibited activity. Reduced form 3- or 2-CHMDB is oxidizable by the cytochrome b6-f complex, but not by the cytochrome b-c1 complex. These results suggest that the quinol oxidizing sites in the cytochrome b6-f complex may differ from those in the mitochondrial cytochrome b-c1 complex.     

Inhibitory effects of dicyclohexylcarbodiimide on spinach cytochrome b6-f complex

The electron transfer activity of purified cytochrome b6-f complex of spinach chloroplast is inhibited by dicyclohexylcarbodiimide (DCCD) in a concentration and incubation time dependent manner. The maximum inhibition of 75% is observed when 300 mole of DCCD per mole of protein (based on cytochrome f) is incubated with cytochrome b6-f complex at room temperature for 40 min. The inhibition of the complex is not due to the formation of cross links between subunits but due to the modification of carboxyls. The amount of DCCD incorporation is directly proportional to the activity loss, suggesting that some carboxyl groups in the complex are directly or indirectly involved in the catalytic function. The incorporated DCCD is located mainly at cytochrome b6 protein. The partially inhibited complex shows the same H+/e-ratio as that of the intact complex when embedded in phospholipid vesicles.     

Differential detergent-solubilization of integral thylakoid membrane complexes in spinach chloroplasts. Localization of photosystem II, cytochrome b6-f complex and photosystem I

Progressive solubilization of spinach chloroplast thylakoids by Triton X-100 was employed to investigate the domain organization of the electron transport complexes in the thylakoid membrane. Triton/chlorophyll ratios of 1:1 were sufficient to disrupt fully the continuity of the thylakoid membrane network, but not sufficient to solubilize either photosystem I (PSI), photosystem II (PSII) or the cytochrome b6-f(Cyt b6-f) complex. Progressive with the Triton concentration increase (Triton/Chl greater than 1:1), a differential solubilization of the three electron transport complexes was observed. Solubilization of the Cyt b6-f complex from the thylakoid membrane preceded that of PSI and apparently occurred early in the solubilization of stroma-exposed segments of the chloroplast lamellae. The initial removal of chlorophyll (up to 40% of the total) occurred upon solubilization of PSI from the stroma-exposed lamella regions in which PSI is localized. The tightly appressed membrane of the grana partition regions was markedly resistant to solubilization by Triton X-100. Thus, solubilization of PSII from this membrane region was initiated only after all Cyt b6-f and PSI complexes were removed from the chloroplast lamellae. The results support the notion of extreme lateral heterogeneity in the organization of the electron transport complexes in higher plant chloroplasts and suggest a Cyt b6-f localization in the membrane of the narrow fret regions which serve as a continuum between the grana and stroma lamellae.     

Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids.

In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.     

The role of the supernumerary subunit of Rhodobacter sphaeroides cytochrome bc1 complex

The smallest molecular weight subunit (subunit IV), which contains no redox prosthetic group, is the only supernumerary subunit in the four-subunit Rhodobacter sphaeroides bc1 complex. This subunit is involved in Q binding and the structural integrity of the complex. When the cytochrome bc1 complex is photoaffinity labeled with [3H]azido-Q derivative, radioactivity is found in subunits IV and I (cytochrome b), indicating that these two subunits are responsible for Q binding in the complex. When the subunit IV gene (fbcQ) is deleted from the R. sphaeroides chromosome, the resulting strain (RSdeltaIV) requires a period of adaptation before the start of photosynthetic growth. The cytochrome bc1 complex in adapted RSdeltaIV chromatophores is labile to detergent treatment (60-75% inactivation), and shows a four-fold increase in the Km for Q2H2. The first two changes indicate a structural role of subunit IV; the third change supports its Q-binding function. Tryptophan-79 is important for structural and Q-binding functions of subunit IV. Subunit IV is overexpressed in Escherichia coli as a GST fusion protein using the constructed expression vector, pGEX/IV. Purified recombinant subunit IV is functionally active as it can restore the bc1 complex activity from the three-subunit core complex to the same level as that of wild-type or complement complex. Three regions in the subunit IV sequence, residues 86-109, 77-85, and 41-55, are essential for interaction with the core complex because deleting one of these regions yields a subunit completely or partially unable to restore cytochrome bc1 from the core complex.     

Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6f complex

Purified detergent-soluble cytochrome b6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300-350 electrons per cyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f alpha-band and the Qy chlorophyll a band in the cyanobacterial complex and an absorbance band in the flavin 450-480-nm region of the chloroplast complex. An additional high molecular weight (M(r) approximately 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of approximately 0.9 (cytochrome f)(-1). The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplast b6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport.     

Oxidation-reduction reactions of cytochrome b6 in a liposome-incorporated cytochrome b6-f complex

The chloroplast cytochrome b6-f complex, incorporated into phospholipid vesicles, shows proton translocation with an observed H+/e- ratio of approximately 2. The oxidation-reduction behavior of cytochrome b6 during electron transport from duroquinol to plastocyanin is affected by incorporation. The most obvious effect of incorporation is an increase in the duration of a steady-state level of cytochrome b6 that persists during electron transport. Reagents that decrease activity increase the duration of the steady state while reagents that stimulate activity decrease this time. Uncoupling conditions yield cytochrome kinetics similar to those in the unincorporated complex. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone and 5-n-undecyl-4,7-dioxobenzothiazole inhibited reduction of cytochrome b6 in the incorporated complex, but this apparent inhibition was due to a rapid oxidation of the cytochrome by these compounds.     

Essentiality of the molecular weight 15,000 protein (subunit IV) in the cytochrome b-c1 complex of Rhodobacter sphaeroides.

The cytochrome b-c1 complex from Rhodobacter sphaeroides was resolved into four protein subunits by a phenyl-Sepharose CL-4B column eluted with different detergents. Individual subunits were purified to homogeneity. Antibodies against subunit IV (Mr = 15,000) were raised and purified. These antibodies had a high titer with isolated subunit IV and with the b-c1 complex from R. sphaeroides. They inhibited 95% of the ubiquinol-cytochrome c reductase activity of the cytochrome b-c1 complex, indicating that subunit IV is essential for the catalytic function of this complex. When detergent-solubilized chromatopores were passed through an anti-subunit IV coupled Affi-Gel 10 column, no no ubiquinol-cytochrome c reductase activity was detected in the effluent, and four proteins, corresponding to the four subunits in the isolated complex, were adsorbed to the column. This indicated that subunit IV in an integral part of the cytochrome b-c1 complex. No change in the apparent Kms for Q2H2 and for cytochrome c was observed with anti-subunit IV treated complex. Antibodies against subunit IV had little effect on the stability of the ubisemiquinone radical in this complex, suggesting that they do not bind to the subunit near its ubiquinone-binding site.     

Spatial organization of the cytochrome b6-f complex within chloroplast thylakoid membranes

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b6-f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3',5,5'-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b6-f antibodies; (4) agglutination of membrane fragments with anti-Cyt b6-f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b6-f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b6-f throughout all thylakoid membranes.     

Interactions between thylakoid electron transfer complexes. I. In vitro kinetic studies with isolated photosystem II and cytochrome b6-f complexes

The cytochrome b6-f complex from spinach thylakoids has been reconstituted with an oxygen-evolving Photosystem II (PSII) preparation isolated from the same source to give oxygenic plastocyanin reductase activity. We observe that (i) mixing of the two complexes in concentrated form prior to dilution with the assay medium is necessary for optimal reconstitution of activity; (ii) incubation for longer times after dilution can also give substantial reconstitution if the two complexes are added separately to the assay mixture; (iii) either monovalent or divalent cations are required for optimum activity in the reconstituted system; (iv) titration of the cytochrome complex with varying amounts of the PSII complex gave a saturation of the plastocyanin reduction activity at a cytochrome complex/PSII ratio of 3-4; (v) kinetic analysis of plastocyanin photoreduction by Photosystem II shows nonlinearity, while first-order reduction kinetics are observed with duroquinol as electron donor; and (vi) as the concentration of plastocyanin is increased, the half-time of the reduction increases. These observations are considered in terms of a functional association between PSII and the cytochrome b6-f complex in this reconstituted system, and the relevance of these observations to the situation in vivo is discussed.     

Nearest-neighbor relationships of the constituent polypeptides in plastoquinol-plastocyanin oxidoreductase

The nearest-neighbor relationship among the constituent polypeptides of the isolated plastoquinol-plastocyanin oxidoreductase from spinach chloroplasts has been investigated. (1) The isolated plastoquinol-plastocyanin oxidoreductase (the b6/f complex) is treated with various concentrations of the cross-linker glutaraldehyde. The treated b6/f complexes are then analyzed by SDS-polyacrylamide gel electrophoresis coupled with the immunodecoration of cross-link products by specific antibodies for each of the four prominent constituent polypeptides. Cytochrome b6 is found to be most resistant to forming any intermolecular cross-link products. At low concentrations of glutaraldehyde, the 'Rieske' iron-sulfur (Fe-S) protein and subunit IV of the b6/f complex, however, appear to form cross-link products with a relative molecular weight of 35 000. Dimers of cytochrome f and cytochrome f/Rieske protein cross-link products can also be detected. (2) When a Rieske Fe-S protein-depleted b6/f complex is used in place of the control b6/f complex, cytochrome b6 is less resistant to intermolecular cross-linking, while subunit IV does not form any 35 kDa cross-link product, unlike the case in control b6/f complex. Subunit IV is concluded to be closely associated with the Rieske Fe-S protein. This provides evidence that subunit IV is a bona fide component of the cytochrome b6/f complex, although no function can yet be assigned to it. The results are discussed in relationship to the spatial and functional relationships among the components of the b6/f complex.     

GSK3 inactivation is involved in mitochondrial complex IV defect in transforming growth factor (TGF) β1-induced senescence

Transforming growth factor β1 (TGF β1) induces Mv1Lu cell senescence by persistently producing mitochondrial reactive oxygen species (ROS) through decreased complex IV activity. Here, we investigated the molecular mechanism underlying the effect of TGF β1 on mitochondrial complex IV activity. TGF β1 progressively phosphorylated the negative regulatory sites of both glycogen synthase kinase 3 (GSK3) α and β, corresponding well to the intracellular ROS generation profile. Pre-treatment of N-acetyl cysteine, an antioxidant, did not alter this GSK3 phosphorylation (inactivation), whereas pharmacological inhibition of GSK3 by SB415286 significantly increased mitochondrial ROS, implying that GSK3 phosphorylation is an upstream event of the ROS generation. GSK3 inhibition by SB415286 decreased complex IV activity and cellular O(2) consumption rate and eventually induced senescence of Mv1Lu cell. Similar results were obtained with siRNA-mediated knockdown of GSK3. Moreover, we found that GSK3 not only exists in cytosol but also in mitochondria of Mv1Lu cell and the mitochondrial GSK3 binds complex IV subunit 6b which has no electron carrier and is topologically located in the mitochondrial intermembrane space. Involvement of subunit 6b in controlling complex IV activity and overall respiration rate was proved with siRNA-mediated knockdown of subunit 6b. Finally, TGF β1 treatment decreased the binding of the subunit 6b to GSK3 and subunit 6b phosphorylation. Taken together, our results suggest that GSK3 inactivation is importantly involved in TGF β1-induced complex IV defects through decreasing phosphorylation of the subunit 6b, thereby contributing to senescence-associated mitochondrial ROS generation.     

Subunit IV of cytochrome bc1 complex from Rhodobacter sphaeroides. Localization of regions essential for interaction with the three-subunit core complex

Recombinant subunit IV mutants which identify the regions essential for restoration of bc(1) activity to the three-subunit core complex of Rhodobacter sphaeroides were generated and characterized. Four C-terminal truncated mutants: IV(1-109), IV(1-85), IV(1-76), and IV(1-40) had 100, 0, 0, and 0% of reconstitutive activity of the wild-type IV, indicating that residues 86-109 are essential. IV(1-109) is associated with the core complex in the same manner as the wild-type IV while mutants IV(1-85), IV(1-76), and IV(1-40) do not associate with the core complex, indicating that subunit IV requires its transmembrane helix region (residues 86-109) for assembly into the bc(1) complex. Since GST-IV(86-109) fusion protein has little reconstitutive activity, some region(s) in residues 1-85 are required for bc(1) activity restoration after subunit IV is incorporated into the complex through the transmembrane helix, presumably by interaction with cytochrome b in the core complex. The interacting regions are identified as residues 41-53 and 77-85, since mutants IV(21-109), IV(41-109), IV(54-109), and IV(77-109) had 95, 98, 53, and 53% of the reconstitutive activity of the wild-type IV. These two interacting regions are on the cytoplasmic side of the chromatophore membrane and closed to the DE loop and helix G of cytochrome b, respectively.     

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and photosystem II.

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.