Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex.

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.

Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex.

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.

On the interaction between cytochrome f and plastocyanin.

The interaction between cytochrome f and its electron acceptor plastocyanin (PC) was studied. To address the question of which specific regions and which of the positively charged residues of cytochrome f are important for the interaction with the negatively charged residues of PC we have used two different experimental approaches. Cytochrome f was proteolytically cleaved and fragments that could bind to a PC-affinity column were isolated. The smallest of these fragments was analysed to give information on the minimum structural requirement for binding to PC. By this procedure, we identified a peptide of approx. 11 kDa, containing the heme binding site, and having an N-terminal sequence identical to that of the mature cytochrome f. This finding suggests that the first 90 amino acids of cytochrome f contain at least some of the residues interacting with PC. The second approach involved modification of Arg residues of cytochrome f with the specific chemical modifier, hydroxyphenylglyoxal (HPG). Cytochrome f modification was performed in the absence of PC to enable identification of residues that are protected from modification when PC is bound to cytochrome f. Two peptides containing Arg residues which are modified in the absence of PC, but are not modified when PC is present, were isolated. Sequence analysis of these two peptides revealed that Arg residues no. 88 and 154 of cytochrome f are the residues that are protected from modification when cytochrome f is bound to PC, suggesting a role for these residues in the binding of cytochrome f to PC.

On the site of function of the Rieske iron-sulfur center in the chloroplast electron transport chain.

A photosynthetic mutant (strain 1073) of Lemna perpusilla was previously shown to have a block in the electron transport chain between plastoquinone and cytochrome f ((1976) Plant Physiol. 57, 577--579). Electron paramagnetic resonance analysis of chloroplasts from this mutant indicates that the g = 1.89 signal of a reduced iron-sulfur center (the 'Rieske' iron-sulfur center) is absent. The absence of this signal indicates the Rieske center is either absent from or defective in the mutant, and this result is consistent with this iron-sulfur center functioning between plastoquinone and cytochrome f in the electron transport chain of chloroplasts.

Nucleotide sequence of the PetM gene encoding a 4 kDa subunit of the cytochrome b6f complex from Chlamydomonas reinhardtii.

We have determined the nucleotide sequence of the PetM gene from the single celled alga Chlamydomonas reinhardtii. The gene encodes a recently characterized, small protein of the cytochrome b6f complex, and based on this sequence, it is proposed that this protein spans the membrane by a single alpha-helix. Comparison of the nucleotide sequence with the deduced amino acid sequence reveals a 60-amino-acid presequence similar to a stroma-targeting peptide.

Iron-sulfur proteins of the green photosynthetic bacterium Chlorobium.

The iron-sulfur proteins of the green photosynthetic bacterium Chlorobium have been characterized by oxidation-reduction potentiometry in conjunction with low-temperature electron paramagnetic resonance spectroscopy. Chlorobium ferredoxin was the only iron-sulfur protein detected in the soluble fraction; no high-potential iron-sulfur protein was observed. In addition, high-potential iron-sulfur protein was not detected in the chromatophores. Four chromatophore-bound iron-sulfur proteins were detected. One is the "Rieske" type iron-sulfur protein with a g-value of 1.90 in the reduced state; the protein has a midpoint potential of + 160 mV (pH 7.0), and this potential is pH dependent. Three g=1.94 chromatophore-bound iron-sulfur proteins were observed, with midpoint potentials of -25, -175, and about -550 mV. A possible role for the latter iron-sulfur protein in the primary photochemical reaction in Chlorobium is considered.

Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast photosystem I at low temperatures.

The extent of P-700 photooxidation at 18 degrees K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700+ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts.

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short-dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photo-reduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.

Duroquinol as an electron donor for chloroplast electron transfer reactions.

Duroquinol (tetramethylhydroquinone) was found to function as an electron donor in chloroplasts. Non-cyclic electron transfer from duroquinol to electron acceptors such as oxygen proceeded at high rates, was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) but was sensitive to the plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB). The electron transport from duroquinol was coupled to the synthesis of ATP. Spectroscopic studies of chloroplast electron carriers in the dark indicated the high-potential "Rieske" iron-sulfur center, cytochrome f, plastocyanin and P-700 were all reduced by duroquinol. The dark reduction of the "Rieske" iron-sulfur center and cytochrome f were inhibited by DBMIB but not by DCMU. These results have been interpreted in terms of a linear sequence of electron carriers in the non-cyclic electron transport chain which includes plastoquinone, the "Rieske" iron-sulfur center, cytochrome f, plastocyanin and P-700.

The relationship between the lifetime and yield of the 735 nm fluorescence of chloroplasts at low temperatures.

The lifetime and relative yield of the 735 nm fluorescence of chloroplasts, over a range of low temperatures (-60 to -196 degrees C) where the yield of fluorescence changes markedly, were found to be directly proportional. It is concluded that the species of chlorophyll responsible for the 735 nm fluorescence, C-705, is present over the entire temperature range but is less fluorescent at the higher temperatures because of greater energy transfer to P-700. It is also concluded from attempts to measure the rise-time of the 735 nm fluorescence at -196 degrees C that the rise-time is less than 50 ps.

Green algal cytochrome b6-f complexes: isolation and characterization from Dunaliella saline, Chlamydomonas reinhardtii and Scenedesmus obliquus.

Cytochrome b6-f complexes have been isolated from Chlamydomonas reinhardtii, Dunaliella saline and Scenedesmus obliquus. Each complex is essentially free of chlorophyll and carotenoids and contains cytochrome b6 and cytochrome f hemes in a 2:1 molar ratio. C. reinhardtii and S. obliquus complexes contain the Rieske iron-sulfur protein (present in approx 1:1 molar ratio to cytochrome f) and each catalyzes a DBMIB- and DNP-INT-sensitive electron transfer from duroquinol to spinach plastocyanin. Immunological assays using antibodies to the peptides from the spinach cytochrome complex show varying cross-reactivity patterns except for the complete absence of binding to the Rieske proteins in any of the three complexes, suggesting little structural similarity between the Rieske proteins of algae with those from higher plants. One complex (D. salina) has been uniformly labeled by growth in NaH14CO3 to determine stoichiometries of constituent polypeptide subunits. Results from these studies indicate that all functionally active cytochrome b6-f complexes contain four subunits which occur in equimolar amounts.

The role of plastoquinone and beta-carotene in the primary reaction of plant photosystem II.

Extraction of Triton Photosystem II chloroplast fragments with 0.2% methanol in hexane for 3 h results in the removal of 90 to 95% of the plastoquinone in the original preparation. The extracted fragments (chlorophyll:plastoquinone ratio, 900: 1) showed no P-680 photooxidation at 15 K after a single laser flash. The extracted fragments also showed no light-induced C-550 absorbance change at 77 K. Reconstitution of the primary reaction of Photosystem II, as evidenced by restoration of low-temperature photooxidation of P-680, could be obtained by the addition of plastoquinone A but not by the addition of beta-carotene. The addition of beta-carotene plus plastoquinone A restored the C-550 absorbance change. These results indicate that plastoquinone functions as the primary electron acceptor of Photosystem II and that beta-carotene does not play a direct role in the primary photochemistry but is required for the C-550 absorbance change.

PRoperties of the low-temperature photosystem I primary reaction in the P-700-chlorophyll alpha-protein.

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15 degrees K in P-700-chlorophyll alpha-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300 degrees K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15 degrees K as well as at 300 degrees K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.

Evidence for the presence of a [2Fe-2S] ferredoxin in bean sprouts.

An iron-sulfur protein with properties similar to those of ferredoxins found in the leaves of higher plants has been isolated from bean sprouts--a non-photosynthetic plant tissue. The bean sprout protein has a molecular mass of 12.5 kDa and appears to contain a single [2Fe-2S] cluster. The absorbance and circular dichroism spectra of the bean sprout protein resemble those of spinach leaf ferredoxin and the bean sprout protein can replace spinach ferredoxin as an electron donor for NADP+ reduction, nitrite reduction and thioredoxin reduction by spinach leaf enzymes. Although the reduced bean sprout protein (Em = -440 mV) is a slightly stronger reductant than spinach ferredoxin and appears to be less acidic than spinach ferredoxin, the two proteins are similar enough so that the bean sprout protein is recognized by an antibody raised against spinach ferredoxin.

Structural characterization of heme sites in spinach cytochrome b6f complexes: a resonance Raman study.

Resonance Raman spectra of cytochrome b6f complexes isolated from spinach chloroplasts have been obtained. Selective resonance enhancements and partial reductions of the complex by redox mediators were used to isolate and identify the contributions of heme b6 and heme f sites to the observed spectra. Corresponding spectra for turnip cytochrome f have also been obtained. Power-dependent photoreduction was observed in cytochrome f of the complex as well as in the isolated cytochrome f during the course of the Raman experiments.

Interaction of stigmatellin and DNP-INT with the Rieske iron-sulfur center of the chloroplast cytochrome b6-f complex.

Stigmatellin and DNP-INT are effective inhibitors of the catalytic activity of the plastoquinol-plastocyanin oxidoreductase complex (cytochrome b6-f complex). Both inhibitors alter the EPR spectrum of the Rieske iron-sulfur center but do not produce band-shifts of cytochrome b-563. The midpoint redox potential of the Rieske center is unaffected by either inhibitor, although both alter the DBMIB-induced g-value shifts of the Rieske center. The results are considered in terms of binding domains for inhibitors in the cytochrome b6-f complex.

Reconstitution of isolated Rieske Fe-S protein into a Rieske-depleted cytochrome b6-f complex.

The Rieske Fe-S protein can be isolated from the cytochrome b6-f complex by means of chromatography on a hydroxyapatite column in the presence of detergent. Depletion of the cytochrome complex from the Rieske protein results in the loss of oxidoreductase activity, as well as the ability to reduce cytochrome b6. The Rieske Fe-S protein can be reconstituted into the Rieske-depleted complex by removal of the Triton X-100 molecules associated with the protein fractions, and their substitution by lipids. Upon reconstitution the complex is reactivated, and the role of the Rieske Fe-S protein in the reduction of both plastocyanin and cytochrome b6 can be demonstrated.

Photosystem I reaction-centre proteins contain leucine zipper motifs. A proposed role in dimer formation.

The photosystem I (PS I) reaction-centre polypeptides, encoded by the psaA and psaB genes, are shown to contain several highly conserved leucine repeats, consisting of a leucine residue every seventh amino acid, similar to the leucine zipper motifs known to mediate DNA-binding polypeptide dimerisation. In each of the PSI reaction-centre subunits the leucine zipper motif precedes highly conserved cysteine residues which have been proposed to ligate the interpolypeptide [4Fe-4S] centre, Fx. We propose that PS I reaction-centre dimerisation and [4Fe-4S] centre formation are mediated through the leucine zipper.