High Yield Non-detergent Isolation of Photosystem I-Light-harvesting Chlorophyll II Membranes from Spinach Thylakoids: IMPLICATIONS FOR THE ORGANIZATION OF THE PS I ANTENNAE IN HIGHER PLANTS.

Styrene-maleic acid copolymer was used to effect a non-detergent partial solubilization of thylakoids from spinach. A high density membrane fraction, which was not solubilized by the copolymer, was isolated and was highly enriched in the Photosystem (PS) I-light-harvesting chlorophyll (LHC) II supercomplex and depleted of PS II, the cytochrome b6/f complex, and ATP synthase. The LHC II associated with the supercomplex appeared to be energetically coupled to PS I based on 77 K fluorescence, P700 photooxidation, and PS I electron transport light saturation experiments. The chlorophyll (Chl) a/b ratio of the PS I-LHC II membranes was 3.2 ± 0.9, indicating that on average, three LHC II trimers may associate with each PS I. The implication of these findings within the context of higher plant PS I antenna organization is discussed.

A map of dielectric heterogeneity in a membrane protein: the hetero-oligomeric cytochrome b6f complex.

The cytochrome b6f complex, a member of the cytochrome bc family that mediates energy transduction in photosynthetic and respiratory membranes, is a hetero-oligomeric complex that utilizes two pairs of b-hemes in a symmetric dimer to accomplish trans-membrane electron transfer, quinone oxidation-reduction, and generation of a proton electrochemical potential. Analysis of electron storage in this pathway, utilizing simultaneous measurement of heme reduction, and of circular dichroism (CD) spectra, to assay heme-heme interactions, implies a heterogeneous distribution of the dielectric constants that mediate electrostatic interactions between the four hemes in the complex. Crystallographic information was used to determine the identity of the interacting hemes. The Soret band CD signal is dominated by excitonic interaction between the intramonomer b-hemes, bn and bp, on the electrochemically negative and positive sides of the complex. Kinetic data imply that the most probable pathway for transfer of the two electrons needed for quinone oxidation-reduction utilizes this intramonomer heme pair, contradicting the expectation based on heme redox potentials and thermodynamics, that the two higher potential hemes bn on different monomers would be preferentially reduced. Energetically preferred intramonomer electron storage of electrons on the intramonomer b-hemes is found to require heterogeneity of interheme dielectric constants. Relative to the medium separating the two higher potential hemes bn, a relatively large dielectric constant must exist between the intramonomer b-hemes, allowing a smaller electrostatic repulsion between the reduced hemes. Heterogeneity of dielectric constants is an additional structure-function parameter of membrane protein complexes.

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b6f complex is active in plastoquinone reduction: implications for cyclic electron transport.

In this study, we have compared three isolation methods of cytochrome b6f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b6f-associated ferredoxin:NADP+ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH-PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b6f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2-n-nonyl-4-hydroxyquinoline N-oxide, an inhibitor of the cytochrome b6 f complex at the Q(i) site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c(i) . The obtained data indicate that FNR associated with the cytochrome b6 f complex can participate in the cyclic electron transport as PSI-PQ or NADPH-PQ oxidoreductase. Moreover, we have shown that PQ can be non-enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non-photochemical reduction pathways of the PQ-pool in chloroplasts.

Purification and crystallization of the cyanobacterial cytochrome b6f complex.

The cytochrome b6f complex from the filamentous cyanobacteria (Mastigocladus laminosus, Nostoc sp. PCC 7120) and spinach chloroplasts has been purified as a homo-dimer. Electrospray ionization mass spectroscopy showed the monomer to contain eight and nine subunits, respectively, and dimeric masses of 217.1, 214.2, and 286.5 kDa for M. laminosus, Nostoc, and the complex from spinach. The core subunits containing or interacting with redox-active prosthetic groups are petA (cytochrome f), B (cytochrome b6, C (Rieske iron-sulfur protein), D (subunit IV), with protein molecular weights of 31.8-32.3, 24.7-24.9, 18.9-19.3, and 17.3-17.5 kDa, and four small 3.2-4.2 kDa polypeptides petG, L, M, and N. A ninth polypeptide, the 35 kDa petH (FNR) polypeptide in the spinach complex, was identified as ferredoxin:NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.8/cytf. The spinach complex contains diaphorase activity diagnostic of FNR and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and ß-carotene at a ratio of approximately one molecule of each per cytochrome f. It also contains bound lipid and detergent, indicating seven lipid-binding sites per monomer. Highly purified complexes are active for approximately 1 week after isolation, transferring 200-300 electrons/cytf s. The M. laminosus complex was shown to be subject to proteolysis and associated loss of activity if incubated for more than 1 week at room temperature. The Nostoc complex is more resistant to proteolysis. Addition of pure synthetic lipid to the cyanobacterial complex, which is mostly delipidated by the isolation procedure, allows rapid formation of large (≥0.2 mm) crystals suitable for X-ray diffraction analysis and structure determination. The crystals made from the cyanobacterial complex diffract to 3.0 Å with R values of 0.222 and 0.230 for M. laminosus and Nostoc, respectively. It has not yet been possible to obtain crystals of the b6f complex from any plant source, specifically spinach or pea, perhaps because of incomplete binding of FNR or other peripheral polypeptides. Well diffracting crystals have been obtained from the green alga, Chlamydomonas reinhardtii (ref. 10).

Isolation of cytochrome b6f complex from grana and stroma membranes from spinach chloroplasts.

The cytochrome b6f complex is located in the appressed granal membranes and nonappressed stroma thylakoids. The procedure presents isolation of the complex from both types of thylakoids by washing with NaBr, detergent treatment, ammonium sulfate fractionation, and sucrose gradient centrifugation. Optimal concentration of the detergent is lower for grana than for stroma vesicles. The cytochrome b6f complex from stroma lamellae locates at a higher density in the sucrose gradient than the granal complex. Electrophoretic analyses indicate that both complexes are dimeric and contain four large subunits and at least three small subunits with masses below 4 kDa. Plastocyanin and 15 kDa protein are also identified in the complexes but in variable amounts.

b6f-Associated chlorophyll: structural and dynamic contribution to the different cytochrome functions.

Cytochromes bc1/b6f complexes catalyze electron transfer from lipid- to water-soluble carriers in both the respiratory and photosynthetic processes. They contain several common redox cofactors, while a chlorophyll a molecule, the function of which is still enigmatic, is only present in b b6f-type complexes. In this work, we describe a mutagenesis approach aimed at characterizing the role of this pigment. Mutants of the binding pocket were constructed to obtain cytochrome (cyt) b6f f complexes altered in chlorophyll position and/or stability. On the basis of a combined biochemical and functional analysis, we conclude that the chlorophyll plays a major structural role in the complex. Moreover, the chlorophyll and its binding pocket may also be implicated in the regulation of photosynthetic state transitions, a function that is specific to cyt b6f complexes.

Proteomic approach to characterize the supramolecular organization of photosystems in higher plants.

A project to investigate the supramolecular structure of photosystems was initiated, which is based on protein solubilizations by digitonin, protein separations by Blue native (BN)-polyacrylamide gel electrophoresis (PAGE) and protein identifications by mass spectrometry (MS). Under the conditions applied, nine photosystem supercomplexes could be described for chloroplasts of Arabidopsis, which have apparent molecular masses between 600 and 3200 kDa on BN gels. Identities of the supercomplexes were determined on the basis of their subunit compositions as documented by 2D BN/SDS-PAGE and BN/BN-PAGE. Two supercomplexes of 1060 and approximately 1600 kDa represent dimeric and trimeric forms of photosystem I (PSI), which include tightly bound LHCI proteins. Compared to monomeric PSI, these protein complexes are of low abundance. In contrast, photosystem II mainly forms part of dominant supercomplexes of 850, 1000, 1050 and 1300 kDa. According to our interpretation, these supercomplexes contain dimeric PSII, 1-4 LHCII trimers and additionally monomeric LHCII proteins. The 1300-kDa PSII supercomplex (containing four LHCII trimers) is partially converted into the 1000-kDa PSII supercomplex (containing two LHCII trimers) in the presence of dodecylmaltoside on 2D BN/BN gels. Analyses of peptides of the trypsinated 1300-kDa PSII supercomplex by mass spectrometry allowed to identify known subunits of the PSII core complex and additionally LHCII proteins encoded by eight different genes in Arabidopsis. Further application of this experimental approach will allow new insights into the supermolecular organization of photosystems in plants.

Purification and crystallization of the cytochrome b6f complex in oxygenic photosynthesis.

The cytochrome b6f complex from the thermophilic cyanobacterium Mastigocladus laminosus and spinach chloroplasts has been purified as a dimeric species. It was found by electrospray ionization mass spectroscopy to contain eight and nine subunits, respectively, and dimeric masses of 217,070 and 286,454 Da. The subunits common to the complex from both sources are petA (cytochrome f), B (cytochrome b6), C (Rieske iron-sulfur protein), D (subunit IV), and small 3.2-4.2 kDa polypeptides petG,L,M, and N. The ninth polypeptide, the 35 kDa petH poly-peptide in the spinach complex, was identified as ferredoxin NADP reductase (FNR), which binds to the complex tightly at a stoichiometry of approx 0.9 (cyt f)-1. The spinach complex contains diaphorase activity diagnostic of FNR, and is active in facilitating ferredoxin-dependent electron transfer from NADPH to the cytochrome b6f complex. The purified cytochrome b6f complex contains stoichiometrically bound chlorophyll a and beta-carotene at a ratio of one per cytochrome f, and bound lipid, in which MGDG and PG are the most abundant species. The delipidated highly purified complexes are active immediately after preparation and for approx 1 wk if left on ice, transferring 300-350 electrons/cyt f/s. Both complexes are subject to proteolysis and associated loss of activity if left for extended periods (>1 wk) at room temperature. Addition of pure synthetic lipid to the delipidated M. laminosus complex (the "lipid augmentation" technique) allows rapid and ready formation of large (>0.2 mm) crystals suitable for x-ray diffraction analysis and structure determination, which diffract with good statistics to 3.0 A.