BBA - Bioenergetics (v.1767, #12)
Editorial Board (ii).
Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis by Ryo Nagao; Akiko Ishii; Osamu Tada; Takehiro Suzuki; Naoshi Dohmae; Akinori Okumura; Masako Iwai; Takeshi Takahashi; Yasuhiro Kashino; Isao Enami (1353-1362).
Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O2/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze–thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O2/mg Chl/h in the absence and presence of CaCl2, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.
Keywords: Diatom; Oxygen evolution; Photosystem II; Extrinsic protein; Chaetoceros gracilis;
Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition by Suleyman I. Allakhverdiev; Dmitry A. Los; Prasanna Mohanty; Yoshitaka Nishiyama; Norio Murata (1363-1371).
Transformation with the bacterial gene codA for choline oxidase allows Synechococcus sp. PCC 7942 cells to accumulate glycinebetaine when choline is supplemented exogenously. First, we observed two types of protective effect of glycinebetaine against heat-induced inactivation of photosystem II (PSII) in darkness; the codA transgene shifted the temperature range of inactivation of the oxygen-evolving complex from 40–52 °C (with half inactivation at 46 °C) to 46–60 °C (with half inactivation at 54 °C) and that of the photochemical reaction center from 44–55 °C (with half inactivation at 51 °C) to 52–63 °C (with half inactivation at 58 °C). However, in light, PSII was more sensitive to heat stress; when moderate heat stress, such as 40 °C, was combined with light stress, PSII was rapidly inactivated, although these stresses, when applied separately, did not inactivate either the oxygen-evolving complex or the photochemical reaction center. Further our studies demonstrated that the moderate heat stress inhibited the repair of PSII during photoinhibition at the site of synthesis de novo of the D1 protein but did not accelerate the photodamage directly. The codA transgene and, thus, the accumulation of glycinebetaine alleviated such an inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein. We propose a hypothetical scheme for the cyanobacterial photosynthesis that moderate heat stress inhibits the translation machinery and glycinebetaine protects it against the heat-induced inactivation.
Keywords: codA gene; Glycinebetaine; Heat stress; Photoinhibition; Photosystem II; Protein synthesis; Repair;
Effects of polyamines on the functionality of photosynthetic membrane in vivo and in vitro by Nikolaos E. Ioannidis; Kiriakos Kotzabasis (1372-1382).
The three major polyamines are normally found in chloroplasts of higher plants and are implicated in plant growth and stress response. We have recently shown that putrescine can increase light energy utilization through stimulation of photophosphorylation [Ioannidis et al., (2006) BBA-Bioenergetics, 1757, 821-828]. We are now to compare the role of the three major polyamines in terms of chloroplast bioenergetics. There is a different mode of action between the diamine putrescine and the higher polyamines (spermidine and spermine). Putrescine is an efficient stimulator of ATP synthesis, better than spermidine and spermine in terms of maximal % stimulation. On the other hand, spermidine and spermine are efficient stimulators of non-photochemical quenching. Spermidine and spermine at high concentrations are efficient uncouplers of photophosphorylation. In addition, the higher the polycationic character of the amine being used, the higher was the effectiveness in PSII efficiency restoration, as well as stacking of low salt thylakoids. Spermine with 50 μM increase F V as efficiently as 100 μM of spermidine or 1000 μM of putrescine or 1000 μM of Mg2+. It is also demonstrated that the increase in F V derives mainly from the contribution of PSIIα centers. These results underline the importance of chloroplastic polyamines in the functionality of the photosynthetic membrane.
Keywords: Non-photochemical quenching; Chloroplast; Cations; Stress; Proton motive force; ATP;
Time-resolved single-turnover of ba 3 oxidase from Thermus thermophilus by Sergey A. Siletsky; Ilya Belevich; Audrius Jasaitis; Alexander A. Konstantinov; Mårten Wikström; Tewfik Soulimane; Michael I. Verkhovsky (1383-1392).
The kinetics of the oxidation of fully-reduced ba 3 cytochrome c oxidase from Thermus thermophilus by oxygen were followed by time-resolved optical spectroscopy and electrometry. Four catalytic intermediates were resolved during this reaction. The chemical nature and the spectral properties of three intermediates (compounds A, P and O) reproduce the general features of aa 3-type oxidases. However the F intermediate in ba 3 oxidase has a spectrum identical to the P state. This indicates that the proton taken up during the P → F transition does not reside in the binuclear site but is rather transferred to the covalently cross-linked tyrosine near that site. The total charge translocation associated with the F → O transition in ba 3 oxidase is close to that observed during the F → O transition in the aa 3 oxidases. However, the P R → F transition is characterized by significantly lower charge translocation, which probably reflects the overall lower measured pumping efficiency during multiple turnovers.
Keywords: Catalytic cycle; Cytochrome c oxidase; Electron transfer; Electric potential generation; Thermus thermophilus;
Fluorescence quenching of IsiA in early stage of iron deficiency and at cryogenic temperatures by Chantal D. van der Weij-de Wit; Janne A. Ihalainen; Edith van de Vijver; Sandrine D'Haene; Hans C.P. Matthijs; Rienk van Grondelle; Jan P. Dekker (1393-1400).
Cyanobacteria respond to iron deficiency during growth by expressing the isiA gene, which produces a chlorophyll–carotenoid protein complex known as IsiA or CP43′. Long-term iron deficiency results in the formation of large IsiA aggregates, some of which associate with photosystem I (PSI) while others are not connected to a photosystem. The fluorescence at room temperature of these unconnected aggregates is strongly quenched, which points to a photoprotective function. In this study, we report time-resolved fluorescence measurements of IsiA aggregates at low temperatures. The average fluorescence lifetimes are estimated to be about 600 ps at 5 K and 150 ps at 80 K. Both lifetimes are much shorter than that of the monomeric complex CP47 at 77 K. We conclude that IsiA aggregates quench fluorescence to a significant extent at cryogenic temperatures. We show by low-temperature fluorescence spectroscopy that unconnected IsiA is present already after two days of growth in an iron-deficient medium, when PSI and PSII are still present in significant amounts and that under these conditions the fluorescence quenching is similar to that after 18 days, when PSI is almost completely absent. We conclude that unconnected IsiA provides photoprotection in all stages of iron deficiency.
Keywords: Fluorescence; IsiA aggregates; Low temperature; Photosystem I; Quenching;
Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H+-pyrophosphatase by Megumi Hirono; Yoichi Nakanishi; Masayoshi Maeshima (1401-1411).
The H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14–17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr409, Val411, and Gly414 showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe388, Thr389, and Val396 in cytoplasmic loop i were essential for efficient H+ translocation. Ala436 and Pro560 in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H+-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme.
Keywords: Energy coupling; H+-pyrophosphatase; Random mutagenesis; Proton pump;
Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein by Regiane Degan Fávaro; Jiri Borecký; Débora Colombi; Aníbal E. Vercesi; Ivan G. Maia (1412-1417).
In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.
Keywords: Uncoupling protein; Arabidopsis thaliana; Site-direct mutagenesis; Proteoliposome; Structure–function relationship;
Processivity of single-headed kinesin motors by Ping Xie; Shuo-Xing Dou; Peng-Ye Wang (1418-1427).
The processive movement of single-headed kinesins is studied by using a ratchet model of non-Markov process, which is built on the experimental evidence that the strong binding of kinesin to microtubule in rigor state induces a large apparent change in the local microtubule conformation. In the model, the microtubule plays a crucial active role in the kinesin movement, in contrast to the previous belief that the microtubule only acts as a passive track for the kinesin motility. The unidirectional movement of single-headed kinesin is resulted from the asymmetric periodic potential between kinesin and microtubule while its processivity is determined by its binding affinity for microtubule in the weak ADP state. Using the model, various experimental results for monomeric kinesin KIF1A, such as the mean step size, the step-size distribution, the long run length and the mean velocity versus load, can be well explained quantitatively. This local conformational change of the microtubule may also play important roles in the processive movement of conventional two-headed kinesins. An experiment to verify the model is suggested.
Keywords: Kinesin; Processivity; Mechanochemistry; Microtubule; Molecular motor;
The monomeric photosystem I-complex of the diatom Phaeodactylum tricornutum binds specific fucoxanthin chlorophyll proteins (FCPs) as light-harvesting complexes by Thomas Veith; Claudia Büchel (1428-1435).
A photosystem I (PSI)–fucoxanthin chlorophyll protein (FCP) complex with a chlorophyll a/P700 ratio of approximately 200:1 was isolated from the diatom Phaeodactylum tricornutum. Spectroscopic analysis proved that the more tightly bound FCP functions as a light-harvesting complex, actively transferring light energy from its accessory pigments chlorophyll c and fucoxanthin to the PSI core. Using an antibody against all FCP polypeptides of Cyclotella cryptica it could be shown that the polypeptides of the major FCP fraction differ from the FCPs found in the PSI fraction. Since these FCPs are tightly bound to PSI, active in energy transfer, and not found in the main FCP fraction, we suppose them to be PSI specific. Blue Native-PAGE, gel filtration and first electron microscopy studies of the PSI–FCP sample revealed a monomeric complex comparable in size and shape to the PSI–LHCI complex of green algae.
Keywords: Photosynthesis; Fluorescence; Diadinoxanthin; Membrane protein; Electron microscopy;
The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction by Kayode S. Oyedotun; Clarissa S. Sit; Bernard D. Lemire (1436-1445).
The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron–sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b 562. Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.
Keywords: Heme; Yeast; Succinate:ubiquinone oxidoreductase; Complex II; Molecular dynamics;
Bioenergetics Cumulative Contents (1446-1452).