BBA - Bioenergetics (v.1767, #1)

A word of farewell and a word of welcome by Dennis Vance; Peter van der Vliet (1-2).

BBA in the year 2007 by Dennis E. Vance (3-4).

The long-lived, light-induced radical YD of the Tyr161 residue in the D2 protein of Photosystem II (PSII) is known to magnetically interact with the CaMn4 cluster, situated ∼ 30 Å away. In this study we report a transient step-change increase in YD EPR intensity upon the application of a single laser flash to S1 state-synchronised PSII-enriched membranes from spinach. This transient effect was observed at room temperature and high applied microwave power (100 mW) in samples containing PpBQ, as well as those containing DCMU. The subsequent decay lifetimes were found to differ depending on the additive used. We propose that this flash-induced signal increase was caused by enhanced spin relaxation of YD by the OEC in the S2 state, as a consequence of the single laser flash turnover. The post-flash decay reflected S2  → S1 back-turnover, as confirmed by their correlations with independent measurements of S2 multiline EPR signal and flash-induced variable fluorescence decay kinetics under corresponding experimental conditions. This flash-induced effect opens up the possibility to study the kinetic behaviour of S-state transitions at room temperature using YD as a probe.
Keywords: Photosystem II; EPR; Oxygen evolving complex; S-state transitions; Tyrosine D; Spin relaxation;

Demonstration of phycobilisome mobility by the time- and space-correlated fluorescence imaging of a cyanobacterial cell by Shuzhen Yang; Zuqi Su; Heng Li; Juanjuan Feng; Jie Xie; Andong Xia; Yandao Gong; Jingquan Zhao (15-21).
The cell-wide mobility of PBSs was confirmed by synchronously monitoring the fluorescence recovery after photobleaching (FRAP) and the fluorescence loss in photobleaching (FLIP). On the other hand, a fluorescence recovery was still observed even if PBSs were immobile (PBSs fixed on the membranes by betaine and isolated PBSs fixed on the agar plate) or PBS mobility was unobservable (cell wholly bleached). Furthermore, it was proved that some artificial factors were involved not only in FRAP but also in FLIP, including renaturation of the reversibly denatured proteins, laser scanning-induced fluorescence loss and photo-damage to the cell. With consideration of the fast renaturation component in fluorescence recovery, the diffusion coefficient was estimated to be tenfold smaller than that without the component. Moreover, it was observed that the fluorescence intensity on the bleached area was always lower than that on the non-bleached area, even after 20 min, while it should be equal if PBSs were mobile freely. Based on the increasing proportion of the PBSs anti-washed to Triton X-100 (1%) with prolonged laser irradiation to the cells locked in light state 1 by PBQ, it was concluded that some PBSs became immobile due to photo-linking to PSII.
Keywords: Mobility; Phycobilisome; FRAP; FLIP; Photosystem I; Photosystem II;

Energy and electron transfer in the photosynthetic reaction center complex of Acidiphilium rubrum containing Zn-bacteriochlorophyll a studied by femtosecond up-conversion spectroscopy by Tetsuo Tomi; Yutaka Shibata; Yuki Ikeda; Seiji Taniguchi; Chosrowjan Haik; Noboru Mataga; Keizo Shimada; Shigeru Itoh (22-30).
A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (PZn), the accessory Zn-BChl a (BZn), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from PZn *, measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from BZn * to PZn, and the latter, the electron transfer from PZn to H. The angle between the transition dipoles of BZn and PZn was estimated to be 36° based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies PZn.
Keywords: Electron transfer; Energy transfer; Fluorescence up-conversion; Purple photosynthetic bacteria; Reaction center; Zn-bacteriochlorophyll a;

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart by Lubica Malekova; Viera Kominkova; Miroslav Ferko; Peter Stefanik; Olga Krizanova; Attila Ziegelhöffer; Adam Szewczyk; Karol Ondrias (31-44).
The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1–100 μM), ATR and CAT (5–100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans–cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.
Keywords: Mitochondrial membrane; Chloride channel; Bongkrekic acid; Atractyloside; Single channel property; Bilayer lipid membrane;

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a 3 slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a 3 and CuB remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.
Keywords: Cytochrome c oxidase; Formamide; Water; Catalytic cycle; Subunit III; Electron transfer; Water cycle; Proton gathering;

On the advantages of using green light to study fluorescence yield changes in leaves by Fabrice Rappaport; Daniel Béal; Anne Joliot; Pierre Joliot (56-65).
In photosynthetic chains, the kinetics of fluorescence yield depends on the photochemical rates at the level of both Photosystem I and II and thus on the absorption cross section of the photosynthetic units as well as on the coupling between light harvesting complexes and photosynthetic traps. A new set-up is described which, at variance with the commonly used set-ups, uses of a weakly absorbed light source (light-emitting diodes with maximum output at 520 nm) to excite the photosynthetic electron chain and probe the resulting fluorescence yield changes and their time course. This approach optimizes the homogeneity of the exciting light throughout the leaf and we show that this homogeneity narrows the distribution of the photochemical rates. Although the exciting light is weakly absorbed, the possibility to tune the intensity of the light emitting diodes allows one to reach photochemical rates ranging from 104 s− 1 to 0.25 s− 1 rendering experimentally accessible different functional regimes. The variations of the fluorescence yield induced by the photosynthetic activity are qualitatively and quantitatively discussed. When illuminating dark-adapted leaves by a weak light, the kinetics of fluorescence changes displays a pronounced plateau which precedes the fluorescence increase reflecting the full reduction of the plastoquinone pool. We ascribe this plateau to the time delay needed to reduce the photosystem I electron acceptors.
Keywords: Photosynthetic chain; Fluorescence; Photochemical quenching; Non-photochemical quenching; Electron transfer;

To investigate whether and how mitochondria can change in plant programmed cell death (PCD), we used the non-photosynthetic Tobacco Bright Yellow 2 (TBY-2) cells. These can be synchronized to high levels, stand out in terms of growth rate and homogeneity and undergo PCD as a result of heat shock. Using these cells we investigated the activity of certain mitochondrial proteins that have a role in providing ATP and/or other nucleoside triphosphates (NTPs). We show that, already after 2 h from the heat shock, when cell viability remains unaffected, the rate of ADP/ATP exchange due to adenine nucleotide translocator (ANT) activity, and the rate of the reactions catalysed by adenylate kinase (ADK; EC and nucleoside diphosphate kinase (NDPK; EC are inhibited in a non-competitive-like manner. In all cases, externally added ascorbate partially prevented the inhibition. These effects occurred in spite of minor (for ANT) or no changes in the mitochondrial protein levels as immunologically investigated. Interestingly, a decrease of both the steady state level of the ascorbate pool and of the activity of l-galactono-γ-lactone dehydrogenase (GLDH) (EC, the mitochondrial enzyme catalysing the last step of ascorbate biosynthesis, were also found.
Keywords: Mitochondria; Plant programmed cell death; Adenine nucleotide translocator; Adenylate kinase; Nucleoside diphosphate kinase; l-galactono-γ-lactone dehydrogenase;

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers by Hiroshi Ishikita; Bernhard Loll; Jacek Biesiadka; Jan Kern; Klaus-Dieter Irrgang; Athina Zouni; Wolfram Saenger; Ernst-Walter Knapp (79-87).
The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E m) of one-electron oxidation for all Car (E m(Car)) by solving the Poisson–Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (CarD1) in van der Waals contact with the chlorin ring of ChlZ(D1). Each CarD1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between CarD1 and plastoquinone/QA may imply a direct charge recombination of Car+QA . The calculated E m(CarD1) and E m(ChlZ(D1)) are, respectively, 83 and 126 mV higher than E m(CarD2) and E m(ChlZ(D2)), which could explain why CarD2 + and ChlZ(D2) + are observed rather than the corresponding CarD1 + and ChlZ(D1) +.
Keywords: Photosystem II; Chlorophyll; β-carotene; Photoprotection; Electron transfer; Redox potential;

Chlorophyll triplet states associated with Photosystem I and Photosystem II in thylakoids of the green alga Chlamydomonas reinhardtii by Stefano Santabarbara; Giancarlo Agostini; Anna Paola Casazza; Christopher D. Syme; P. Heathcote; Felix Böhles; Michael C.W. Evans; Robert C. Jennings; Donatella Carbonera (88-105).
The analysis of FDMR spectra, recorded at multiple emission wavelengths, by a global decomposition technique, has allowed us to characterise the triplet populations associated with Photosystem I and Photosystem II of thylakoids in the green alga Chlamydomonas reinhardtii. Three triplet populations are observed at fluorescence emissions characteristic of Photosystem II, and their zero field splitting parameters have been determined. These are similar to the zero field parameters for the three Photosystem II triplets previously reported for spinach thylakoids, suggesting that they have a widespread occurrence in nature. None of these triplets have the zero field splitting parameters characteristic of the Photosystem II recombination triplet observed only under reducing conditions. Because these triplets are generated under non-reducing redox conditions, when the recombination triplet is undetectable, it is suggested that they may be involved in the photoinhibition of Photosystem II. At emission wavelengths characteristic of Photosystem I, three triplet populations are observed, two of which are attributed to the P700 recombination triplet frozen in two different conformations, based on the microwave-induced fluorescence emission spectra and the triplet minus singlet difference spectra. The third triplet population detected at Photosystem I emission wavelengths, which was previously unresolved, is proposed to originate from the antenna chlorophyll of the core or the unusually blue-shifted outer antenna complexes of this organism.
Keywords: Triplet states; Photoinhibition; Non-photochemical quenching; Photosystem I; Photosystem II; Chlamydomonas;

Variable fluorescence in green sulfur bacteria by Martin F. Hohmann-Marriott; Robert E. Blankenship (106-113).
Green sulfur bacteria possess a complex photosynthetic machinery. The dominant light harvesting systems are chlorosomes, which consist of bacteriochlorophyll c, d or e oligomers with small amounts of protein. The chlorosomes are energetically coupled to the membrane-embedded iron sulfur-type reaction center via a bacteriochlorophyll a-containing baseplate protein and the Fenna–Matthews–Olson (FMO) antenna protein. The fluorescence yield and spectral properties of these photosynthetic complexes were investigated in intact cells of several species of green sulfur bacteria under physiological, anaerobic conditions. Surprisingly, green sulfur bacteria show a complex modulation of fluorescence yield upon illumination that is very similar to that observed in oxygenic phototrophs. Within a few seconds of illumination, the fluorescence reaches a maximum, which decreases within a minute of illumination to a lower steady state. Fluorescence spectroscopy reveals that the fluorescence yield during both processes is primarily modulated on the FMO-protein level, while the emission from chlorosomes remains mostly unchanged. The two most likely candidates that modulate bacteriochlorophyll fluorescence are (1) direct excitation quenching at the FMO-protein level and (2) indirect modulation of FMO-protein fluorescence by the reduction state of electron carriers that are part of the reaction center.
Keywords: Green sulfur bacteria; Bacteriochlorophyll fluorescence; Chlorosome; FMO-protein;

We studied binding of ATP and of the ATP analogs adenosine 5′-(β,γ-methylene)triphosphate (AMPCP) and β,γ-imidoadenosine 5′-triphosphate (AMPPNP) to the Ca2+–ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca2E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca2+ concentration (10 mM). They are likely due to a different position of the γ-phosphate which affects the β-sheet in the P domain.
Keywords: Infrared spectroscopy; Ca2+ pump; SERCA1a; Caged ATP; ATP binding; Nucleotide binding;

Acknowledgement (124-126).