BBA - Bioenergetics (v.1659, #1)

RecA-like motor ATPases—lessons from structures by Jiqing Ye; Andrew R. Osborne; Michael Groll; Tom A. Rapoport (1-18).
A large class of ATPases contains a RecA-like structural domain and uses the energy of nucleotide binding and hydrolysis to perform mechanical work, for example, to move polypeptides or nucleic acids. These ATPases include helicases, ABC transporters, clamp loaders, and proteases. The functional units of the ATPases contain different numbers of RecA-like domains, but the nucleotide is always bound at the interface between two adjacent RecA-like folds and the two domains move relative to one another during the ATPase cycle. The structures determined for different RecA-like motor ATPases begin to reveal how they move macromolecules.
Keywords: ATPase; RecA; Structure; Motor protein; Oligomerization;

Molecular interference of Cd2+ with Photosystem II by Kajsa G.V. Sigfridsson; Gábor Bernát; Fikret Mamedov; Stenbjörn Styring (19-31).
Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd2+ is known to exchange, with high affinity in a slow reaction, for the Ca2+ cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd2+ binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd2+-binding to those sites, we have studied how Cd2+ affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd2+ with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd2+ were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from YZ to P680 + was inhibited; (ii) QA to QB electron transfer was slowed down; (iii) the S2 state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from QA Fe2+ was modified but its amplitude was not sensitive to the presence of Cd2+. In addition, the presence of both Ca2+ and DCMU abolished Cd2+-induced effects partially and in different sites. The number of sites for Cd2+ binding and the possible nature of these sites are discussed.
Keywords: Cadmium; Calcium; DCMU; EPR; Fluorescence; Photosystem II;

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.
Keywords: Respiration; Nitrogen fixation; Cytochrome c oxidase; Quinol oxidase; Symbiosis;

Effect of deuterium oxide on contraction characteristics and ATPase activity in glycerinated single rabbit skeletal muscle fibers by Takakazu Kobayashi; Yasutake Saeki; Shigeru Chaen; Ibuki Shirakawa; Haruo Sugi (46-51).
We studied the effect of deuterium oxide (D2O) on contraction characteristics and ATPase activity of single glycerinated muscle fibers of rabbit psoas. D2O increased the maximum isometric force P 0 by about 20%, while the force versus stiffness relation did not change appreciably. The maximum shortening velocity under zero load V max did not change appreciably in D2O, so that the force-velocity (PV) curve was scaled depending on the value of P 0. The Mg-ATPase activity of the fibers during generation of steady isometric force P 0 was reduced by about 50% in D2O. Based on the Huxley contraction model, these results can be accounted for in terms of D2O-induced changes in the rate constants f 1 and g 1 for making and breaking actin–myosin linkages in the isometric condition, in such a way that f 1/(f 1+g 1) increases by about 20%, while (f 1+g 1) remains unchanged. The D2O effect at the molecular level is discussed in connection with biochemical studies on actomyosin ATPase.
Keywords: Deuterium oxide; Muscle contraction; ATPase activity; Huxley contraction model; Skeletal muscle fiber;

In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart by Francesca Di Pancrazio; Irene Mavelli; Miriam Isola; Gianni Losano; Pasquale Pagliaro; David A. Harris; Giovanna Lippe (52-62).
A method has been developed to allow the level of F0F1ATP synthase capacity and the quantity of IF1 bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF1 content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF1 antibodies.Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF1 content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF1. In addition, both in vivo and in vitro, 1.3–1.4 mol of IF1 was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF1 in the F0 sector.
Keywords: Inhibitor protein IF1; Mitochondrial F0F1ATP synthase; Ischemic preconditioning; Goat heart; Reactive hyperemia; Blue native polyacrylamide electrophoresis;

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain by Helmut Kirchhoff; Mark Aurel Schöttler; Julia Maurer; Engelbert Weis (63-72).
Reduction kinetics of cytochrome f, plastocyanin (PC) and P700 (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810–860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P700. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P700. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.
Keywords: Plastocyanin; Redox equilibrium; Thylakoids; Diffusion;

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix–helix distances were investigated by the introduction of one or two cysteines in helix–helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α2β2 tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α2β2 tetramer. Each β subunit in the α2β2 tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.
Keywords: Transhydrogenase; Proton-pumping; NAD; NADP; Membrane protein; Transmembrane helix; Helix–helix interaction;

The Na+/Ca2+ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na+ or Ca2+. Na+/Ca2+ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca2+ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K m for Ca2+. When present on the same side as Ca2+, K+ activated exchange by lowering the K m for Ca2+ from 2  to 0.9 μM. The K m for Na+ was 8 mM. In the absence of Ca2+, the exchanger catalyzed high rates of Na+/Li+ and Na+/K+ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na+/Ca2+ and Na+/K+ exchange with IC50 values of 10 and 0.6 μM, respectively. The V max for Na+/Ca2+ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.
Keywords: Na+/Ca2+ exchange; Cardiac mitochondria; ATP production;

The stoichiometry of the chloroplast ATP synthase oligomer III in Chlamydomonas reinhardtii is not affected by the metabolic state by Jürgen M.W. Meyer zu Tittingdorf; Sascha Rexroth; Eva Schäfer; Ralf Schlichting; Christoph Giersch; Norbert A. Dencher; Holger Seelert (92-99).
The chloroplast H+-ATP synthase is a key component for the energy supply of higher plants and green algae. An oligomer of identical protein subunits III is responsible for the conversion of an electrochemical proton gradient into rotational motion. It is highly controversial if the oligomer III stoichiometry is affected by the metabolic state of any organism. Here, the intact oligomer III of the ATP synthase from Chlamydomonas reinhardtii has been isolated for the first time. Due to the importance of the subunit III stoichiometry for energy conversion, a gradient gel system was established to distinguish oligomers with different stoichiometries. With this methodology, a possible alterability of the stoichiometry in respect to the metabolic state of the cells was examined. Several growth parameters, i.e., light intensity, pH value, carbon source, and CO2 concentration, were varied to determine their effects on the stoichiometry. Contrary to previous suggestions for E. coli, the oligomer III of the chloroplast H+-ATP synthase always consists of a constant number of monomers over a wide range of metabolic states. Furthermore, mass spectrometry indicates that subunit III from C. reinhardtii is not modified posttranslationally. Data suggest a subunit III stoichiometry of the algae ATP synthase divergent from higher plants.
Keywords: ATP synthase; Oligomer III; Stoichiometry; Chloroplast; Metabolic state;

Dissipation of excess energy triggered by blue light in cyanobacteria with CP43′ (isiA) by Jean-Charles Cadoret; Raphaël Demoulière; Johann Lavaud; Hans J. van Gorkom; Jean Houmard; Anne-Lise Etienne (100-104).
The chlorophyll-protein CP43′ (isiA gene) induced by stress conditions in cyanobacteria is shown to serve as an antenna for Photosystem II (PSII), in addition to its known role as an antenna for Photosystem I (PSI). At high light intensity, this antenna is converted to an efficient trap for chlorophyll excitations that protects system II from photo-inhibition. In contrast to the ‘energy-dependent non-photochemical quenching’ (NPQ) in chloroplasts, this photoprotective energy dissipation in cyanobacteria is triggered by blue light. The induction is proportional to light intensity. Induction and decay of the quenching exhibit the same large temperature-dependence.
Keywords: Non-photochemical quenching; Iron-stress; Chlorophyll antenna; Photoprotection; Photosystems;