BBA - Bioenergetics (v.1797, #8)

The mechanism of rotating proton pumping ATPases by Mayumi Nakanishi-Matsui; Mizuki Sekiya; Robert K. Nakamoto; Masamitsu Futai (1343-1352).
Two proton pumps, the F-ATPase (ATP synthase, FoF1) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F1 or V1) and a membrane intrinsic (Fo or Vo) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40–60 nm, the E. coli F1 sector was found to rotate at surprisingly high speeds (> 400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the β subunits, or between the β and γ subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F1-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.
Keywords: ATP synthase; F-ATPase; V-ATPase; Subunit rotation; Single molecule observation; Thermodynamic analysis;

Oscillations in energy metabolism by Stefano Iotti; Marco Borsari; David Bendahan (1353-1361).
Organisation of mitochondrial metabolism is a quintessential example of a complex dissipative system which can display dynamic instabilities. Several findings have indicated that the conditions inducing instabilities are within the physiological range and that mild perturbations could elicit oscillations. Different mathematical models have been put forth in order to explain the genesis of oscillations in energy metabolism. One model considers mitochondria as an organised network of oscillators and indicates that communication between mitochondria involves mitochondrial reactive oxygen species (ROS) production acting as synchronisers of the energy status of the whole population of mitochondria. An alternative model proposes that extramitochondrial pH variations could lead to mitochondrial oscillations. Oscillatory phenomena in energy metabolism have also been investigated in vivo on the basis of 31P magnetic resonance spectroscopy (MRS) measurements of phosphocreatine post-exercise recovery in human and animal skeletal muscle. The corresponding results provide experimental evidences about the role exerted by cytosolic pH on oscillations. Finally a new simple non-linear mathematical model describing the overall chemical reaction of phosphocreatine recovery predicting oscillatory recovery pattern under certain experimental conditions is presented and discussed in the light of the experimental results reported so far.
Keywords: Mitochondrial oscillation; Energy metabolism; Magnetic resonance spectroscopy; Metabolic dynamics; Mathematical model; Dissipative system; Metabolic instability;

F1F0-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations by David B. Hicks; Jun Liu; Makoto Fujisawa; Terry A. Krulwich (1362-1377).
This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H+-coupled ATP synthesis at external pH values > 10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na+- instead of H+-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H+ transfers to ATP synthases via membrane-associated microcircuits between the H+ pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.
Keywords: ATPase; Oxidative phosphorylation; c-ring; Bacillus pseudofirmus OF4; Bacillus TA2.A1; Spirulina platensis;

The human mitochondrial replication fork in health and disease by Sjoerd Wanrooij; Maria Falkenberg (1378-1388).
Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.
Keywords: mtDNA; DNA replication; Mitochondria; POLRMT; Twinkle; Mitochondrial disease;

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated ‘energy-wasting’ process during CR.
Keywords: Aging; Calorie restriction; Mitochondrial uncoupling; Proton leak; Oxidative stress; Indirect calorimetry; Rodent models of aging; Bongkrekate;

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO by Maria Merkulova; Anastasia Bakulina; Youg Raj Thaker; Gerhard Grüber; Vladimir Marshansky (1398-1409).
We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant K D  = 3.44 × 10 7  M, similar to the K D  = 3.13 × 10 7  M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser392. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser392 phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.
Keywords: V-type ATPase; Arf-GEF ARNO; Sec7-domain; PB-domain; BIAcore; NMR; Peptide structure; Phosphorylation;

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I by Ivan V. Shelaev; Fedor E. Gostev; Mahir D. Mamedov; Oleg M. Sarkisov; Victor A. Nadtochenko; Vladimir A. Shuvalov; Alexey Yu. Semenov (1410-1420).
The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A0 (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump–probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A1 (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700+A0A1) PS I reaction center (RC) from that of the open (state P700A0A1) RC reveals the pure spectrum of the P700+A0 ion–radical pair. The experimental data were analyzed using a simple kinetic scheme: An* → k 1 [(PA0)*A1 → < 100 fs P+A0 A1] → k 2 P+A0A1 , and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700+A0 A1 state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A0A1 in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700+A0A1 .
Keywords: Femtosecond pump–probe spectroscopy; Photosystem I; Reaction center; Electron transfer; Radical pairs;

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl replacement by Br in active PSII. In Br substituted samples, Cl is effectively replaced by Br in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. The following results have been obtained. i) The oxygen-evolving activities of the Br-PSII samples are significantly lower than that of the Cl-PSII samples; ii) The same S2 multiline EPR signals are observed in both Br and Cl-PSII samples; iii) The amplitudes of the visible light induced S1TyrZ and S2TyrZ EPR signals are significantly decreased after Br substitution; the S1TyrZ EPR signal is up-shifted about 8 G, whereas the S2TyrZ signal is down-shifted about 12 G after Br substitution. These results imply that the redox properties of TyrZ and spin interactions between TyrZ and Mn-cluster could be significantly modified due to Br substitution. It is suggested that Cl/Br probably coordinates to the Ca2+ ion of the Mn-cluster in active photosystem II.
Keywords: Photosystem II; Tyrosine Z; Mn-cluster; Chloride; Bromide substitution; Electron paramagnetic resonance;

Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: Physiological and chlorophyll a fluorescence measurements by Mohd. Aslam Yusuf; Deepak Kumar; Ravi Rajwanshi; Reto Jörg Strasser; Merope Tsimilli-Michael; Govindjee; Neera Bhalla Sarin (1428-1438).
Tocopherols (vitamin E) are lipid soluble antioxidants synthesized by plants and some cyanobacteria. We have earlier reported that overexpression of the γ-tocopherol methyl transferase (γ-TMT) gene from Arabidopsis thaliana in transgenic Brassica juncea plants resulted in an over six-fold increase in the level of α-tocopherol, the most active form of all the tocopherols. Tocopherol levels have been shown to increase in response to a variety of abiotic stresses. In the present study on Brassica juncea, we found that salt, heavy metal and osmotic stress induced an increase in the total tocopherol levels. Measurements of seed germination, shoot growth and leaf disc senescence showed that transgenic Brassica juncea plants overexpressing the γ-TMT gene had enhanced tolerance to the induced stresses. Analysis of the chlorophyll a fluorescence rise kinetics, from the initial “O” level to the “P” (the peak) level, showed that there were differential effects of the applied stresses on different sites of the photosynthetic machinery; further, these effects were alleviated in the transgenic (line 16.1) Brassica juncea plants. We show that α-tocopherol plays an important role in the alleviation of stress induced by salt, heavy metal and osmoticum in Brassica juncea.
Keywords: α-Tocopherol; Brassica juncea; Chlorophyll fluorescence; JIP-test; OJIP fluorescence transient; Stress alleviation;

Subunit–subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp. by Araceli Cano-Estrada; Miriam Vázquez-Acevedo; Alexa Villavicencio-Queijeiro; Francisco Figueroa-Martínez; Héctor Miranda-Astudillo; Yraima Cordeiro; Julio A. Mignaco; Debora Foguel; Pierre Cardol; Marie Lapaille; Claire Remacle; Stephan Wilkens; Diego González-Halphen (1439-1448).
Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1–9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.
Keywords: Oxidative phosphorylation; F1F0-ATP synthase; Dimeric mitochondrial complex V; Chlorophycean algae; Stator stalk; Chlamydomonas reinhardtii; Polytomella sp.; ASA subunits;

As an important component of marine phytoplankton, diatoms must be able to cope with large changes in illumination on a daily basis. They have an active xanthophyll cycle and non-photochemical quenching (NPQ), but no homolog has been detected for the gene encoding the PsbS protein required for NPQ in plants. However, diatoms do have a branch of the light-harvesting complex superfamily, the Lhcx clade, which is most closely related to the LI818 (LhcSR) genes of the green alga Chlamydomonas, known to be upregulated in response to a variety of stresses. When cultures of the diatom T. pseudonana grown under low light (40 µmol photons m 2 s 1) were exposed to high light stress (HL, 700 µmol photons m 2 s 1), transcripts of three of these genes (Lhcx1, Lhcx4, Lhcx6) were transiently accumulated. The amount of Lhcx6 protein was low under low light, but increased continuously during 10 h of HL exposure, then slowly dropped to background levels in the dark. However, HL had little effect on the Lhcx1 protein, which was present under low light and only doubled after HL exposure. Diatoxanthin levels increased throughout the HL period with no change in diadinoxanthin. The fraction of NPQ attributable to photoinhibitory quenching (qI) also increased throughout the HL exposure. Taken together, the Lhcx6 protein could be associated with diatoxanthin binding and play a direct role in excess energy dissipation via sustained quenching during acclimation to prolonged HL stress, while the Lhcx1 protein may play a more structural role in thylakoid membrane organization under all conditions.
Keywords: Fucoxanthin chlorophyll a/c proteins; High light stress; LI818; NPQ; Photoprotection; Thalassiosira pseudonana;

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri by Wesley D. Swingley; Masakazu Iwai; Yang Chen; Shin-ichiro Ozawa; Kenji Takizawa; Yuichiro Takahashi; Jun Minagawa (1458-1464).
Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment–protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715–730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.
Keywords: Evolution; Light-harvesting complex; Photosystem I; Prasinophytes;

Spectral dependence of energy transfer in wild-type peripheral light-harvesting complexes of photosynthetic bacteria by Andrew Gall; Egidijus Sogaila; Vidmantas Gulbinas; Oana Ilioaia; Bruno Robert; Leonas Valkunas (1465-1469).
The precise position of the upper exciton component and relevant vibronic transitions of the B850 ring in peripheral light-harvesting complexes from purple photosynthetic bacteria are important values for determining the exciton bandwidth and electronic structure of the B850 ring. To determine the presence of these components in wild-type LH2 complexes the pump-probe femtosecond transient spectra obtained with excitation into the 730–840 nm spectral range are analyzed. We show that at excitation wavelengths less than 780 nm B850 absorption bands are present and that, in accordance with exciton theory, these bands peak further in the blue when the lowest optically allowed transition is more red-shifted.
Keywords: B850; Exciton theory; LH2; Photosynthesis; Pump-probe transient spectroscopy;

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.
Keywords: Mitochondria; Reactive oxygen species; Brown adipose tissue; Uncoupling protein 1;

An alternative complex III (ACIII) is a respiratory complex with quinol:electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc 1 complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa 3 oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidences, taken together with mass spectrometry revealed a structural association between ACIII and caa 3 oxygen reductase. The association was also shown to be functional, since quinol:oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.
Keywords: Alternative complex III; bc 1 Complex; caa 3 Oxygen reductase; Quinol; HQNO; Complex iron–sulfur molybdoenzyme family;

Synthesis of fatty acids de novo is required for photosynthetic acclimation of Synechocystis sp. PCC 6803 to high temperature by Yohei Nanjo; Naoki Mizusawa; Hajime Wada; Antoni R. Slabas; Hidenori Hayashi; Yoshitaka Nishiyama (1483-1490).
The role of fatty acid synthesis in the acclimation of the photosynthetic machinery to high temperature was investigated in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that had a lower than wild-type level of enoyl-(acyl-carrier-protein) reductase FabI, a key component of the type-II fatty acid synthase system. The mutant exhibited marked impairment in the tolerance and acclimation of cells to high temperature: photoautotrophic growth of the mutant was severely inhibited at 40 °C. Moreover, mutant cells were unable to achieve wild-type enhancement of the thermal stability of photosystem II (PSII) when the growth temperature was raised from 25 °C to 38 °C. Enhancement of the thermal stability of PSII was abolished when wild-type cells were treated with triclosan, a specific inhibitor of FabI, and the enhancement of thermal stability was also blocked in darkness and in the presence of chloramphenicol. Analysis of fatty acids in thylakoid membranes revealed that levels of unsaturated fatty acids did not differ between mutant and wild-type cells, indicating that the saturation of fatty acids in membrane lipids might not be responsible for the enhancement of thermal stability at elevated temperatures. Our observations suggest that the synthesis de novo of fatty acids, as well as proteins, is required for the enhancement of the thermal stability of PSII during the acclimation of Synechocystis cells to high temperature.
Keywords: Acclimation; Fatty acid synthesis; High-temperature stress; Photosystem II; Synechocystis; Thermotolerance;

Energetics in Photosystem II from Thermosynechococcus elongatus with a D1 protein encoded by either the psbA 1 or psbA 3 gene by Miwa Sugiura; Yuki Kato; Ryouta Takahashi; Hiroyuki Suzuki; Tadashi Watanabe; Takumi Noguchi; Fabrice Rappaport; Alain Boussac (1491-1499).
The main cofactors involved in the function of Photosystem II (PSII) are borne by the D1 and D2 proteins. In some cyanobacteria, the D1 protein is encoded by different psbA genes. In Thermosynechococcus elongatus the amino acid sequence deduced from the psbA 3 gene compared to that deduced from the psbA 1 gene points a difference of 21 residues. In this work, PSII isolated from a wild type T. elongatus strain expressing PsbA1 or from a strain in which both the psbA 1 and psbA 2 genes have been deleted were studied by a range of spectroscopies in the absence or the presence of either a urea type herbicide, DCMU, or a phenolic type herbicide, bromoxynil. Spectro-electrochemical measurements show that the redox potential of PheoD1 is increased by 17 mV from −522 mV in PsbA1-PSII to −505 mV in PsbA3-PSII. This increase is about half that found upon the D1-Q130E single site directed mutagenesis in Synechocystis PCC 6803. This suggests that the effects of the D1-Q130E substitution are, at least partly, compensated for by some of the additional amino-acid changes associated with the PsbA3 for PsbA1 substitution. The thermoluminescence from the S2QA −• charge recombination and the C ≡ N vibrational modes of bromoxynil detected in the non-heme iron FTIR difference spectra support two binding sites (or one site with two conformations) for bromoxynil in PsbA3-PSII instead of one in PsbA1-PSII which suggests differences in the QB pocket. The temperature dependences of the S2QA −• charge recombination show that the strength of the H-bond to PheoD1 is not the only functionally relevant difference between the PsbA3-PSII and PsbA1-PSII and that the environment of QA (and, as a consequence, its redox potential) is modified as well. The electron transfer rate between P680 +• and YZ is found faster in PsbA3 than in PsbA1 which suggests that the redox potential of the P680/P680 +• couple (and hence that of 1P680 */P680 +•) is tuned as well when shifting from PsbA1 to PsbA3. In addition to D1-Q130E, the non-conservative amongst the 21 amino acid substitutions, D1-S270A and D1-S153A, are proposed to be involved in some of the observed changes.
Keywords: Photosystem II; D1 protein; psbA gene; Electron transfer; Thermosynechococcus elongatus; Pheophytin; Site-directed mutagenesis;

Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes by Emilie Lagoutte; Sabria Mimoun; Mireille Andriamihaja; Catherine Chaumontet; François Blachier; Frédéric Bouillaud (1500-1511).
Sulfide (H2S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.
Keywords: Mitochondria; Sulfide quinone reductase; Mitochondrial complex I; Detoxification; Stoichiometry;

Redox-coupled proton transfer in the active site of cytochrome cbb 3 by Vivek Sharma; Mårten Wikström; Ville R.I. Kaila (1512-1520).
Cytochrome cbb 3 is a distinct member of the superfamily of respiratory heme-copper oxidases, and is responsible for driving the respiratory chain in many pathogenic bacteria. Like the canonical heme-copper oxidases, cytochrome cbb 3 reduces oxygen to water and couples the released energy to pump protons across the bacterial membrane. Homology modeling and recent electron paramagnetic resonance (EPR) studies on wild type and a mutant cbb 3 enzyme [V. Rauhamäki et al. J. Biol. Chem. 284 (2009) 11301-11308] have led us to perform high-level quantum chemical calculations on the active site. These calculations bring molecular insight into the unique hydrogen bonding between the proximal histidine ligand of heme b 3 and a conserved glutamate, and indicate that the catalytic mechanism involves redox-coupled proton transfer between these residues. The calculated spin densities give insight in the difference in EPR spectra for the wild type and a recently studied E383Q-mutant cbb 3-enzyme. Furthermore, we show that the redox-coupled proton movement in the proximal cavity of cbb 3-enzymes contributes to the low redox potential of heme b 3, and suggest its potential implications for the high apparent oxygen affinity of these enzymes.
Keywords: Heme-copper oxidases; Proton-coupled electron transfer (PCET); Electron transfer; Proton transfer; Density functional theory (DFT); Electron paramagnetic resonance (EPR);

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA 810, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700 oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700+ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700+ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q r site of the cytb6f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.
Keywords: Cyclic electron transport; Photosystem I; Photo-electrochemical control; P700 oxidation; Kinetic model;

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas by Marie Lapaille; Marc Thiry; Emilie Perez; Diego González-Halphen; Claire Remacle; Pierre Cardol (1533-1539).
Mitochondrial F1FO ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.
Keywords: Mitochondrial ATP synthase; ATP2 mutant; Chlamydomonas; Beta subunit; Organelle interaction;

Investigation of the enzymatic activity of the Na+,K+-ATPase via isothermal titration microcalorimetry by Raimund Noske; Flemming Cornelius; Ronald J. Clarke (1540-1545).
Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na+,K+-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of −51 (± 1) kJ mol 1. This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na+ and K+ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis–Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K m and K I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays.
Keywords: Steady-state kinetics; ATP hydrolysis; ADP inhibition; Thermodynamic efficiency; Pig kidney; Shark rectal gland;

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus by Miwa Sugiura; Sayo Harada; Takashi Manabe; Hidenori Hayashi; Yasuhiro Kashino; Alain Boussac (1546-1554).
A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA 3 gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b 559 that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ∆Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b 559 into the low potential form. Structural reasons for such effects are discussed.
Keywords: Photosystem II; Psb30; Cytochrome b 559; Small subunit; Trans-membrane protein; Side-path electron transfer; Thermosynechococcus elongatus;

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition by Ramzi J. Khairallah; Genevieve C. Sparagna; Nishanth Khanna; Karen M. O'Shea; Peter A. Hecker; Tibor Kristian; Gary Fiskum; Christine Des Rosiers; Brian M. Polster; William C. Stanley (1555-1562).
Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca2+-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca2+-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca2+-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca2+ retention capacity and decreased Ca2+-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca2+-induced MPTP opening.
Keywords: Cardiac; Eicosapentaenoic acid; Docosahexaenoic acid; Fish oil; Heart; Mitochondrial Permeability transition pore;