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BBA - Bioenergetics (v.1767, #11)

Editorial Board (pp. ii).

Ycf12 is a core subunit in the photosystem II complex by Yasuhiro Kashino; Takeshi Takahashi; Natsuko Inoue-Kashino; Akiko Ban; Yohei Ikeda; Kazuhiko Satoh; Miwa Sugiura (pp. 1269-1275).
The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of ∼5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.

Keywords: Abbreviations; Chl; chlorophyll; GRAVY; grand average values of hydropathicity; LMW; low molecular weight; MES; 2-(; N; -morpholino)ethanesulfonic acid; PS II; photosystem II; ycf; hypothetical chloroplast reading frameLow molecular weight polypeptide; Photosystem II; Psb30; Thermosynechococcus elongatus; ycf12


Reduced creatine-stimulated respiration in doxorubicin challenged mitochondria: Particular sensitivity of the heart by Malgorzata Tokarska-Schlattner; Max Dolder; Isabelle Gerber; Oliver Speer; Theo Wallimann; Uwe Schlattner (pp. 1276-1284).
Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its use is limited by a risk of cardiotoxicity, which is not completely understood. Recently, we have shown that DXR impairs essential properties of purified mitochondrial creatine kinase (MtCK), with cardiac isoenzyme (sMtCK) being particularly sensitive. In this study we assessed the effects of DXR on respiration of isolated structurally and functionally intact heart mitochondria, containing sMtCK, in the presence and absence of externally added creatine (Cr), and compared these effects with the response of brain mitochondria expressing uMtCK, the ubiquitous, non-muscle MtCK isoenzyme. DXR impaired respiration of isolated heart mitochondria already after short-term exposure (minutes), affecting both ADP- and Cr-stimulated respiration. During a first short time span (minutes to 1 h), detachment of MtCK from membranes occurred, while a decrease of MtCK activity related to oxidative damage was only observed after longer exposure (several hours). The early inhibition of Cr-stimulated respiration, in addition to impairment of components of the respiratory chain involves a partial disturbance of functional coupling between MtCK and ANT, likely due to interaction of DXR with cardiolipin leading to competitive inhibition of MtCK/membrane binding. The relevance of these findings for the regulation of mitochondrial energy production in the heart, as well as the obvious differences of DXR action in the heart as compared to brain tissue, is discussed.

Keywords: Anthracycline; Creatine kinase; Cardiotoxicity; Isolated mitochondria; Creatine-simulated respiration


Transport and metabolism ofl-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis by Anna Atlante; Lidia de Bari; Antonella Bobba; Ersilia Marra; Salvatore Passarella (pp. 1285-1299).
Having confirmed that externally addedl-lactate can enter cerebellar granule cells, we investigated whether and howl-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions.(1)l-lactate enters mitochondria, perhaps via anl-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of anl-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis.(2)In both normal and apoptotic cell homogenates, externally addedl-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirroredl-lactate uptake by mitochondria and occurred with a hyperbolic dependence onl-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition ofl-lactate. The rate of the process depended onl-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellularl-lactate/pyruvate shuttle, whose activity was limited by the putativel-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier.(3)l-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putativel-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

Keywords: Abbreviations; ANT; adenine nucleotide translocator; ARS; arsenite; DIV; days; in vitro; BME; basal medium Eagle; α-CCN; α-cyanocinnamate; CGC; cerebellar granule cells; COX; cytochrome oxidase; d; -LAC; d; -lactate; l; -LAC; l; -lactate; cLDH; cytosolic; l; -lactate dehydrogenase; mLDH; mitochondrial; l; -lactate dehydrogenase; OUA; ouabain; OXA; oxamate; PBS; phosphate buffer saline medium; Pi; phosphate; PDS; pyruvate detecting system; PheSUC; phenylsuccinate; PYR; pyruvate; ROT; rotenone; S.D.; standard deviation; SUCC; succinate; S-K25 cells; control cells; S-K5 cells; apoptotic cells; TX-100; Triton-X-100mitochondria; l; -lactate; l; -lactate dehydrogenase; transport; metabolism; apoptosis


Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center by Hiroshi Ishikita; Artur Galstyan; Ernst-Walter Knapp (pp. 1300-1309).
In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (BA), bacteriopheopytin (HA), the primary quinone (QA) to the secondary quinone (QB). Although the non-heme iron complex (Fe complex) is located between QA and QB, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637–644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459–4465]. In this study, we calculated redox potentials of QA/B ( Em(QA/B)) and the Fe complex ( Em(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. Em(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic Em(Fe) downshift by 230 mV upon formation of QA− (but not QB−) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.

Keywords: Non-heme iron; Redox potential; Quinone; Electron transfer; Bacterial photosynthetic reaction center; Photosystem II


Proton paths in the sarcoplasmic reticulum Ca2+-ATPase by Eeva-Liisa Karjalainen; Karin Hauser; Andreas Barth (pp. 1310-1318).
The sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) pumps Ca2+ and countertransport protons. Proton pathways in the Ca2+ bound and Ca2+-free states are suggested based on an analysis of crystal structures to which water molecules were added. The pathways are indicated by chains of water molecules that interact favorably with the protein. In the Ca2+ bound state Ca2E1, one of the proposed Ca2+ entry paths is suggested to operate additionally or alternatively as proton pathway. In analogs of the ADP-insensitive phosphoenzyme E2P and in the Ca2+-free state E2, the proton path leads between transmembrane helices M5 to M8 from the lumenal side of the protein to the Ca2+ binding residues Glu-771, Asp-800 and Glu-908. The proton path is different from suggested Ca2+ dissociation pathways. We suggest that separate proton and Ca2+ pathways enable rapid (partial) neutralization of the empty cation binding sites. For this reason, transient protonation of empty cation binding sites and separate pathways for different ions are advantageous for P-type ATPases in general.

Keywords: Abbreviations; BHQ; 2,5-di-; tert; -butyl-1,4-dihydroxybenzene; Ca; 2; E1P; ADP-sensitive phosphoenzyme; E2(TG); Ca; 2+; -free ATPase with thapsigargin bound; E2(TG; +; BHQ); Ca; 2+; -free ATPase with thapsigargin and 2,5-di-; tert; -butyl-1,4-dihydroxybenzene bound; E2P; ADP-insensitive phosphoenzyme; E2(TG; +; MgF; 4; 2−; ); E2 complex with thapsigargin and MgF; 4; 2−; E2(TG; +; AlF; 4; −; ); E2 complex with thapsigargin and AlF; 4; −; MCCE; multiconformation continuum electrostatics; SR; sarcoplasmic reticulum; TG; thapsigarginSERCA1a; Ca; 2+; pump; Na,K-ATPase; Proton countertransport; Proton pathway


Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force by Boris A. Feniouk; Alberto Rebecchi; Donatella Giovannini; Sofie Anefors; Armen Y. Mulkidjanian; Wolfgang Junge; Paola Turina; B. Andrea Melandri (pp. 1319-1330).
H+-FOF1-ATP synthase couples proton flow through its membrane portion, FO, to the synthesis of ATP in its headpiece, F1. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.

Keywords: Abbreviations; F; O; F; 1; H; +; transporting F; O; F; 1; -ATP synthase; Δ; ∼; μ; H; +; transmembrane difference of proton electrochemical potential; Δ; ψ; transmembrane difference of electrical potential; BChl; bacteriochlorophyll; ACMA; 9-amino-6-chloro-2-methoxy-acrydine; Pi; inorganic phosphateATP synthase; Subunit gamma; ADP inhibition; Activation; Met23Lys; Rhodobacter capsulatus


Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents by Nicola Giangregorio; Annamaria Tonazzi; Cesare Indiveri; Ferdinando Palmieri (pp. 1331-1339).
During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.

Keywords: Abbreviations; CAC; Carnitine/acylcarnitine carrier; MTSES; Sodium(2-sulfonatoethyl)-methane-thiosulfonate; MTSEA; Sodium(2-aminoethyl)-methane-thiosulfonate; NEM; N-ethylmaleimide; Pipes; 1,4-piperazinediethanesulfonic acid; SDS-PAGE; Sodium dodecyl sulfate polyacrylamide gel electrophoresisMitochondria; Carnitine/acylcarnitine carrier; Site-directed mutagenesis; Membrane transport; Thiol reagent


Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution by Francesca Gubellini; Francesco Francia; Paola Turina; Daniel Lévy; Giovanni Venturoli; B. Andrea Melandri (pp. 1340-1352).
The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.

Keywords: Abbreviations; ICM; intracytoplasmic membranes; Bchl; bacteriochlorophyll; ATPase; ATP synthase (EC 3.6.3.14); F; 1; soluble portion of the ATP synthase; LH1, LH2; light harvesting complex 1, 2; RC; reaction center; PL; phospholipid; EM; electron microscopy; QELS; quasi elastic light scattering; LDAO; lauryldimethylamine oxide; Δ; ψ; transmembrane difference of electrical potential; ΔpH; transmembrane difference of pH; Tricine; N; -[2-hydroxy-1,1-; bis; (hydroxymethyl)ethyl] glycine; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis Rhodobacter capsulatus; ATP synthase; Intracytoplasmic membrane; Size distribution; Heterogeneity
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