BBA - Bioenergetics (v.1817, #11)

Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders by A.M. Voets; P.J. Lindsey; S.J. Vanherle; E.D. Timmer; J.J. Esseling; W.J.H. Koopman; P.H.G.M. Willems; G.C. Schoonderwoerd; D. De Groote; B.T. Poll-The; I.F.M. de Coo; H.J.M. Smeets (1971-1978).
Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A > G or m.3302A > G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.► The role of oxidative stress and antioxidant therapy in OXPHOS disorders is unclear. ► ROS, antioxidant and oxidative damage levels determine oxidative stress status together. ► Almost all primary fibroblast cell lines of OXPHOS patients showed increased ROS levels. ► Adaptive processes could prevent the accumulation of oxidative protein damage. ► Oxidative stress status is not mutation- but patient-specific.
Keywords: Oxidative stress; POLG1; CI deficiency; MELAS; ROS; Protein carbonyls;

Enhanced sensitivity and characterization of photosystem II in transgenic tobacco plants with decreased chloroplast glutathione reductase under chilling stress by Shunhua Ding; Ming Lei; Qingtao Lu; Aihong Zhang; Yan Yin; Xiaogang Wen; Lixin Zhang; Congming Lu (1979-1991).
Chloroplast glutathione reductase (GR) plays an important role in protecting photosynthesis against oxidative stress. We used transgenic tobacco (Nicotiana tabacum) plants with severely decreased GR activities by using a gene encoding tobacco chloroplast GR for the RNAi construct to investigate the possible mechanisms of chloroplast GR in protecting photosynthesis against chilling stress. Transgenic plants were highly sensitive to chilling stress and accumulated high levels of H2O2 in chloroplasts. Spectroscopic analysis and electron transport measurements show that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements demonstrate that there was a slow electron transfer between QA and QB and decreased redox potential of QB in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses illustrate that PSII protein accumulation was decreased greatly in transgenic plants. Our results suggest that chloroplast GR plays an important role in protecting PSII function by maintaining the electron transport in PSII acceptor side and stabilizing PSII complexes under chilling stress. Our results also suggest that the recycling of ascorbate from dehydroascorbate in the ascorbate–glutathione cycle in the chloroplast plays an essential role in protecting PSII against chilling stress.► Role of GR in protecting photosynthesis against chilling stress was investigated. ► Transgenic tobacco plants with decreased chloroplast GR by RNAi were used. ► PSII activity was significantly reduced in transgenic plants. ► GR plays an important role in protecting PSII function against chilling stress.
Keywords: Chilling stress; Chlorophyll fluorescence; Glutathione reductase; Photosystem II; Transformed tobacco (Nicotiana tabacum); Thermoluminescence;

The marine cyanobacterium Prochloron is a unique photosynthetic organism that lives in obligate symbiosis with colonial ascidians. We compared Prochloron harbored in four different host species and cultured Prochlorothrix by means of spectroscopic measurements, including time-resolved fluorescence, to investigate host-induced differences in light-harvesting strategies between the cyanobacteria. The light-harvesting efficiency of photosystems including antenna Pcb, PS II–PS I connection, and pigment status, especially that of PS I Red Chls, were different among the four samples. We also discuss relationships between these observed characteristics and the light conditions, to which Prochloron cells are exposed, influenced by distribution pattern in the host colonies, presence or absence of tunic spicules, and microenvironments within the ascidians' habitat.► We examined excitation–relaxation dynamics in the cyanobacteria, Prochloron and Prochlorothrix. ► The sample Prochloron were harbored by four different ascidians. ► Efficiencies of the light-harvesting systems were different among samples. ► PS II–PS I connections and energy traps were different among samples.
Keywords: Prochloron; Prochlorothrix; Symbiosis; Time-resolved fluorescence; In hospite spectroscopy;

Influence of the PsbA1/PsbA3, Ca2+/Sr2+ and Cl/Br exchanges on the redox potential of the primary quinone QA in Photosystem II from Thermosynechococcus elongatus as revealed by spectroelectrochemistry by Yuki Kato; Tadao Shibamoto; Shoichi Yamamoto; Tadashi Watanabe; Naoko Ishida; Miwa Sugiura; Fabrice Rappaport; Alain Boussac (1998-2004).
Ca2+ and Cl ions are essential elements for the oxygen evolution activity of photosystem II (PSII). It has been demonstrated that these ions can be exchanged with Sr2+ and Br, respectively, and that these ion exchanges modify the kinetics of some electron transfer reactions at the Mn4Ca cluster level (Ishida et al., J. Biol. Chem. 283 (2008) 13330–13340). It has been proposed from thermoluminescence experiments that the kinetic effects arise, at least in part, from a decrease in the free energy level of the Mn4Ca cluster in the S3 state though some changes on the acceptor side were also observed. Therefore, in the present work, by using thin-layer cell spectroelectrochemistry, the effects of the Ca2+/Sr2+ and Cl/Br exchanges on the redox potential of the primary quinone electron acceptor QA, E m(QA/QA ), were investigated. Since the previous studies on the Ca2+/Sr2+ and Cl/Br exchanges were performed in PsbA3-containing PSII purified from the thermophilic cyanobacterium Thermosynechococcus elongatus, we first investigated the influences of the PsbA1/PsbA3 exchange on E m(QA/QA ). Here we show that i) the E m(QA/QA ) was up-shifted by ca. + 38 mV in PsbA3-PSII when compared to PsbA1-PSII and ii) the Ca2+/Sr2+ exchange up-shifted the E m(QA/QA ) by ca. + 27 mV, whereas the Cl/Br exchange hardly influenced E m(QA/QA ). On the basis of the results of E m(QA/QA ) together with previous thermoluminescence measurements, the ion-exchange effects on the energetics in PSII are discussed.Display Omitted► PsbA1/A3 exchange shifts the redox potential of QA of PSII by ca. + 38 mV. ► Biosynthetic Ca2+/Sr2+ exchange shifts the redox potential of QA by ca. + 27 mV. ► Cl/Br exchange hardly influences the redox potential of QA. ► The findings show that tuning of the cofactors' potentials is exquisitely subtle.
Keywords: Photosystem II; Redox potential; Electron transfer; D1 protein; PsbA protein;

The ScoI homologue SenC is a copper binding protein that interacts directly with the cbb 3-type cytochrome oxidase in Rhodobacter capsulatus by Eva Lohmeyer; Sebastian Schröder; Grzegorz Pawlik; Petru-Iulian Trasnea; Annette Peters; Fevzi Daldal; Hans-Georg Koch (2005-2015).
Sco proteins are widespread assembly factors for the CuA centre of aa 3-type cytochrome oxidases in eukaryotic and prokaryotic organisms. However, Sco homologues are also found in bacteria like Rhodobacter capsulatus which lack aa 3-type cytochrome oxidases and instead use a cbb 3-type cytochrome oxidase (cbb 3 Cox) without a CuA centre as a terminal oxidase. In the current study, we have analyzed the role of Sco (SenC) during cbb 3 Cox assembly in R. capsulatus. In agreement with earlier works, we found a strong cbb 3 Cox defect in the absence of SenC that impairs the steady-state stability of the CcoN, CcoO and CcoP core subunits, without the accumulation of detectable assembly intermediates. In vivo cross-linking results demonstrate that SenC is in close proximity to the CcoP and CcoH subunits of cbb 3 Cox, suggesting that SenC interacts directly with cbb 3 Cox during its assembly. SenC binds copper and the cbb 3 Cox assembly defect in the absence of SenC can be rescued by the addition of least 0.5 μM Cu. Neither copper nor SenC influenced the transcription of the ccoNOQP operon encoding for cbb 3 Cox. Transcription of senC itself was also not influenced by Cu unless the putative Cu-export ATPase CcoI was absent. As CcoI is specifically required for the cbb 3 Cox assembly, these data provide a direct link between Cu delivery to cbb 3 Cox and SenC function.► SenC is required for cbb 3 Cox assembly at low Cu2+ concentrations/high growth rates. ► SenC interacts directly with the CcoP and CcoH subunits of R. capsulatus cbb 3 Cox. ► SenC is regulated by Cu availability in the absence of the Cu export ATPase CcoI.
Keywords: Sco proteins; Copper insertion; Cytochrome c oxidase; Copper transport; Respiratory growth; Rhodobacter capsulatus;

Inactivation of the conserved open reading frame ycf34 of Synechocystis sp. PCC 6803 interferes with the photosynthetic electron transport chain by Thomas Wallner; Yoshinori Hagiwara; Gábor Bernát; Roman Sobotka; Edward J. Reijerse; Nicole Frankenberg-Dinkel; Annegret Wilde (2016-2026).
Ycf34 is a hypothetical chloroplast open reading frame that is present in the chloroplast genomes of several non-green algae. Ycf34 homologues are also encoded in all sequenced genomes of cyanobacteria. To evaluate the role of Ycf34 we have constructed and analysed a cyanobacterial mutant strain. Inactivation of ycf34 in Synechocystis sp. PCC 6803 showed no obvious phenotype under normal light intensity growth conditions. However, when the cells were grown under low light intensity they contained less and smaller phycobilisome antennae and showed a strongly retarded growth, suggesting an essential role of the Ycf34 polypeptide under light limiting conditions. Northern blot analysis revealed a very weak expression of the phycocyanin operon in the ycf34 mutant under light limiting growth in contrast to the wild type and to normal light conditions. Oxygen evolution and P700 measurements showed impaired electron flow between photosystem II and photosystem I under these conditions which suggest that the impaired antenna size is most likely due to a highly reduced plastoquinone pool which triggers regulation on a transcriptional level. Using a FLAG-tagged Ycf34 we found that this protein is tightly bound to the thylakoid membranes. UV–vis and Mössbauer spectroscopy of the recombinant Ycf34 protein demonstrate the presence of an iron–sulphur cluster. Since Ycf34 lacks homology to known iron–sulphur cluster containing proteins, it might constitute a new type of iron–sulphur protein implicated in redox signalling or in optimising the photosynthetic electron transport chain.► Cyanobacterial homologue of the chloroplast ORF ycf34 is a novel iron–sulphur protein. ► Ycf34 is required for growth under light limitation. ► Synechocystis mutants lacking Ycf34 are impaired in phycocyanin accumulation. ► Inactivation of ycf34 alters the photosynthetic electron transport chain.
Keywords: Hypothetical chloroplast open reading frame; Phycobilisome; Iron–sulphur protein; Electron transport chain; Photosynthesis; Synechocystis sp. PCC 6803;

Composition of the mitochondrial electron transport chain in Acanthamoeba castellanii: Structural and evolutionary insights by Ryan M.R. Gawryluk; Kenneth A. Chisholm; Devanand M. Pinto; Michael W. Gray (2027-2037).
The mitochondrion, derived in evolution from an α-proteobacterial progenitor, plays a key metabolic role in eukaryotes. Mitochondria house the electron transport chain (ETC) that couples oxidation of organic substrates and electron transfer to proton pumping and synthesis of ATP. The ETC comprises several multiprotein enzyme complexes, all of which have counterparts in bacteria. However, mitochondrial ETC assemblies from animals, plants and fungi are generally more complex than their bacterial counterparts, with a number of ‘supernumerary’ subunits appearing early in eukaryotic evolution. Little is known, however, about the ETC of unicellular eukaryotes (protists), which are key to understanding the evolution of mitochondria and the ETC. We present an analysis of the ETC proteome from Acanthamoeba castellanii, an ecologically, medically and evolutionarily important member of Amoebozoa (sister to Opisthokonta). Data obtained from tandem mass spectrometric (MS/MS) analyses of purified mitochondria as well as ETC complexes isolated via blue native polyacrylamide gel electrophoresis are combined with the results of bioinformatic queries of sequence databases. Our bioinformatic analyses have identified most of the ETC subunits found in other eukaryotes, confirming and extending previous observations. The assignment of proteins as ETC subunits by MS/MS provides important insights into the primary structures of ETC proteins and makes possible, through the use of sensitive profile-based similarity searches, the identification of novel constituents of the ETC along with the annotation of highly divergent but phylogenetically conserved ETC subunits.► We studied in detail the electron transport chain (ETC) of Acanthamoeba castellanii. ► This is the first broad proteomic analysis of the ETC for a member of Amoebozoa. ► We used profile-based bioinformatics to annotate divergent ETC subunits. ► We identified several novel subunits in the highly stable dimeric ATP synthase. ► Proteomic analysis confirmed novel structural features of several ETC proteins.
Keywords: Mitochondrion; Protist; Electron transport chain; Mass spectrometry;

Reactive oxygen species affect ATP hydrolysis by targeting a highly conserved amino acid cluster in the thylakoid ATP synthase γ subunit by Felix Buchert; Yvonne Schober; Andreas Römpp; Mark L. Richter; Christoph Forreiter (2038-2048).
The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.► H2O2 and singlet oxygen affect the chloroplast ATPase, in particular the γ-subunit. ► Main ROS targets are amino acids within a conserved met/cys cluster of the γ-subunit. ► Both ROS have a detrimental effect on the CF1 activity. ► Amino acid substitution resulted in increased tolerance against both ROS types.
Keywords: (Chloroplast) F-type ATP synthase; Singlet oxygen; Hydrogen peroxide; Reactive oxygen species;

Tyrosine nitration of voltage-dependent anion channels in cardiac ischemia-reperfusion: reduction by peroxynitrite scavenging by Meiying Yang; Amadou K.S. Camara; Bassam T. Wakim; Yifan Zhou; Ashish K. Gadicherla; Wai-Meng Kwok; David F. Stowe (2049-2059).
Excess superoxide (O2 •−) and nitric oxide (NO•) forms peroxynitrite (ONOO) during cardiac ischemia reperfusion (IR) injury, which in turn induces protein tyrosine nitration (tyr-N). Mitochondria are both a source of and target for ONOO. Our aim was to identify specific mitochondrial proteins that display enhanced tyr-N after cardiac IR injury, and to explore whether inhibiting O2 •−/ONOO during IR decreases mitochondrial protein tyr-N and consequently improves cardiac function. We show here that IR increased tyr-N of 35 and 15 kDa mitochondrial proteins using Western blot analysis with 3-nitrotyrosine antibody. Immunoprecipitation (IP) followed by LC–MS/MS identified 13 protein candidates for tyr-N. IP and Western blot identified and confirmed that the 35 kDa tyr-N protein is the voltage-dependent anion channel (VDAC). Tyr-N of native cardiac VDAC with IR was verified on recombinant (r) VDAC with exogenous ONOO. We also found that ONOO directly enhanced rVDAC channel activity, and rVDAC tyr-N induced by ONOO formed oligomers. Resveratrol (RES), a scavenger of O2 •−/ONOO, reduced the tyr-N levels of both native and recombinant VDAC, while L-NAME, which inhibits NO• generation, only reduced tyr-N levels of native VDAC. O2 •− and ONOO levels were reduced in perfused hearts during IR by RES and L-NAME and this was accompanied by improved cardiac function. These results identify tyr-N of VDAC and show that reducing ONOO during cardiac IR injury can attenuate tyr-N of VDAC and improve cardiac function.► IR enhances tyr-N of mitochondrial protein, especially VDAC. ► RES and L-NAME reduce tyr-N of VDAC through inhibiting or scavenging O2 •−/ONOO generated during IR. ► Tyr-N of VDAC affects its structure and function.
Keywords: Mitochondria; Protein tyrosine nitration; Cardiac injury; VDAC; ROS/RNS scavengers;

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.► A spent-media methodology is demonstrated to reveal metabolic alterations in cancer. ► c-Myc, KLF4 and Oct1 differentially modulate the cellular energy metabolism. ► Isoleucine, AADA and GABA are correlated with cancer cell's bioenergetics profile. ► Isoleucine, AADA and GABA could serve as biomarkers of oncogene activation.
Keywords: Metabolism; Bioenergetics; Secretomics; Oxidative phosphorylation; Glycolysis;

The mitochondrial permeability transition pore (PTP) — An example of multiple molecular exaptation? by Angelo Vianello; Valentino Casolo; Elisa Petrussa; Carlo Peresson; Sonia Patui; Alberto Bertolini; Sabina Passamonti; Enrico Braidot; Marco Zancani (2072-2086).
The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500 Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins.In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role.Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.
Keywords: Permeability transition; Exaptation; Evolution; Eukarya; Mitochondria;

Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans by Alexander M. Arutyunyan; Junshi Sakamoto; Mai Inadome; Yoshiki Kabashima; Vitaliy B. Borisov (2087-2094).
Cytochromes bd are terminal oxidases in the respiratory chains of many prokaryotic organisms. They reduce O2 to 2H2O at the expense of electrons extracted from quinol. The oxidases can be divided into two subfamilies, L and S, based on the presence of either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I designated as ‘Q-loop’. The L-subfamily members, e.g. the enzyme from Escherichia coli, are relatively well-studied and were shown to generate proton-motive force. The S-subfamily comprises the majority of cytochromes bd including the enzyme from Geobacillus thermodenitrificans but is very poor studied. We compared the properties of cytochromes bd from G. thermodenitrificans and E. coli at room temperature using a combination of absorption, CD and MCD spectroscopy. The G. thermodenitrificans enzyme does contain the high-spin heme b HS (“b 595”) despite the fact that its characteristic Q00-band (“α”-band) at 595 nm is not seen in the absorption spectra; stoichiometry of hemes b LS, b HS and d per the enzyme complex is suggested to be 1:1:1. At 1 mM CO, 20–25% of ferrous heme b HS in the G. thermodenitrificans oxidase binds the ligand, while in case of the E. coli enzyme such a reaction is minor. In the G. thermodenitrificans oxidase, the excitonic interaction between ferrous hemes b HS and d decreased as compared to that in the E. coli bd. The latter may suggest that the two enzymes differ in the distance between heme d and heme b HS and/or in the angle between their porphyrin planes.► G. thermodenitrificans cytochrome bd studied by absorption, CD and MCD spectroscopy. ► Purified enzyme retains high-spin heme b HS (previously called “b 595”). ► At 1 mM CO, about 20–25% of high-spin ferroheme b HS binds the ligand. ► Compared to E. coli, excitonic interaction between ferrohemes b HS and d decreased.
Keywords: Metabolism; Molecular bioenergetics; Spectroscopy; Excitonic coupling; Chlorin; Thermophilic bacterium;