BBA - Bioenergetics (v.1787, #12)

The Ca2+- and oxidative stress-induced mitochondrial permeability transition (MPT) plays an important role in phenomena ranging from tissue damage upon infarction to muscle wasting in some forms of dystrophy. The process is due to the activation of a large pore in the inner mitochondrial membrane. Anti-oxidants are considered a preventive and remedial tool, and mitochondria-targeted redox-active compounds have been developed. Plant polyphenols are generally considered as anti-oxidants, and thus candidates to the role of mitochondria-protecting agents. In patch-clamp experiments, easily oxidizable polyphenols induced closure of the MPT channel. In swelling experiments with suspensions of mitochondria, high (20–50 μM) concentrations of quercetin, the most efficient inhibitor, promoted instead the onset of the MPT. Chelators of Fe2+/3+ and Cu+/2+ ions counteracted this effect. Fluorescent indicators of superoxide production confirmed that quercetin potentiates O2 •− generation by isolated mitochondria and cultured cells. Since this was not affected by chelating Fe and Cu ions, the MPT-inducing effect can be ascribed to a “secondary”, metal ion-catalyzed production of ROS. These results are a direct demonstration of the ambivalent redox character of polyphenols. Their mode of action in vivo cannot be taken for granted, but needs to be experimentally verified.
Keywords: Mitochondrial permeability transition pore; Polyphenol; Quercetin; Reactive oxygen species (ROS); Patch-clamp;

Hypoxia and the metabolic phenotype of prostate cancer cells by L.H. Higgins; H.G. Withers; A. Garbens; H.D. Love; L. Magnoni; S.W. Hayward; C.D. Moyes (1433-1443).
Many cancer cells have an unusual ability to grow in hypoxia, but the origins of this metabolic phenotype remain unclear. We compared the metabolic phenotypes of three common prostate cancer cell models (LNCaP, DU145, PC3), assessing energy metabolism, metabolic gene expression, and the response to various culture contexts (in vitro and xenografts). LNCaP cells had a more oxidative phenotype than PC3 and DU145 cells based upon respiration, lactate production, [ATP], metabolic gene expression, and sensitivity of these parameters to hypoxia. PC3 and DU145 cells possessed similar Complex II and mtDNA levels, but lower Complex III and IV activities, and were unresponsive to dinitrophenol or dichloroacetate, suggesting that their glycolytic phenotype is due to mitochondrial dysfunction rather than regulation. High passage under normoxia converted LNCaP from oxidative to glycolytic cells (based on respiration and lactate production), and altered metabolic gene expression. Though LNCaP-derived cells differed from the parental line in mitochondrial enzyme activities, none differed in mitochondrial content (assessed as cardiolipin levels). When LNCaP-derived cells were grown as xenografts in immunodeficient mice, there were elements of a hypoxic response (e.g., elevated VEGF mRNA) but line-specific changes in expression of select glycolytic, mitochondrial and fatty acid metabolic genes. Low oxygen in vitro did not influence the mRNA levels of SREBP axis, nor did it significantly alter triglyceride production in any of the cell lines suggesting that the pathway of de novo fatty acid synthesis is not directly upregulated by hypoxic conditions. Collectively, these studies demonstrate important differences in the metabolism of these prostate cancer models. Such metabolic differences would have important ramifications for therapeutic strategies involving metabolic targets.
Keywords: Hypoxia-inducible factor; HIF-1; Aerobic glycolysis; Mitochondrial dysfunction; LNCaP; DU145; PC3;

The kinetics and thermodynamics of forward and reverse electron transfer around the reaction center of purple bacterium Rhodobacter sphaeroides were studied in vivo by flash-excited delayed fluorescence, prompt fluorescence (induction) and kinetic difference absorption. By protection of the photomultiplier from intense bacteriochlorophyll prompt fluorescence evoked by laser excitation, the time resolution of the fluorometer was reduced typically 10 μs. Two precursor states of the delayed fluorescence were identified: P+QA and cyt c 2 3+QA whose enthalpy levels were 340 meV and 1020 meV below A⁎, respectively. The free energy of the P+QA state relative to A⁎ was − 870 meV in whole cells. Similar values were obtained earlier for isolated reaction center and chromatophore. The free energies of cyt c 2 3+QA and P+QA states showed no or very weak (− 6 meV/pH unit) pH-dependence, respectively, supporting the concept of pH-independent redox midpoint potential of QA/QA in intact cells. In accordance with the multiphasic kinetics of delayed fluorescence, the kinetics of re-opening of the closed reaction center is also complex (it extends up to 1 s) as a consequence of acceptor and donor-side reactions. The control of charge export from the reaction center by light regime, redox agents and inhibitors is investigated. The complex kinetics may arise from the distribution of quinones in different redox states on the acceptor side (QB binding site and pool) and from organization of electron transfer components in supercomplexes.
Keywords: Photosynthesis; Delayed fluorescence; Reaction center; Redox chemistry; Quinone; Cytochrome;

Glucose intolerance in C57Bl/6 mice has been associated with mutations in the nicotinamide nucleotide transhydrogenase (Nnt) gene. It has been proposed that the absence of NNT from mitochondria leads to increased mitochondrial reactive oxygen species production and subsequent activation of uncoupling protein 2 (UCP2). Activation of UCP2 has been suggested to uncouple electron transport from ATP synthesis in pancreatic beta cell mitochondria thereby decreasing glucose tolerance due to decreased insulin secretion through lower ATP/ADP ratios. The hypothesis tested in this paper is that UCP2 function is required for the dysregulation of glucose homeostasis observed in NNT ablated mice. Single and double Nnt and Ucp2 knockout mouse lines were used to measure glucose tolerance, whole animal energy balance and biochemical characteristics of mitochondrial uncoupling. As expected, glucose tolerance was diminished in mice lacking NNT. This was independent of UCP2 as it was observed either in the presence or absence of UCP2. The range of metabolic parameters examined in the mice and the proton conductance of isolated mitochondria remained unaltered in this double NNT and UCP2 knockout model. Ablation of UCP2 did not itself affect glucose tolerance and therefore previous observations of increased glucose tolerance of mice lacking UCP2 were not confirmed. We conclude that the decreased glucose tolerance in Nnt knockout mice observed in our experiments does not require UCP2.
Keywords: Uncoupling protein 2 (UCP2); Nicotinamide nucleotide transhydrogenase (NNT); Proton leak; Glucose tolerance; Glucose stimulated insulin secretion (GSIS);

The two open reading frames in the Synechocystis sp. PCC 6803 genome, sll1214 and sll1874, here designated cycI and cycII, respectively, encode similar proteins, which are involved in the Mg protoporphyrin monomethylester (MgProtoME) cyclase reaction. The impairment of tetrapyrrole biosynthesis was examined by separate inactivation of both cyclase encoding genes followed by analysis of chlorophyll contents, MgProtoME levels and several enzyme activities of tetrapyrrole biosynthesis. We additionally addressed the question, whether the two isoforms can complement cyclase deficiency under normal aerobic and micro-oxic growth conditions in light. A cycII knock-out mutant grew without any adverse symptoms at normal air conditions, but showed MgProtoME accumulation at growth under low oxygen conditions. A complete deletion of cycI failed in spite of mixotrophic growth and low light at both ambient and low oxygen, but resulted in accumulation of 150 and 28 times more MgProtoME, respectively, and circa 60% of the wild-type chlorophyll content. The CycI deficiency induced a feedback-controlled limitation of the metabolic flow in the tetrapyrrole biosynthetic pathway by reduced ALA synthesis and Fe chelatase activity. Ectopic expression of the CycI protein restored the wild-type phenotype in cycI mutant cells under ambient air as well as micro-oxic growth conditions. Overexpressed CycII protein could not compensate for cycI mutation under micro-oxic and aerobic growth conditions, but complemented the cycII knock-out mutant as indicated by wild-type MgProtoME and chlorophyll levels. Our findings indicate the essential contribution of CycI to the cyclase reaction at ambient and low oxygen conditions, while low oxygen conditions additionally require CycII for the cyclase activity.
Keywords: Tetrapyrrole biosynthesis; Chlorophyll; Photosynthesis; Cyanobacterium synechocystis; Photosensitization;

Photochemical and photoelectrochemical quenching of chlorophyll fluorescence in photosystem II by Wim Vredenberg; Milan Durchan; Ondřej Prášil (1468-1478).
This paper deals with kinetics and properties of variable fluorescence in leaves and thylakoids upon excitation with low intensity multi-turnover actinic light pulses corresponding with an excitation rate of about 10 Hz. These show a relatively small and amply documented rise in the sub-s time range towards the plateau level F pl followed by a delayed and S-shaped rise towards a steady state level F m which is between three and four fold the initial dark fluorescence level F o. Properties of this retarded slow rise are i) rate of dark recovery is (1–6 s)− 1, ii) suppression by low concentration of protonophores, iii) responsiveness to complementary single turnover flash excitation with transient amplitude towards a level F m which is between five and six fold the initial dark fluorescence level F o and iv) in harmony with and quantitatively interpretable in terms of a release of photoelectrochemical quenching controlled by the trans-thylakoid proton pump powered by the light-driven Q cycle. Data show evidence for a sizeable fluorescence increase upon release of (photo) electrochemical quenching, defined as qPE. Release of qPE occurs independent of photochemical quenching defined here as qPP even under conditions at which qPP = 1. The term photochemical quenching, hitherto symbolized by qP, will require a new definition, because it incorporates in its present form a sizeable photoelectrochemical component. The same is likely to be true for definition and use of qN as an indicator of non photochemical quenching.
Keywords: Chlorophyll a fluorescence; Photosystem II; Heterogeneity; Photochemical quenching; Photoelectrochemical quenching; Kinetic model;

Localization of cytochrome b 6 f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803 by Matthias Schultze; Björn Forberich; Sascha Rexroth; Nina Gwendolyn Dyczmons; Matthias Roegner; Jens Appel (1479-1485).
The cytochrome b 6 f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b 6 f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.
Keywords: Thylakoids; Plasma membrane; Rieske protein; Hydrogenase; Cyanobacteria; Quinol oxidase;

Ca2+-induced permeability transition can be observed even in yeast mitochondria under optimized experimental conditions by Akiko Yamada; Takenori Yamamoto; Yuya Yoshimura; Shunichi Gouda; Satoshi Kawashima; Naoshi Yamazaki; Kikuji Yamashita; Masatoshi Kataoka; Toshihiko Nagata; Hiroshi Terada; Douglas R. Pfeiffer; Yasuo Shinohara (1486-1491).
Yeast mitochondria have generally been believed not to undergo the permeability transition (PT) by the accumulation of Ca2+ within the mitochondrial matrix, unlike mammalian mitochondria. However, the reason why the yeast PT is not induced by Ca2+ has remained obscure. In this study, we examined in detail the effects of Ca2+ on yeast mitochondria under various conditions. As a result, we discovered that the PT could be induced even in yeast mitochondria by externally added Ca2+ under optimized experimental conditions. The 2 essential parameters for proper observation of the PT-inducing effects of Ca2+ were the concentrations of the respiratory substrate and that of inorganic phosphate (Pi) in the incubation medium. The yeast mitochondrial PT induced by Ca2+ was found to be insensitive to cyclosporin A and suppressed in the presence of a high concentration of Pi. Furthermore, when the PT was induced in yeast mitochondria by Ca2+, the release of cytochrome c from mitochondria was also observed.
Keywords: Permeability transition; Cytochrome c; Mitochondrion; Yeast; Apoptosis; Ca2+;

Investigation of the low-affinity oxidation site for exogenous electron donors in the Mn-depleted photosystem II complexes by V.N. Kurashov; E.R. Lovyagina; D.Yu. Shkolnikov; M.K. Solntsev; M.D. Mamedov; B.K. Semin (1492-1498).
In the manganese-depleted photosystem II (PSII[−Mn]) preparations, oxidation of exogenous electron donors is carried out through the high-affinity (HA) and the low-affinity (LA) sites. This paper investigates the LA oxidation site in the PSII(−Mn) preparations where the HA, Mn-binding site was blocked with ferric cations [[11] B.K. Semin, M.L. Ghirardi, M. Seibert, Blocking of electron donation by Mn(II) to YZ following incubation of Mn-depleted photosystem II membranes with Fe(II) in the light, Biochemistry 41 (2002) 5854–5864.]. In blocked (PSII[−Mn,+Fe]) preparations electron donation by Mn(II) cations to YZ was not detected at Mn(II) concentration 10 μM (corresponds to K m for Mn(II) oxidation at the HA site), but detected at Mn concentration 100 μM (corresponds to K m for the LA site) by fluorescence measurements. Comparison of pH-dependencies of electron donation by Mn(II) through the HA and the LA sites revealed the similar рKа equal to 6.8. Comparison of K m for diphenylcarbazide (DPC) oxidation at the LA site and K d for AT thermoluminescence band suppression by DPC in PSII(−Mn,+Fe) samples suggests that there is relationship between the LA site and AT band formation. The role of D1-His190 as an oxidant of exogenous electron donors at the LA site is discussed. In contrast to electrogenic electron transfer from Mn(II) at the HA site to YZ , photovoltage due to Mn(II) oxidation in iron-blocked PSII(−Mn) core particles was not detected.
Keywords: Chlorophyll fluorescence; Iron; Manganese; Oxygen-evolving complex; Photosystem II; Photovoltage; Thermoluminescence;

Filling the “green gap” of the major light-harvesting chlorophyll a/b complex by covalent attachment of Rhodamine Red by Kristina Gundlach; Mara Werwie; Sabine Wiegand; Harald Paulsen (1499-1504).
The major light-harvesting chlorophyll a/b complex (LHCII) greatly enhances the efficiency of photosynthesis in green plants. Recombinant LHCII can be assembled in vitro from its denatured, bacterially expressed apoprotein and plant pigments. This makes it an interesting candidate for biomimetic light-harvesting in photovoltaic applications. Due to its almost 20 pigments bound per apoprotein, LHCII absorbs efficiently in the blue and red spectral domains of visible light but less efficiently in the green domain, the so-called “green gap” in its absorption spectrum. Here we present a hybrid complex of recombinant LHCII with organic dyes that add to LHCII absorption in the green spectral region. One or three Rhodamine Red dye molecules were site-specifically attached to cysteine side chains in the apoprotein and did not interfere with LHCII assembly, function and stability. The dyes transferred their excitation energy virtually completely to the chlorophylls in LHCII, partially filling in the green gap. Thus, organic dyes can be used to increase the absorption cross section and, thus, the light-harvesting efficiency of recombinant LHCII.
Keywords: FRET (Förster resonance energy transfer); LHCII; Maleimide dye; Photosynthesis; Site-specific labeling; Solar spectrum;

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria by Hélène Cheap; Sophie Bernad; Valérie Derrien; László Gerencsér; Julia Tandori; Pedro de Oliveira; Deborah K. Hanson; Péter Maróti; Pierre Sebban (1505-1515).
Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely QA and QB. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P+QA state (P is the primary electron donor) and temperature dependence of the rate of P+QA charge recombination that are in good agreement show that in the two RC variants, both QA and QB are destabilized by about the same free energy amount: respectively ∼ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of QA and QB . This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.
Keywords: Photosynthesis; Electron transfer; Proton transfer; Reaction center; Bioenergetics; Hydrogen-bond network; Reaction center;

Sulfite oxidation in Sinorhizobium meliloti by Jeremy J. Wilson; Ulrike Kappler (1516-1525).
Sulfite-oxidizing enzymes (SOEs) are crucial for the metabolism of many cells and are particularly important in bacteria oxidizing inorganic or organic sulfur compounds. However, little is known about SOE diversity and metabolic roles. Sinorhizobium meliloti contains four candidate genes encoding SOEs of three different types, and in this work we have investigated the role of SOEs in S. meliloti and their possible link to the metabolism of the organosulfonate taurine. Low level SOE activity (∼ 1.4 U/mg) was present under all conditions tested while growth on taurine and thiosulfate induced high activities (5.5–8.8 U/mg) although S. meliloti cannot metabolize thiosulfate. Protein purification showed that although expression of two candidate genes matched SOE activity patterns, only a single group 2 SOE, SorT (SMc04049), is responsible for this activity. SorT is a heme-free, periplasmic homodimer (78 kDa) that has low homology to other bacterial SOEs. SorT has an apparent k cat of 343 s1 and high affinities for both sulfite (K Mapp_pH8 15.5 μM) and ferricyanide (K Mapp_pH8 3.44 μM), but not cytochrome c, suggesting a need for a high redox potential natural electron acceptor. K Mapp_sulfite was nearly invariant with pH which is in contrast to all other well characterized SOEs. SorT is part of an operon (SMc04049-04047) also containing a gene for a cytochrome c and an azurin, and these might be the natural electron acceptors for the enzyme. Phylogenetic analysis of SorT-related SOEs and enzymes of taurine degradation indicate that there is no link between the two processes.
Keywords: Sulfite oxidation; Metalloenzyme; Taurine metabolism; Energy generation; Gene expression; Enzyme;