BBA - Bioenergetics (v.1767, #7)

Exposing the Complex III Qo semiquinone radical by Haibo Zhang; Artur Osyczka; P. Leslie Dutton; Christopher C. Moser (883-887).
Complex III Qo site semiquinone has been assigned pivotal roles in productive energy-conversion and destructive superoxide generation. After a 30-year search, a genetic heme bH knockout arrests this transient semiquinone EPR radical, revealing the natural engineering balance pitting energy-conserving, short-circuit minimizing, split electron transfer and catalytic speed against damaging oxygen reduction.
Keywords: Complex III; Cytochrome bc1; Semiquinone; Qo; Superoxide; Electron transfer;

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats by Li-Ju Huang; Chin Hsu; Tsen-Ni Tsai; Shu-Jung Wang; Rei-Cheng Yang (888-896).
Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F0F1-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F1-ATPase β-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F0F1-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.
Keywords: Sepsis; Mitochondrial F0F1-ATPase; IF1; Liver;

Myoglobin with chlorophyllous chromophores: Influence on protein stability by Dejan Marković; Stefanie Pröll; Claudia Bubenzer; Hugo Scheer (897-904).
The stabilities of myoglobin, apo-myoglobin, and of two myoglobins with chlorophyllous chromophores (Zn-pheophorbide a and Zn-bacteriopheophorbide a), have been studied by thermal and chemical denaturation. With guanidinium chloride, the stability order is myoglobin > Zn-pheophorbide-myoglobin > Zn-bacteriopheophorbide-myoglobin ∼ apo-myoglobin. The thermal behavior is more complex. The transition temperature of thermal unfolding of the apoprotein (62.4 °C) is increased by Zn-pheophorbide a (83.9 °C) and Zn-bacteriopheophorbide a (82.6 °C) to a similar degree as by the native chromophore, heme (83.5 °C). The recovery with Zn-pheophorbide (92–98%) is even higher than with heme (74–76%), while with Zn-bacteriopheophorbide (40%) it is as low as with the apoprotein (42%). Recovery also depends on the rates of heating, and in particular the time spent at high temperatures. It is concluded that irreversibility of unfolding is related to loss of the chromophores, which are required for proper re-folding.
Keywords: Myoglobin; Heat denaturation; Chemical denaturation; Reversibility of unfolding; Chromophore exchange; Chlorophyllide; Bacteriochlorophyllide;

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, QA and QB, and the S2 state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of QA reoxidation.
Keywords: Electron transport; Photosystem II; Oxygen evolution; Fluorescence; Extrinsic polypeptides; Polyamines;

Biochemical phenotypes associated with the mitochondrial ATP6 gene mutations at nt8993 by Alessandra Baracca; Gianluca Sgarbi; Marina Mattiazzi; Gabriella Casalena; Eleonora Pagnotta; Maria L. Valentino; Maurizio Moggio; Giorgio Lenaz; Valerio Carelli; Giancarlo Solaini (913-919).
Two point mutations (T > G and T > C) at the same 8993 nucleotide of mitochondrial DNA (at comparable mutant load), affecting the ATPase 6 subunit of the F1F0-ATPase, result in neurological phenotypes of variable severity in humans. We have investigated mitochondrial function in lymphocytes from individuals carrying the 8993T > C mutation: the results were compared with data from five 8993T > G NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) patients. Both 8993T > G and 8993T > C mutations led to energy deprivation and ROS overproduction. However, the relative contribution of the two pathogenic components is different depending on the mutation considered. The 8993T > G change mainly induces an energy deficiency, whereas the 8993T > C favours an increased ROS production. These results possibly highlight the different pathogenic mechanism generated by the two mutations at position 8993 and provide useful information to better characterize the biochemical role of the highly conserved Leu-156 in ATPase 6 subunit of the mitochondrial ATP synthase complex.
Keywords: mtDNA; T8993C; ATP synthase; Membrane potential; ROS; Mitochondria;

Chlorophyll synthesis and degradation were analyzed in the cyanobacterium Synechocystis sp. PCC 6803 by incubating cells in the presence of 13C-labeled glucose or 15N-containing salts. Upon mass spectral analysis of chlorophyll isolated from cells grown in the presence of 13C-glucose for different time periods, four chlorophyll pools were detected that differed markedly in the amount of 13C incorporated into the porphyrin (Por) and phytol (Phy) moieties of the molecule. These four pools represent (i) unlabeled chlorophyll (12Por12Phy), (ii) 13C-labeled chlorophyll (13Por13Phy), and (iii, iv) chlorophyll, in which either the porphyrin or the phytol moiety was 13C-labeled, whereas the other constituent of the molecule remained unlabeled (13Por12Phy and 12Por13Phy). The kinetics of 12Por12Phy disappearance, presumably due to chlorophyll de-esterification, and of 13Por12Phy, 12Por13Phy, and 13Por13Phy accumulation due to chlorophyll synthesis provided evidence for continuous chlorophyll turnover in Synechocystis cells. The loss of 12Por12Phy was three-fold faster in a photosystem I-less strain than in a photosystem II-less strain and was accelerated in wild-type cells upon exposure to strong light. These data suggest that most chlorophyll appears to be de-esterified in Synechocystis upon dissociation and repair of damaged photosystem II. A substantial part of chlorophyllide and phytol released upon the de-esterification of chlorophyll can be recycled for the biosynthesis of new chlorophyll molecules contributing to the formation of 13Por12Phy and 12Por13Phy chlorophyll pools. The phytol kinase, Slr1652, plays a significant but not absolutely critical role in this recycling process.
Keywords: Chlorophyll biosynthesis; Chlorophyll degradation; Mass spectrometry; Stable isotope labeling; Cyanobacteria;

The H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14−17 transmembrane domains, and is found in a range of organisms. We focused on the second quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains long conserved cytoplasmic loops. We prepared a library of 1536 mutants that were assayed for pyrophosphate hydrolysis and proton translocation. Mutant enzymes with low substrate hydrolysis and proton-pump activities were selected and their DNAs sequenced. Of these, 34 were single-residue substitution mutants. We generated 29 site-directed mutant enzymes and assayed their activity. The mutation of 10 residues in the fifth transmembrane domain resulted in low coupling efficiencies, and a mutation of Gly198 showed neither hydrolysis nor pumping activity. Four residues in cytoplasmic loop e were essential for substrate hydrolysis and efficient H+ translocation. Pro189, Asp281, and Val351 in the periplasmic loops were critical for enzyme function. Mutation of Ala357 in periplasmic loop h caused a selective reduction of proton-pump activity. These low-efficiency mutants reflect dysfunction of the energy-conversion and/or proton-translocation activities of H+-pyrophosphatase. Four critical residues were also found in transmembrane domain 6, three in transmembrane domain 7, and five in transmembrane domains 8 and 9. These results suggest that transmembrane domain 5 is involved in enzyme function, and that energy coupling is affected by several residues in the transmembrane domains, as well as in the cytoplasmic and periplasmic loops. H+-pyrophosphatase activity might involve dynamic linkage between the hydrophilic and transmembrane domains.
Keywords: Energy coupling; H+-pyrophosphatase; Random mutagenesis; Proton pump;

Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy by Werner J.H. Koopman; Mark A. Hink; Sjoerd Verkaart; Henk-Jan Visch; Jan A.M. Smeitink; Peter H.G.M. Willems (940-947).
We previously reported that inhibition of mitochondrial complex I (CI) by rotenone induces marked increases in mitochondrial length and degree of branching, thus revealing a relationship between mitochondrial function and shape. We here describe the first time use of fluorescence correlation spectroscopy (FCS) to simultaneously probe mitochondrial mobility and intra-matrix protein diffusion, with the aim to investigate the effects of chronic CI inhibition on the latter two parameters. To this end, EYFP was expressed in the mitochondrial matrix of human skin fibroblasts (mitoEYFP) using baculoviral transduction and its diffusion monitored by FCS. This approach revealed the coexistence of moving and stationary mitochondria within the same cell and enabled simultaneous quantification of mitochondrial velocity and mitoEYFP diffusion. When CI activity was chronically reduced by 80% using rotenone treatment, the percentage of moving mitochondria and their velocity decreased by 30%. MitoEYFP diffusion did not differ between moving and stationary mitochondria but was increased 2-fold in both groups of mitochondria following rotenone treatment. We propose that the increase in matrix protein diffusion together with the increase in mitochondrial length and degree of branching constitutes part of an adaptive response which serves to compensate for the reduction in CI activity and mitochondrial motility.
Keywords: Complex I deficiency; EYFP; Rotenone;

Changes in the LHCII-mediated energy utilization and dissipation adjust the methanol-induced biomass increase by E. Navakoudis; N.E. Ioannidis; D. Dörnemann; K. Kotzabasis (948-955).
Considerably low methanol concentrations of 0.5% (v/v), induce an immense increase in biomass production in cultures of the unicellular green alga Scenedesmus obliquus compared to controls without additional methanol. The effect is light-regulated and it mimics high-CO2 induced changes of the molecular structure and function of the photosynthetic apparatus. There is evidence that methanol enhances under high light conditions by molecular changes in the LHCII – a decrease of the functional antenna-size per active reaction center – the photochemical effectiveness of the absorbed energy. This means that the non-photochemical quenching (NPQ) is minimized and thereby the overall dissipation energy. Experiments with mutants of Scenedesmus Wt produced evidence that the LHCII is the locus of the mechanism which regulates the methanol effect. The employed mutants were Wt-LHC, lacking a functioning LHCII, the light-dependent greening mutant C-2A′, and the double mutant C-2A′-LHC, combining both mutations.
Keywords: Biomass production; Light harvesting complex II (LHCII); Methanol metabolism; Non-photochemical quenching (NPQ); Photosynthetic apparatus; Scenedesmus obliquus (mutants);

Monitoring fluorescence of individual chromophores in peridinin–chlorophyll–protein complex using single molecule spectroscopy by S. Wörmke; S. Mackowski; T.H.P. Brotosudarmo; C. Jung; A. Zumbusch; M. Ehrl; H. Scheer; E. Hofmann; R.G. Hiller; C. Bräuchle (956-964).
Single molecule spectroscopy experiments are reported for native peridinin–chlorophyll a–protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm− 1. In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.
Keywords: Light-harvesting complexes; Fluorescence; Single molecule spectroscopy; Chromophore interaction;

Functional roles of arginine residues in mung bean vacuolar H+-pyrophosphatase by Yi-Yuong Hsiao; Yih-Jiuan Pan; Shen-Hsing Hsu; Yun-Tzu Huang; Tseng-Huang Liu; Ching-Hung Lee; Chien-Hsien Lee; Pei-Feng Liu; Wen-Chi Chang; Yung-Kai Wang; Lee-Feng Chien; Rong-Long Pan (965-973).
Plant vacuolar H+-translocating inorganic pyrophosphatase (V-PPase EC 3.6.1.1) utilizes inorganic pyrophosphate (PPi) as an energy source to generate a H+ gradient potential for the secondary transport of ions and metabolites across the vacuole membrane. In this study, functional roles of arginine residues in mung bean V-PPase were determined by site-directed mutagenesis. Alignment of amino-acid sequence of K+-dependent V-PPases from several organisms showed that 11 of all 15 arginine residues were highly conserved. Arginine residues were individually substituted by alanine residues to produce R → A-substituted V-PPases, which were then heterologously expressed in yeast. The characteristics of mutant variants were subsequently scrutinized. As a result, most R → A-substituted V-PPases exhibited similar enzymatic activities to the wild-type with exception that R242A, R523A, and R609A mutants markedly lost their abilities of PPi hydrolysis and associated H+-translocation. Moreover, mutation on these three arginines altered the optimal pH and significantly reduced K+-stimulation for enzymatic activities, implying a conformational change or a modification in enzymatic reaction upon substitution. In particular, R242A performed striking resistance to specific arginine-modifiers, 2,3-butanedione and phenylglyoxal, revealing that Arg242 is most likely the primary target residue for these two reagents. The mutation at Arg242 also removed F inhibition that is presumably derived from the interfering in the formation of substrate complex Mg2+–PPi. Our results suggest accordingly that active pocket of V-PPase probably contains the essential Arg242 which is embedded in a more hydrophobic environment.
Keywords: Proton translocation; Tonoplast; Vacuole; Site-directed mutagenesis; Vacuolar H+-pyrophosphatase;

Electron transport, the proton gradient and ATP synthesis were determined in thylakoids that had been briefly exposed to a low concentration of trypsin during illumination. This treatment cleaves the γ subunit of the ATP synthase into two large fragments that remain associated with the enzyme. Higher rates of electron transport are required to generate a given value of the proton gradient in the trypsin-treated membranes than in control membranes, indicating that the treated membranes are proton leaky. Since venturicidin restores electron transport and the proton gradient to control levels, the proton leak is through the ATP synthase. Remarkably, the synthesis of ATP by the trypsin-treated membranes at saturating light intensities is only slightly inhibited even though the proton gradient is significantly lower in the treated thylakoids. ATP synthesis and the proton gradient were determined as a function of light intensity in control and trypsin-treated thylakoids. The trypsin-treated membranes synthesized ATP at lower values of the proton gradient than the control membranes. Cleavage of the γ subunit abrogates inhibition of the activity of the chloroplast ATP synthase by the ε subunit. Our results suggest that overcoming inhibition by the ε subunit costs energy.
Keywords: Chloroplast ATP synthase; Thylakoid membranes; Electron transport; ATP synthesis; Proton flux; γ subunit; ε subunit;

Assessing the molecular basis for rat-selective induction of the mitochondrial permeability transition by norbormide by Alessandra Zulian; Valeria Petronilli; Sergio Bova; Federica Dabbeni-Sala; Gabriella Cargnelli; Maurizio Cavalli; David Rennison; Jessica Stäb; Olivia Laita; Dong Jun Lee; Margaret A. Brimble; Brian Hopkins; Paolo Bernardi; Fernanda Ricchelli (980-988).
It was recently demonstrated that the rat-selective toxicant norbormide also induces rat-selective opening of the permeability transition pore (PTP) in isolated mitochondria. Norbormide is a mixture of endo and exo stereoisomers; however, only the endo forms are lethal to rats. In the present study we tested both endo and exo isomers as well as neutral and cationic derivatives of norbormide to: (i) verify if the PTP-regulatory activity by norbormide is stereospecific; (ii) define the structural features of norbormide responsible for PTP-activation, (iii) elucidate the basis for the drug species-specificity. Our results show that: (i) norbormide isomers affect PTP in a rat-selective fashion; however, no relevant differences between lethal and non-lethal forms are observed suggesting that drug regulation of PTP-activity and lethality in rats are unrelated phenomena; (ii) a (phenylvinyl)pyridine moiety represents the key element conferring the PTP-activating effect; (iii) cationic derivatives of rat-active compounds accumulate in the matrix via the membrane potential and activate the PTP also in mouse and guinea pig mitochondria. These findings suggest that the norbormide-sensitive PTP-target is present in all species examined, and is presumably located on the matrix side. The species-selectivity may depend on the unique properties of a transport system allowing drug internalisation in rat mitochondria.
Keywords: Norbormide; Rat-toxicant; Mitochondria; Permeability transition; Species-specificity;

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria by Marek Vrbacký; Zdeněk Drahota; Tomáš Mráček; Alena Vojtíšková; Pavel Ješina; Pavel Stopka; Josef Houštěk (989-997).
Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.
Keywords: Brown adipose tissue mitochondria; Glycerophosphate dehydrogenase; Reactive oxygen species; Fluorescent probes; Oxygraphy; EPR;

Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase by Lars Langemeyer; Siegfried Engelbrecht (998-1005).
FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((αβ)3γδε) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10γε relative to (αβ)3δab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but “horizontal” repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of “vertical” repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.
Keywords: ATP; Synthesis; FoF1 (EC 3.6.3.14); Proton; Translocation;