BBA - Bioenergetics (v.1757, #1)

Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant by Ulf Dühring; Klaus-Dieter Irrgang; Katja Lünser; Julia Kehr; Annegret Wilde (3-11).
The Ycf37 protein has been suggested to be involved in the biogenesis and/or stability of the cyanobacterial photosystem I (PSI) [A. Wilde, K. Lünser, F. Ossenbühl, J. Nickelsen, T. Börner, Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content, Biochem. J. 357 (2001) 211–216]. With Ycf37 specific antibodies, we analyzed the localization of Ycf37 within the thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803. Inspection of a sucrose gradient profile indicated that small amounts of Ycf37 co-fractionated with monomeric photosynthetic complexes, but not with trimeric PSI. Isolating 3xFLAG epitope-tagged Ycf37 by affinity-tag purification rendered several PSI subunits that specifically co-precipitated with this protein. Blue-native PAGE newly revealed two monomeric PSI complexes (PSI and PSI*) in wild-type thylakoids. The lower amount of PsaK present in PSI* may explain its higher electrophoretic mobility. PSI* was more prominent in high-light grown cells and interestingly proved absent in the Δycf37 mutant. PSI* appeared again when the mutant was complemented in trans with the wild-type ycf37 gene. In the Δycf37 mutant the amount of trimeric PSI complexes was reduced to about 70% of the wild-type level with no significant changes in photochemical activity and subunit composition of the remaining photosystems. Our results indicate that Ycf37 plays a specific role in the preservation of PSI* and the biogenesis of PSI trimers.
Keywords: Chloroplast open reading frame; Photosystem I; Synechocystis sp. PCC 6803; Thylakoid;

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2–2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100–1800 μmol photons m−2 s−1). Simultaneous measurements of FI and 820-nm transmission kinetics (I820) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2–0.4 s) is accompanied by a decrease of I820, i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4–2 s) of the dip is not paralleled by the change in I820. These results were compared with that measured with pea leaves—representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I–P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 μM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I820 kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin–NADP+–oxidoreductase or Mehler–peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.
Keywords: Chlorophyll fluorescence; OJIP-transient; Lichen; 820-nm transmission; Photobiont; Photosystem; Thylakoid membrane; Trebouxia; Lasallia pustulata; Umbilicaria hirsuta; Hypogymnia physodes;

Dinitrophenol-induced mitochondrial uncoupling in vivo triggers respiratory adaptation in HepG2 cells by Valérie Desquiret; Dominique Loiseau; Caroline Jacques; Olivier Douay; Yves Malthièry; Patrick Ritz; Damien Roussel (21-30).
Here, we show that 3 days of mitochondrial uncoupling, induced by low concentrations of dinitrophenol (10 and 50 μM) in cultured human HepG2 cells, triggers cellular metabolic adaptation towards oxidative metabolism. Chronic respiratory uncoupling of HepG2 cells induced an increase in cellular oxygen consumption, oxidative capacity and cytochrome c oxidase activity. This was associated with an upregulation of COXIV and ANT3 gene expression, two nuclear genes that encode mitochondrial proteins involved in oxidative phosphorylation. Glucose consumption, lactate and pyruvate production and growth rate were unaffected, indicating that metabolic adaptation of HepG2 cells undergoing chronic respiratory uncoupling allows continuous and efficient mitochondrial ATP production without the need to increase glycolytic activity. In contrast, 3 days of dinitrophenol treatment did not change the oxidative capacity of human 143B.TK cells, but it increased glucose consumption, lactate and pyruvate production. Despite a large increase in glycolytic metabolism, the growth rate of 143B.TK cells was significantly reduced by dinitrophenol-induced mitochondrial uncoupling. We propose that chronic respiratory uncoupling may constitute an internal bioenergetic signal, which would initiate a coordinated increase in nuclear respiratory gene expression, which ultimately drives mitochondrial metabolic adaptation within cells.
Keywords: Human cell line; Mitochondria; Energy metabolism; Cytochrome-c oxidase; Glycolysis;

A theoretical study on nitric oxide reductase activity in a ba 3-type heme-copper oxidase by L. Mattias Blomberg; Margareta R.A. Blomberg; Per E.M. Siegbahn (31-46).
The mechanism of nitric oxide reduction in a ba 3-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a 3 and is partly reduced having some nitroxyl anion character (3NO), and it is thus activated toward the attack by the second NO. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu B is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the N―O bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the N―O bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the N―O bond and form N2O at a feasible rate. The cleavage of the N―O bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.
Keywords: Nitric oxide reductase; NOR; Heme-copper oxidase; Cytochrome c oxidase; CcO; Reduction; Electron transfer; Nitrous oxide; Nitric oxide; DFT; B3LYP;

Cyanobacterial psbA families in Anabaena and Synechocystis encode trace, constitutive and UVB-induced D1 isoforms by Cosmin I. Sicora; Sarah E. Appleton; Christopher M. Brown; Jonathon Chung; Jillian Chandler; Amanda M. Cockshutt; Imre Vass; Douglas A. Campbell (47-56).
Cyanobacteria cope with UVB induced photoinhibition of Photosystem II by regulating multiple psbA genes to boost the expression of D1 protein (in Synechocystis sp. PCC6803), or to exchange the constitutive D1:1 protein to an alternate D1:2 isoform (in Synechococcus sp. PCC7942). To define more general patterns of cyanobacterial psbA expression, we applied moderately photoinhibitory UVB to Anabaena sp. PCC7120 and tracked the expression of its five psbA genes. psbAI, encoding a D1:1 protein isoform characterized by a Gln130, represented the majority of the psbA transcript pool under control conditions. psbAI transcripts decreased upon UVB treatment but the total psbA transcript pool increased 3.5 fold within 90 min as a result of sharply increased psbAII, psbAIV and psbAIII transcripts encoding an alternate D1:2 protein isoform characterized by Glu130, similar to that of Synechococcus. Upon UVB treatment the relaxation of flash induced chlorophyll fluorescence showed a characteristic acceleration of a decay phase likely associated with the exchange from the D1:1 protein isoform encoded by psbAI to the alternate D1:2 isoform encoded by psbAIV, psbAII and psbAIII. Throughout the UVB treatment the divergent psbA0 made only a trace contribution to the total psbA transcript pool. This suggests a similarity to the divergent psbAI gene from Synechocystis, whose natural expression we demonstrate for the first time at a trace level similar to psbA0 in Anabaena. These trace-expressed psbA genes in two different cyanobacteria raise questions concerning the functions of these divergent genes.
Keywords: Anabaena; D1; Photoinhibition; Photosystem II; psbA; Synechocystis; UVB;

Acute effect of fatty acids on metabolism and mitochondrial coupling in skeletal muscle by Sandro M. Hirabara; Leonardo R. Silveira; Luciane C. Alberici; Carol V.G. Leandro; Rafael H. Lambertucci; Gisele C. Polimeno; Maria F. Cury Boaventura; Joaquim Procopio; Anibal E. Vercesi; Rui Curi (57-66).
Acute effects of free fatty acids (FFA) were investigated on: (1) glucose oxidation, and UCP-2 and -3 mRNA and protein levels in 1 h incubated rat soleus and extensor digitorium longus (EDL) muscles, (2) mitochondrial membrane potential in cultured skeletal muscle cells, (3) respiratory activity and transmembrane electrical potential in mitochondria isolated from rat skeletal muscle, and (4) oxygen consumption by anesthetized rats. Long-chain FFA increased both basal and insulin-stimulated glucose oxidation in incubated rat soleus and EDL muscles and reduced mitochondrial membrane potential in C2C12 myotubes and rat skeletal muscle cells. Caprylic, palmitic, oleic, and linoleic acid increased O2 consumption and decreased electrical membrane potential in isolated mitochondria from rat skeletal muscles. FFA did not alter UCP-2 and -3 mRNA and protein levels in rat soleus and EDL muscles. Palmitic acid increased oxygen consumption by anesthetized rats. These results suggest that long-chain FFA acutely lead to mitochondrial uncoupling in skeletal muscle.
Keywords: Long-chain fatty acid; Skeletal muscle; Mitochondrial uncoupling; Glucose metabolism; Mitochondria;

Spectral analysis of the bc 1 complex components in situ: Beyond the traditional difference approach by Vladimir P. Shinkarev; Antony R. Crofts; Colin A. Wraight (67-77).
The cytochrome (cyt) bc 1 complex (ubiquinol: cytochrome c oxidoreductase) is the central enzyme of mitochondrial and bacterial electron-transport chains. It is rich in prosthetic groups, many of which have significant but overlapping absorption bands in the visible spectrum. The kinetics of the cytochrome components of the bc 1 complex are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. This difference-wavelength (DW) approach has been used extensively in the development and testing of the Q-cycle mechanism of the bc 1 complex in Rhodobacter sphaeroides chromatophores. However, the DW approach does not fully compensate for spectral interference from other components, which can significantly distort both amplitudes and kinetics. Mechanistic elaboration of cyt bc 1 turnover requires an approach that overcomes this limitation. Here, we compare the traditional DW approach to a least squares (LS) analysis of electron transport, based on newly determined difference spectra of all individual components of cyclic electron transport in chromatophores. Multiple sets of kinetic traces, measured at different wavelengths in the absence and presence of specific inhibitors, were analyzed by both LS and DW approaches. Comparison of the two methods showed that the DW approach did not adequately correct for the spectral overlap among the components, and was generally unreliable when amplitude changes for a component of interest were small. In particular, it was unable to correct for extraneous contributions to the amplitudes and kinetics of cyt b L. From LS analysis of the chromophoric components (RC, c tot, b H and b L), we show that while the Q-cycle model remains firmly grounded, quantitative reevaluation of rates, amplitudes, delays, etc., of individual components is necessary. We conclude that further exploration of mechanisms of the bc 1 complex, will require LS deconvolution for reliable measurement of the kinetics of individual components of the complex in situ.
Keywords: bc 1 complex; Q-cycle; Electron transfer; Spectral deconvolution; Kinetics; Least squares; Rhodobacter sphaeroides;