BBA - Bioenergetics (v.1457, #3)

First photosystem II crystals capable of water oxidation by Athina Zouni; Rafael Jordan; Eberhard Schlodder; Petra Fromme; Horst Tobias Witt (103-105).
Oxygen evolution and proton release of crystallised photosystem II core complexes isolated from Synechococcus elongatus were measured. The yields show that the crystals themselves are capable of highly active water oxidation. This opens the possibility for the structural analysis of the outstanding water-oxidising apparatus.
Keywords: Photosynthesis; Photosystem II; Crystallisation; Oxygen evolution; Proton release; Synechococcus elongatus;

Interaction of photosynthetic pigments with various organic solvents 2 by Mitsuo Umetsu; Zheng-Yu Wang; Kenzi Yoza; Masayuki Kobayashi; Tsunenori Nozawa (106-117).
Magnetic circular dichroism (MCD) and absorption spectra of metal bacteriochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q y(0–0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q x(0–0), and the ratio of MCD Q y(0–0) intensity to the dipole strength (B/D) was inversely proportional to the difference in energy between the Q x(0–0) and Q y(0–0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl4), however, the correlation did not hold because of the existence of aggregates. Hence, the correlation between the values of B/D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl β-D-glucopyranoside.
Keywords: Magnetic circular dichroism; Bacteriochlorophyll a; Coordination state; Light-harvesting complex 1; n-Octyl-β-D-glucopyranoside (β-OG) treatment;

Morphological and functional changes of mitochondria from density separated trout erythrocytes by Luca Tiano; Patrizia Ballarini; Giorgio Santoni; Michal Wozniak; Giancarlo Falcioni (118-128).
Density separated trout erythrocytes, using a discontinuous Percoll gradient, yielded three distinct subfractions (top, middle and bottom) since older cells are characterized by increasing density. Cells from each subfraction were incubated with mitochondria-specific fluorescent probe Mitotracker and JC-1 in order to assess mitochondrial mass and membrane potential by means of cytofluorimetric analysis, confocal microscopy and subsequent computer-aided image analysis allowing a detailed investigation at single cell level. Both cytofluorimetric data and image analysis revealed changes in size and redistribution of mitochondria starting from the light fraction to the bottom. In particular in young erythrocytes small mitochondria were detected localized exclusively around the nucleus in a crown-like shape, the middle fraction revealed enlarged mitochondria partially scattered throughout the cytosol, whereas the last fraction represented again mitochondria with reduced size being distinctly dispersed throughout the cytosol in the cells. Concerning membrane potential considerations, our study revealed a dramatic decrease of ΔΨm in the bottom layer cell mitochondria compared to the top and unusual membrane potential increase of a subpopulation of enlarged mitochondria. ΔpH was also investigated in the three fractions by pretreating the cells with nigericin, allowing to confirm a mitochondrial energetic impairment in older cells.
Keywords: Trout erythrocyte; Mitochondria; Aging; Oxidative stress; Membrane potential; JC-1;

Relative to ferredoxin:NADP+ reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700+, suggest lack of function of the truncated FNR in the plastoquinone–cytochrome b 6 f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated ΔpetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275–4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b 6 f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed.
Keywords: Ferredoxin:NADP+ reductase; Cyclic electron transport; Photosystem I; Green fluorescent protein; petH; (Synechocystis PCC 6803);

Comparative study of the g=4.1 EPR signals in the S2 state of photosystem II by Alain Boussac; A.William Rutherford (145-156).
The Mn4 complex which is involved in water oxidation in photosystem II is known to exhibit three types of EPR signals in the S2 state, one of the five redox states of the enzyme cycle: a multiline signal (spin 1/2), signals at g>5 (spin 5/2) and a signal at g=4.1 (or g=4.25). The g=4.1 signal could be generated under two distinct sets of conditions: either by illumination at room temperature or at 200 K in certain experimental conditions (g4S signal) or by near-infrared illumination between ≈77 and ≈160 K of the S2-multiline state (g4IR signal). The two g=4.1 signals arise from states which have quite different stability in terms of temperature. In the present work we have compared these two signals in order to test if they originate from the same or from different chemical origins. The microwave power saturation properties of the two signals measured at 4.2 K were found to be virtually identical. Their temperature dependencies measured at non-saturating powers were also identical. The presence of Curie law behavior for the g4S and g4IR signals indicates that the states responsible for both signals are ground states. The orientation dependence, anisotropy and resolved hyperfine structure of the two g4 signals were also found to be virtually indistinguishable. We have been unable to confirm the behavior reported earlier indicating that the g4S signal is an excited state, nor were we able to confirm the presence of signal from a higher excited state in samples containing the g4S, nor a radical signal in samples containing the g4IR. These findings are best interpreted assuming that the two signals have a common origin i.e. a spin 5/2 ground state arising from a magnetically coupled Mn-cluster of 4 Mn ions.
Keywords: Near-infrared; Oxygen evolution; Mn cluster; Spin state transition;

The YZ-tyrosine radical was trapped by freezing immediately after illumination in Ca2+-depleted Photosystem II (PS II) membranes and the pH-dependent characteristics of the radical were investigated using CW-EPR and pulsed ENDOR. The spectrum of the Y Z radical trapped in the Y ZS1 state at pH 5.5 was cation-like as reported in Mn-depleted PS II (H. Mino et al., Spectrochim. Acta A 53 (1997) 1465–1483). By illuminating the PS II-retaining S2 state, the Y Z radical and a broad doublet signal formed in the g≈2 region were trapped concomitantly. The spectrum of the trapped Y Z radical in the Y ZS2 state was cation-like at pH 5.5 but the pulsed ENDOR measurements reveals the involvement of the neutral Y Z radical in the doublet signal. At pH 7.0, the resulting Y Z signal was the mixture of the cation-like and neutral radical spectra, and considerably different from the neutral radical found in Mn-depleted PS II. pH-Dependent changes in the properties of the Y Z radical are discussed in relation to the redox events occurring in Ca2+-depleted PS II.
Keywords: Photosystem II; Tyrosine Z; ENDOR; Ca2+ depletion; Doublet signal; pH dependence;

Mitochondrial protonophoric activity induced by a thyromimetic fatty acid analogue by Orit Hermesh; Bella Kalderon; Benjamin Berman; Jacob Bar-Tana (166-174).
Calcium-dependent uncoupling of liver mitochondrial oxidative phosphorylation by a non-metabolizable long chain fatty acyl analogue was compared with uncoupling induced by in vivo thyroid hormone treatment. β,β′-Methyl-substituted hexadecane α,ω-dioic acid (Medica 16) is reported here to induce a saturable 20–30% decrease in liver mitochondrial ΔΨ, ΔpH and protonmotive force which proceeds in the presence of added Ca2+ to cyclosporin A-sensitive mitochondrial permeabilization. Ca2+-dependent uncoupling by Medica 16 was accompanied by atractylate-enhanced, bongkrekic-inhibited activation of mitochondrial Ca2+ efflux. The direct mitochondrial effect exerted in vitro by Medica 16 is similar to that induced by in vivo thyroid hormone treatment. Hence, the thyromimetic protonophoric activity of Medica 16 and the uncoupling activity of TH converge onto components of the mitochondrial permeabilization transition pore.
Keywords: Mitochondria; Uncoupling; Fatty acid; Thyroid hormone;

Rapid spectrophotometric method for quantitation of cytochrome c release from isolated mitochondria or permeabilized cells revisited by Florence Appaix; Marie-Nathalie Minatchy; Catherine Riva-Lavieille; Jose Olivares; Bruno Antonsson; Valdur A. Saks (175-181).
This paper recalls the earlier work by Keilin, Margoliash and others at the beginning of the 20th century and shows how their results can be used for the rapid solution of new problems of modern science. It describes a rapid and simple spectrophotometric method for quantitative determination of cytochrome c release from isolated mitochondria or permeabilized cells induced by proapoptotic proteins. For this, the Soret (γ) peak at 414 nm in the spectrum of cytochrome c is used. The results of spectrophotometric assay of cytochrome c release are in accord with those of oxygraphic determination of cytochrome c-dependent respiration of isolated mitochondria and permeabilized cardiomyocytes.

Brain mitochondrial carrier protein-1 (BMCP1), a new member of the mitochondrial uncoupling carrier, has been shown to be expressed predominantly in the brain of the mice and humans. We cloned rat BMCP1 cDNA and investigated its mRNA level during postnatal development and under various metabolic conditions. The nucleotide sequence of the cDNA revealed that rat BMCP1 protein was composed of 322 amino acid residues, and was 99 and 96% identical to the mouse and human proteins and 29, 33 and 35% identical to rat uncoupling protein (UCP) 1, UCP2 and UCP3, respectively. The molecular weight was predicted to be 36 017 Da and the protein of this size was detectable when the cDNA was expressed in vitro. Using Northern blot analysis, the corresponding mRNA, approximately 1.8-kb in size, was found expressed predominantly in the cerebrum, cerebellum and hypothalamus. A unique developmental pattern was identified in the brain, where BMCP1 expression was low in their fetal life, but significantly elevated in the first postnatal week. Thereafter BMCP1 mRNA was maintained to be gradually increased. In 48-h fasted or insulin-induced hypoglycemic rats, BMCP1 mRNA expression in the hypothalamus slightly, but significantly, decreased compared with that in their appropriate controls. The present results indicate that BMCP1 may be involved in pathogenesis of mitochondrial dysfunction in neurons induced by aging or neurodegenerative disorders, and perhaps in energy balance in the brain.
Keywords: cDNA cloning; Amino acid sequence; Gene expression; Brain mitochondrial carrier protein-1; Developmental alteration;

In order to obtain information on the organization of the pigment molecules in chlorophyll (Chl) a/b/c-containing organisms, we have carried out circular dichroism (CD), linear dichroism (LD) and absorption spectroscopic measurements on intact cells, isolated thylakoids and purified light-harvesting complexes (LHCs) of the prasinophycean alga Mantoniella squamata. The CD spectra of the intact cells and isolated thylakoids were predominated by the excitonic bands of the Chl a/b/c LHC. However, some anomalous bands indicated the existence of chiral macrodomains, which could be correlated with the multilayered membrane system in the intact cells. In the red, the thylakoid membranes and the LHC exhibited a well-discernible CD band originating from Chl c, but otherwise the CD spectra were similar to that of non-aggregated LHC II, the main Chl a/b LHC in higher plants. In the Soret region, however, an unusually intense (+) 441 nm band was observed, which was accompanied by negative bands between 465 and 510 nm. It is proposed that these bands originate from intense excitonic interactions between Chl a and carotenoid molecules. LD measurements revealed that the QY dipoles of Chl a in Mantoniella thylakoids are preferentially oriented in the plane of the membrane, with orientation angles tilting out more at shorter than at longer wavelengths (9° at 677 nm, 20° at 670 nm and 26° at 662 nm); the QY dipole of Chl c was found to be oriented at 29° with respect to the membrane plane. These data and the LD spectrum of the LHC, apart from the presence of Chl c, suggest an orientation pattern of dipoles similar to those of higher plant thylakoids and LHC II. However, the tendency of the QY dipoles of Chl b to lie preferentially in the plane of the membrane (23° at 653 nm and 30° at 646 nm) is markedly different from the orientation pattern in higher plant membranes and LHC II. Hence, our CD and LD data show that the molecular organization of the Chl a/b/c LHC, despite evident similarities, differs significantly from that of LHC II.
Keywords: Alga; Carotenoid; Light-harvesting complex; Linear dichroism; Circular dichroism; Photosynthesis;

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena by Rafael Moreno-Sánchez; Raúl Covián; Ricardo Jasso-Chávez; Sara Rodrı́guez-Enrı́quez; Fermı́n Pacheco-Moisés; M.Eugenia Torres-Márquez (200-210).
The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H+ gradient (>60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc 1 complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc 1 complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.
Keywords: ATP synthesis; Antimycin; Cyanide-resistant respiration; Euglena;

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties 1 1 This review is dedicated to the memory of Professor Lars Ernster. by Tania Bizouarn; Ola Fjellström; Johan Meuller; Magnus Axelsson; Anders Bergkvist; Carina Johansson; B. Göran Karlsson; Jan Rydström (211-228).
Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP+ to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP+ by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site.
Keywords: Transhydrogenase; Proton pump; NADP; NAD; Membrane protein;

Photosystem II fluorescence quenching in the cyanobacterium Synechocystis PCC 6803: involvement of two different mechanisms by K. El Bissati; E. Delphin; N. Murata; A.-L. Etienne; D. Kirilovsky (229-242).
The structural changes associated to non-photochemical quenching in cyanobacteria is still a matter of discussion. The role of phycobilisome and/or photosystem mobility in this mechanism is a point of interest to be elucidated. Changes in photosystem II fluorescence induced by different quality of illumination (state transitions) or by strong light were characterized at different temperatures in wild-type and mutant cells, that lacked polyunsaturated fatty acids, of the cyanobacterium Synechocystis PCC 6803. The amplitude and the rate of state transitions decreased by lowering temperature in both strains. Our results support the hypothesis that a movement of membrane complexes and/or changes in the oligomerization state of these complexes are involved in the mechanism of state transitions. The quenching induced by strong blue light which was not associated to D1 damage and photoinhibition, did not depend on temperature or on the membrane state. Thus, the mechanism involved in the formation of this type of quenching seems to be unrelated to the movement of membrane complexes. Our results strongly support the idea that the mechanism involved in the fluorescence quenching induced by light 2 is different from that involved in strong blue light induced quenching.
Keywords: State transition; Fluorescence quenching; Photosystem II; Cyanobacterium; Synechocystis;

The study of turnover of two distinct forms of the photosystem II (PSII) D1 protein in cells of the cyanobacterium Synechococcus PCC 7942 showed that the ‘high-light’ form D1:2 is degraded significantly faster at 500 μE m−2 s−1 as compared with 50 μE m−2 s−1 while the degradation rates of the ‘low-light’ form D1:1 under low and high irradiance are not substantially different. Consequently, the D1:1 turnover does not match photoinactivation of PSII under increased irradiance and therefore the cells containing this D1 form exhibit a decrease in the PSII activity. Monitoring of the content of each D1 form during a recovery from growth–temperature photoinhibition showed a good correlation between the synthesis of D1:2 and restoration of the PSII activity. In contrast, when photoinhibitory treatment was conducted at low temperature, a fast recovery was not accompanied by the D1:2 accumulation. The data suggest that photoinactivation at growth temperature results in a modification of PSII that inhibits insertion of D1:1 and, therefore, for restoration of the photochemical activity in the photoinactivated PSII complexes the D1:2 synthesis is needed. This may represent the primary reason for the requirement of psbAII/psbAIII expression under increased irradiance.
Keywords: Cyanobacterium; D1 protein; Photoinhibition; Photosynthesis; Photosystem II; Synechococcus PCC 7942;

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin by Heinz-Jürgen Steinhoff; Anton Savitsky; Christoph Wegener; Matthias Pfeiffer; Martin Plato; Klaus Möbius (253-262).
Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The gxx component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of gxx versus Azz of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the MN photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the MN intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.
Keywords: Electron paramagnetic resonance; Site-directed spin labeling; Retinal protein;

Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure by Takiko Daikoku; Yasuo Shinohara; Atsushi Shima; Naoshi Yamazaki; Hiroshi Terada (263-272).
To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of β-adrenergic receptor (β-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of β1-AR and C/EBPβ in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.
Keywords: Brown adipose tissue; White adipose tissue; Uncoupling protein; Energy metabolism;

Here, we describe an assay for the tissue content of purinogen, a highly phosphorylated labile polymer containing ATP and phosphoglycerate found in the mitochondrial intermembrane space. We report the purinogen content (as adenine nucleotide equivalents) of rat heart and, for the first time, of rat liver, kidney, brain and mixed skeletal muscle. The findings show that purinogen contains very significant proportions of cell adenine nucleotides ranging from 25% of the free pool in brain and skeletal muscle to 135% of it in kidney. The evidence that purinogen may form a controlled intracellular reservoir of inorganic phosphate is briefly discussed.
Keywords: Brain purinogen; Heart purinogen; Kidney purinogen; Liver purinogen; Muscle purinogen; ATP polymer;