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BBA - Bioenergetics (v.1708, #3)

Editorial Board (pp. ii).

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron–sulfur cluster F_X by Stefano Santabarbara; Peter Heathcote; Michael C.W. Evans (pp. 283-310).

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron–sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron–sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.

Keywords: Photosystem I; Electron transfer; Phylloquione A; ₁; Iron–sulphur centre (F; _X; , F; _A; , F; _B; ); Redox potential

Photosynthetic activity of far-red light in green plants by Hugo Pettai; Vello Oja; Arvi Freiberg; Agu Laisk (pp. 311-321).

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q_A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8±0.5 kJ mol⁻¹ and 12.5±0.7 kJ mol⁻¹ have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol⁻¹ between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.

Keywords: Abbreviations; CP; chlorophyll protein; F; ₀; ,; F; _m; chlorophyll fluorescence yield, minimum with open reaction centres and maximum with closed reaction centres; FWHM; full width at half maximum; LED; light-emitting diode; LHC; light-harvesting complex; PSI; PSII, photosystem I and II; PQ; plastoquinone; P680; PSII reaction centre pigment; Q; _A; PSII primary acceptor quinone; Y; (; λ; ); quantum yield of electron transport at wavelength; λQuantum yield of photosynthesis; O; ₂; evolution; Photosystem II; Far-red chlorophyll

Inhibitor studies on non-photochemical plastoquinone reduction and H₂ photoproduction in Chlamydomonas reinhardtii by Florence Mus; Laurent Cournac; V�ronique Cardettini; Amandine Caruana; Gilles Peltier (pp. 322-332).

In the absence of PSII, non-photochemical reduction of plastoquinones (PQs) occurs following NADH or NADPH addition in thylakoid membranes of the green alga Chlamydomonas reinhardtii. The nature of the enzyme involved in this reaction has been investigated in vitro by measuring chlorophyll fluorescence increase in anoxia and light-dependent O₂ uptake in the presence of methyl viologen. Based on the insensitivity of these reactions to rotenone, a type-I NADH dehydrogenase (NDH-1) inhibitor, and their sensitivity to flavoenzyme inhibitors and thiol blocking agents, we conclude to the involvement of a type-II NADH dehydrogenase (NDH-2) in PQ reduction. Intact Chlamydomonas cells placed in anoxia have the property to produce H₂ in the light by a Fe-hydrogenase which uses reduced ferredoxin as an electron donor. H₂ production also occurs in the absence of PSII thanks to the existence of a non-photochemical pathway of PQ reduction. From inhibitors effects, we suggest the involvement of a plastidial NDH-2 in PSII-independent H₂ production in Chlamydomonas. These results are discussed in relation to the absence of ndh genes in Chlamydomonas plastid genome and to the existence of 7 ORFs homologous to type-II NDHs in its nuclear genome.

Keywords: Hydrogen production; Photosystem II; NADH dehydrogenase; Plastoquinone; Chlamydomonas reinhardtii

Extinction coefficients of cytochromes b559 and c550 of Thermosynechococcus elongatus and Cyt b559/PS II stoichiometry of higher plants by Olga Kaminskaya; Jan Kern; Vladimir A. Shuvalov; Gernot Renger (pp. 333-341).

“Reduced minus oxidized�? difference extinction coefficients Δ ɛ in the α-bands of Cyt b559 and Cyt c550 were determined by using functionally and structurally well-characterized PS II core complexes from the thermophilic cyanobacterium Thermosynechococcus elongatus. Values of 25.1±1.0 mM⁻¹ cm⁻¹ and 27.0±1.0 mM⁻¹ cm⁻¹ were obtained for Cyt b559 and Cyt c550, respectively. Anaerobic redox titrations covering the wide range from −250 up to +450 mV revealed that the heme groups of both Cyt b559 and Cyt c550 exhibit homogenous redox properties in the sample preparation used, with E_m values at pH 6.5 of 244±11 mV and −94±21 mV, respectively. No HP form of Cyt b559 could be detected. Experiments performed on PS II membrane fragments of higher plants where the content of the high potential form of Cyt b559 was varied by special treatments (pH, heat) have shown that the α-band extinction of Cyt b559 does not depend on the redox form of the heme group. Based on the results of this study the Cyt b559/PSII stoichiometry is inferred to be 1:1 not only in thermophilic cyanobacteria as known from the crystal structure but also in PSII of plants. Possible interrelationships between the structure of the Q_B site and the microenvironment of the heme group of Cyt b559 are discussed.

Keywords: Abbreviations; PS II; photosystem II; Cyt; cytochrome; WOC; water-oxidizing complex; Chl; chlorophyll; Car; carotenoid; Pheo; pheophytin; E; _m; midpoint potential; HP; high potential; IP; intermediate potential; LP; low potential; Δ; ɛ; difference “reduced minus oxidized�? extinction coefficient; MES; 2-[N-Morpholino]ethanesulfonic acid; β-DM; n-dodecyl β-; d; -maltoside; FWHM; full width at half maximumPhotosystem II; Cyt b559; Cyt c550; Redox potential; Extinction coefficient

Irreversible photoinhibition of photosystem II is caused by exposure of Synechocystis cells to strong light for a prolonged period by Suleyman I. Allakhverdiev; Nelly Tsvetkova; Prasanna Mohanty; Bal�sz Szalontai; Byoung Yong Moon; M�nika Debreczeny; Norio Murata (pp. 342-351).

Irreversible photoinhibition of photosystem II (PSII) occurred when Synechocystis sp. PCC 6803 cells were exposed to very strong light for a prolonged period. When wild-type cells were illuminated at 20 °C for 2 h with light at an intensity of 2,500 μmol photons m⁻² s⁻¹, the oxygen-evolving activity of PSII was almost entirely and irreversibly lost, whereas the photochemical reaction center in PSII was inactivated only reversibly. The extent of irreversible photoinhibition was enhanced at lower temperatures and by the genetically engineered rigidification of membrane lipids. Western and Northern blotting demonstrated that, after cells had undergone irreversible photoinhibition, the precursor to D1 protein in PSII was synthesized but not processed properly. These observations may suggest that exposure of Synechocystis cells to strong light results in the irreversible photoinhibition of the oxygen-evolving activity of PSII via impairment of the processing of pre-D1 and that this effect of strong light is enhanced by the rigidification of membrane lipids.

Keywords: Abbreviations; BQ; 1,4-benzoquinone; CCCP; carbonyl cyanide; m; -chlorophenylhydrazone; Chl; chlorophyll; DCMU; 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; PSII; photosystem IIPhotodamage; Photosystem II; D1 protein; S; ynechocystis

Interruption of the Calvin cycle inhibits the repair of Photosystem II from photodamage by Shunichi Takahashi; Norio Murata (pp. 352-361).

In photosynthetic organisms, impairment of the activities of enzymes in the Calvin cycle enhances the extent of photoinactivation of Photosystem II (PSII). We investigated the molecular mechanism responsible for this phenomenon in the unicellular green alga Chlamydomonas reinhardtii. When the Calvin cycle was interrupted by glycolaldehyde, which is known to inhibit phosphoribulokinase, the extent of photoinactivation of PSII was enhanced. The effect of glycolaldehyde was very similar to that of chloramphenicol, which inhibits protein synthesis de novo in chloroplasts. The interruption of the Calvin cycle by the introduction of a missense mutation into the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) also enhanced the extent of photoinactivation of PSII. In such mutant 10-6C cells, neither glycolaldehyde nor chloramphenicol has any additional effect on photoinactivation. When wild-type cells were incubated under weak light after photodamage to PSII, the activity of PSII recovered gradually and reached a level close to the initial level. However, recovery was inhibited in wild-type cells by glycolaldehyde and was also inhibited in 10-6C cells. Radioactive labelling and Northern blotting demonstrated that the interruption of the Calvin cycle suppressed the synthesis de novo of chloroplast proteins, such as the D1 and D2 proteins, but did not affect the levels of psbA and psbD mRNAs. Our results suggest that the photoinactivation of PSII that is associated with the interruption of the Calvin cycle is attributable primarily to the inhibition of the protein synthesis-dependent repair of PSII at the level of translation in chloroplasts.

Keywords: Abbreviations; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylure; PSII; Photosystem II; pre-D1; precursor to D1 protein; Rubisco; ribulose-1,5-bisphosphate carboxylase/oxygenase; TAP; Tris–acetate phosphateCalvin cycle; Photoinactivation; Photoinhibition; Photosystem II; Repair cycle

Coherent phase microscopy in cell biology: visualization of metabolic states by Vladimir P. Tychinsky; Alexander V. Kretushev; Tatyana V. Vyshenskaya; Alexander N. Tikhonov (pp. 362-366).

Visualization of functional properties of individual cells and intracellular organelles still remains an experimental challenge in cell biology. The coherent phase microscopy (CPM) provides a convenient and non-invasive tool for imaging cells and intracellular organelles. In this work, we report results of statistical analysis of CPM images of cyanobacterial cells ( Synechocystis sp. PCC 6803) and spores ( Bacillus licheniformis). It has been shown that CPM images of cyanobacterial cells and spores are sensitive to variations of their metabolic states. We found a correlation between one of optical parameters of the CPM image (‘phase thicknesses’ Δ h) and cell energization. It was demonstrated that the phase thickness Δ h decreased after cell treatment with the uncoupler CCCP or inhibitors of electron transport (KCN or DCMU). Statistical analysis of distributions of parameter Δ h and cell diameter d demonstrated that a decrease in the phase thickness Δ h could not be attributed entirely to a decrease in geometrical sizes of cells. This finding demonstrates that the CPM technique may be a convenient tool for fast and non-invasive diagnosis of metabolic states of individual cells and intracellular organelles.

Keywords: Abbreviations; CPM; coherent phase microscopy; DCMU; 3-(3,4-dichlorophenyl)-1,1-dimethylurea; CCCP; carbonylcyanide-3-chlorophenyl hydrazoneCoherent phase microscopy; Cyanobacteria; Spores; Membrane potential

Iron deficiency induces a chlorophyll d-binding Pcb antenna system around Photosystem I in Acaryochloris marina by Min Chen; Thomas S. Bibby; Jon Nield; Anthony Larkum; James Barber (pp. 367-374).

The prochlorophyte-like cyanobacterium Acaryochloris marina contains two pcb genes, pcbA and pcbC, which encode chlorophyll (Chl) d-binding antenna proteins PcbA and PcbC, respectively. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR), it is shown that when Acaryochloris cells are grown in an iron-deficient medium, the transcription of the pcbC gene is up-regulated compared to that of pcbA. Biochemical and immunological analyses indicated that under the same iron-deficient conditions, the level of Photosystem I (PSI) decreased compared with that of Photosystem II (PSII). Electron microscopy revealed that concomitant with these changes was the formation of Pcb–PSI supercomplexes which, in their largest form, were composed of 18 Pcb subunits forming a ring around the trimeric PSI reaction centre core. Mass spectrometry indicated that the PcbC protein is the main constituent of this outer PSI antenna system. It is therefore concluded that in Acaryochloris, the PcbC protein forms an antenna for PSI when iron levels become limiting and in this way compensates for the drop in the level of PSI relative to PSII which occurs under these conditions.

Keywords: Acaryochloris; Iron stress; Photosynthesis; Pcb protein; Photosystem; Gene transcript level

Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition by Boris F. Krasnikov; Dmitry B. Zorov; Yuri N. Antonenko; Andrey A. Zaspa; Igor V. Kulikov; Bruce S. Kristal; Arthur J.L. Cooper; Abraham M. Brown (pp. 375-392).

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca²⁺, phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.

Keywords: Abbreviations; MPT; membrane permeability transition; Δ; Ψ; mitochondrial membrane potential; PhAsO; phenylarsine oxide; TPP; ⁺; tetraphenyl-phosphonium; PC; personal computer; LED; light-emitting diode; PD; photodiode; ADC; analog–digital converter; DAC; digital–analog converter; BSA; bovine serum albumin; RLM; rat liver mitochondria; RCR; respiratory control ratio; CsA; cyclosporin AMitochondria; Permeability transition; Multiparameter recording; Kinetic analysis; Ion leak

Oligomerization of H⁺-pyrophosphatase and its structural and functional consequences by Hisatoshi Mimura; Yoichi Nakanishi; Masayoshi Maeshima (pp. 393-403).

The H⁺-pyrophosphatase (H⁺-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H⁺-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H⁺-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H⁺-PPase is present as an oligomer made up of at least two or three sets of dimers.

Keywords: Abbreviations; H; ⁺; -PPase; H; ⁺; -translocating pyrophosphatase; PP; _i; inorganic pyrophosphate; ScPP; S. coelicolor; H; ⁺; -PPase; BM; 3-(; N; -maleimidylpropionyl)biocytin; CuPh; Cu(II)-(1,10-phenanthroline); ₃; DTT; dithiothreitol; 2-ME; 2-mercaptoethanol; ECL; enhanced chemiluminescenceDisulfide bond; H; ⁺; -pyrophosphatase; Oligomerization; Proton pump

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli by Tania Bizouarn; Gijs I. van Boxel; Tina Bhakta; J. Baz Jackson (pp. 404-410).

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants ( K_d values) for NADPH (0.87 μM), NADP⁺ (16 μM), NADH (50 μM), and NAD⁺ (100–500 μM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K_d values for NAD⁺ and NADH are similar to those previously reported with isolated dI, but the K_d values for NADP⁺ and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised.

Keywords: Abbreviations; dI; the NAD(H)-binding component of transhydrogenase; dIII; the NADP(H)-binding component; dII; the membrane-spanning componentTranshydrogenase; Proton pump; Membrane protein; Nicotinamide nucleotide; Calorimetry

Inhibition of plasma membrane Ca²⁺-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca²⁺-occluded state by Otacilio C. Moreira; Priscila F. Rios; Hector Barrabin (pp. 411-419).

The bidentate complex of ATP with Cr³⁺, CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca²⁺-ATPase and the Na⁺,K⁺-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca²⁺ and Na⁺, respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca²⁺-ATPase. The complex inhibited with similar efficiency the Ca²⁺-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly ( T_1/2=30 min at 37 °C) with a K_i=28±9 μM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg²⁺ but unaltered when Ca²⁺ was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca²⁺ occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La³⁺ with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca²⁺ at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca²⁺ promoted by the plasma membrane Ca²⁺-ATPase goes through an enzymatic phospho-intermediate that maintains Ca²⁺ ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.

Keywords: Abbreviations; SERCA; sarcoplasmic reticulum Ca; ²⁺; -ATPase; pNPP; p; -nitrophenyl phosphate; PMCA; plasma membrane Ca; ²⁺; -ATPase; EGTA; ethylene glycol bis (β-aminoethyl ether),; N; ,; N; ,; N′; ,; N′; -tetraacetic acid; CrATP; bidentate chromium(III) ATP complex; HEPES; (; N; -[2-Hydroxyethyl] piperazine-; N′; - [2-ethanesulfonic acid]); PMSF; p; -methyl sulfonylfluoride; DTT; dithiothreitolPMCA; Erythrocyte; Chromium; CrATP; Lanthanum; Occlusion; Calcium

Bioenergetics Cumulative Contents (pp. 420-422).

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BBA - Bioenergetics (v.1708, #3)