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BBA - Bioenergetics (v.1797, #1)

Reviewer Acknowledgement (pp. iii-iv).

Editorial Board (pp. i).

Emerging roles of mitochondrial proteases in neurodegeneration by Paola Martinelli; Elena I. Rugarli (pp. 1-10).

Fine tuning of integrated mitochondrial functions is essential in neurons and rationalizes why mitochondrial dysfunction plays an important pathogenic role in neurodegeneration. Mitochondria can contribute to neuronal cell death and axonal dysfunction through a plethora of mechanisms, including low ATP levels, increased reactive oxygen species, defective calcium regulation, and impairment of dynamics and transport. Recently, mitochondrial proteases in the inner mitochondrial membrane have emerged as culprits in several human neurodegenerative diseases. Mitochondrial proteases degrade misfolded and non-assembled polypeptides, thus performing quality control surveillance in the organelle. Moreover, they regulate the activity of specific substrates by mediating essential processing steps. Mitochondrial proteases may be directly involved in neurodegenerative diseases, as recently shown for the m-AAA protease, or may regulate crucial mitochondrial molecules, such as OPA1, which in turn is implicated in human disease. The mitochondrial proteases HTRA2 and PARL increase the susceptibility of neurons to apoptotic cell death. Here we review our current knowledge on how disturbances of the mitochondrial proteolytic system affect neuronal maintenance and axonal function.

Keywords: Mitochondrial proteases; Paraplegin; AFG3L2; Neuronal and axonal degeneration; Hereditary spastic paraplegia; Spinocerebellar ataxia

D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy by Joseph L. Hughes; Nicholas Cox; A. William Rutherford; Elmars Krausz; Thanh-Lan Lai; Alain Boussac; Miwa Sugiura (pp. 11-19).

In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA₃ gene over the psbA₁ gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at <10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Q_x band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the Pheo_D1 Q_x band induced by Q_A⁻ (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable ( t_1/2>50 ms) Q_A⁻ yield was ∼95% in D1:3, and ∼60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Q_x band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 13¹-keto of Pheo_D1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of P_D1, in particular Ser153Ala. Photo-induced Q_A⁻ formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P⁺.

Keywords: PheoD1; Primary donor; Side-pathway; Charge transfer transition

A novel functional element in the N-terminal region of Arum concinnatum alternative oxidase is indispensable for catalytic activity of the enzyme in HeLa cells by Yusuke Kakizaki; Roger S. Seymour; Kikukatsu Ito (pp. 20-28).

Alternative oxidase (AOX) is a quinol-oxygen oxidoreductase, which is known to possess a dicarboxylate diiron reaction center held in structurally postulated α-helical bundle. However, little is known about the structural or functional features of its N-terminal region in any organism, with the exception of a regulatory cysteine residue (CysI) in angiosperm plants. Here, we show that transcripts of two AOX1 isozymes ( AcoAOX1a and AcoAOX1b) are coexpressed in thermogenic appendices of Arum concinnatum, while their enzymatic activities seem to be distinct. Namely, AcoAOX1a, an abundantly expressed transcript in vivo, shows an apparent cyanide-insensitive and n-propyl gallate-sensitive respiration during ectopic expression of the protein in HeLa cells, whereas AcoAOX1b exhibits a lower transcript expression, and appears to be totally inactive as AOX at the protein level. Our functional analyses further reveal that an E83K substitution in AcoAOX1b, which is located far upstream of CysI in the N-terminal region, is the cause of this loss of function. These results suggest the presence of a naturally occurring inactive AOX homologue in thermogenic plants. Accordingly, our results further imply that the N-terminal region of the AOX protein functionally contributes to the dynamic activities of respiratory control within the mitochondria.

Keywords: Alternative oxidase; Arum lily; Thermogenesis; Inactive isoform; N-terminus; Loss of function

Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids by Yewei Ma; Ren-Kui Bai; Robert Trieu; Lee-Jun C. Wong (pp. 29-37).

Somatic mitochondrial DNA alterations have been found in all types of cancer. To better understand the role of mitochondria and their involvement in the pathogenic mechanisms of cancer development, the effects of cancer mitochondria were investigated in a defined nuclear background using a transmitochondrial cybrid system. Our results demonstrated that cancer mitochondria confer a significant reduction in cell growth when cells are metabolically stressed in a galactose medium. Activities of the respiratory chain complexes, cellular oxygen consumption, and ATP synthesis rates were found to be much lower in breast cancer cells, than those in normal breast epithelial cells of MCF-10A (10A). These results suggest that there is reduced mitochondrial function in the studied breast cancer cell lines. Similarly reduced mitochondrial function was observed in cybrids containing cancer mitochondria. Novel tRNA mutations were also identified in two breast cancer cell lines, possibly responsible for the observed mitochondrial dysfunction. We conclude that altered mitochondria in cancer cells may play a crucial role in tumor development.

Keywords: Breast cancer; Transmitochondrial cybrid; Mitochondrial tRNA mutation; Defective oxidative phosphorylation; P53

Probing binding determinants in center P of the cytochrome bc₁ complex using novel hydroxy-naphthoquinones by Louise M. Hughes; Raul Covian; Gordon W. Gribble; Bernard L. Trumpower (pp. 38-43).

Atovaquone is a substituted 2-hydroxy-naphthoquinone used therapeutically against Plasmodium falciparum (malaria) and Pneumocystis pathogens. It acts by inhibiting the cytochrome bc₁ complex via interactions with the Rieske iron–sulfur protein and cytochrome b in the ubiquinol oxidation pocket. As the targeted pathogens have developed resistance to this drug there is an urgent need for new alternatives. To better understand the determinants of inhibitor binding in the ubiquinol oxidation pocket of the bc₁ complex we synthesized a series of hydroxy-naphthoquinones bearing a methyl group on the benzene ring that is predicted to interact with the nuclear encoded Rieske iron–sulfur protein. We have also attempted to overcome the metabolic instability of a potent cytochrome bc₁ complex inhibitor, a 2-hydroxy-naphthoquinone with a branched side chain, by fluorinating the terminal methyl group. We have tested these new 2-hydroxy-naphthoquinones against yeast and bovine cytochrome bc₁ complexes to model the interaction with pathogen and human enzymes and determine parameters that affect efficacy of binding of these inhibitors. We identified a hydroxy-naphthoquinone with a trifluoromethyl function that has potential for development as an anti-fungal and anti-parasitic therapeutic.

Keywords: Abbreviations; nHDBT; nonyl-hydroxy-dioxobenzoxythiazole; NQ; naphthoquinoneHydroxy-naphthoquinones; Cytochrome; bc; ₁; complex; Malaria; Plasmodium; Pneumocystis; Atovaquone

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions by Jean Alric; Jérôme Lavergne; Fabrice Rappaport (pp. 44-51).

Assimilation of atmospheric CO₂ by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.

Keywords: Abbreviations; PSII; photosystem II; PSI; photosystem I; P; ₇₀₀; primary electron donor of PSI (reduced form); P; ₇₀₀; ⁺; primary electron donor of PSI (oxidized form); DCMU; 3(3,4-dichlorophenyl)-1,1-dimethylurea; HA; hydroxylamine; DBMIB; 2,5-dibromo-3-methyl-6-isopropyl-; p; -benzoquinone; MV; methylviologenElectron transfer; Green algae; Chlamydomonas reinhardtii; Photosystem I; Cytochrome; b; ₆; f

Dissimilar mechanisms of cytochrome c release induced by octyl glucoside-activated BAX and by BAX activated with truncated BID by Tsyregma Li; Tatiana Brustovetsky; Bruno Antonsson; Nickolay Brustovetsky (pp. 52-62).

In the present study, we compared alkali-resistant BAX insertion into the outer mitochondrial membrane, mitochondrial remodeling, mitochondrial membrane potential changes, and cytochrome c (Cyt c) release from isolated brain mitochondria triggered by recombinant BAX oligomerized with 1% octyl glucoside (BAX_oligo) and by a combination of monomeric BAX (BAX_mono) and caspase 8-cleaved C-terminal fragment of recombinant BID (truncated BID, t^cBID). We also examined whether the effects induced by BAX_oligo or by BAX_mono activated with t^cBID depended on induction of the mitochondrial permeability transition. The results obtained in this study revealed that t^cBID plus BAX_mono produced BAX insertion and Cyt c release without overt changes in mitochondrial morphology. On the contrary, treatment of mitochondria with BAX_oligo resulted in BAX insertion and Cyt c release, which were accompanied by gross distortion of mitochondrial morphology. The effects of BAX_oligo could be at least partially suppressed by mitochondrial depolarization. The effects of t^cBID plus BAX_mono were insensitive to depolarization. BAX_oligo produced similar BAX insertion, mitochondrial remodeling, and Cyt c release in KCl- and in N-methyl-d-glucamine-based incubation media indicating a non-essential role for K⁺ influx into mitochondria in these processes. A combination of cyclosporin A and ADP, inhibitors of the mitochondrial permeability transition, attenuated Cyt c release, mitochondrial remodeling, and depolarization induced by BAX_oligo, but failed to influence the effects produced by t^cBID plus BAX_mono. Thus, our results suggest a significant difference in the mechanisms of the outer mitochondrial membrane permeabilization and Cyt c release induced by detergent-oligomerized BAX_oligo and by BAX activated with t^cBID.

Keywords: Abbreviations; BAX; _mono; monomeric BAX; BAX; _oligo; monomeric BAX oligomerized in the presence of 1% octyl glucoside; tBID; truncated BID; t; ^c; BID; C-terminal fragment of BID generated by cutting BID with caspase 8 and subsequently separated from the N-terminal fragment and caspase; mPT; mitochondrial permeability transition; OMM; outer mitochondrial membrane; IMM; inner mitochondrial membrane; Δ; ψ; mitochondrial membrane potential; COX IV; cytochrome oxidase subunit IV; TPP; ⁺; tetraphenyl phosphonium cationApoptosis; BID; BAX; Mitochondria; Morphology; Brain

Heat-induced disassembly and degradation of chlorophyll-containing protein complexes in vivo by Lenka Lípová; Pavel Krchňák; Josef Komenda; Petr Ilík (pp. 63-70).

Gradual heating of green leaves up to non-physiological temperatures is often used to estimate thermal stability of photosynthetic apparatus. However, a complete sequence of heat-induced disassembly and denaturation of chlorophyll-containing protein complexes (CPCs) has not been reported yet. In this work, we heated (1 °C·min−¹) barley leaves to temperatures selected according to the changes in the chlorophyll fluorescence temperature curve (FTC) and we analyzed CPC stability by two-dimensional native Deriphat/SDS–PAGE . The first distinct change in both structure and function of photosystem II (PSII) appeared at 40–50 °C. PSII core (CCII) dimers began to dissociate monomers, which was accompanied by a decrease in PSII photochemistry and reflected in FTC as the first fluorescence increase. Further changes in CPCs appeared at 57–60 °C, when FTC increases to its second maximum. Photosystem I (PSI) cores (CCI) partially dissociated from light-harvesting complexes of PSI (LHCI) and formed aggregates. The rest of CCI–LHCI complexes, as well as the CCI aggregates, degraded to the PSI-A/B heterodimer in leaves heated to 70 °C. Heating to these temperatures led to a complete degradation of CCII components and corresponding loss of PSII photochemistry. Trimeric light-harvesting complexes of PSII (LHCII) markedly dissociated to monomers and denatured, as evidenced by a release of large amount of free chlorophylls. Between 70 and 80 °C, a complete degradation of LHCII occurred, leaving the PSI-A/B heterodimer as the only detectable CPC in the membrane. This most thermostable CPC disappeared after heating to 90 °C, which corresponded to a loss of PSI photochemistry.

Keywords: Chlorophyll-containing protein complex; Chlorophyll fluorescence temperature curve, Disassembly; Heat denaturation; High temperature; Native electrophoresis

Energetic performance is improved by specific activation of K⁺ fluxes through K_Ca channels in heart mitochondria by Miguel A. Aon; Sonia Cortassa; An-Chi Wei; Morten Grunnet; Brian O'Rourke (pp. 71-80).

Mitochondrial volume regulation depends on K⁺ movement across the inner membrane and a mitochondrial Ca²⁺-dependent K⁺ channel (mitoK_Ca) reportedly contributes to mitochondrial K⁺ uniporter activity. Here we utilize a novel K_Ca channel activator, NS11021, to examine the role of mitoK_Ca in regulating mitochondrial function by measuring K⁺ flux, membrane potential (Δ Ψ_m), light scattering, and respiration in guinea pig heart mitochondria. K⁺ uptake and the influence of anions were assessed in mitochondria loaded with the K⁺ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH₂PO₄) salts of K⁺ to energized mitochondria in a sucrose-based medium. K⁺ fluxes saturated at ∼10 mM for each salt, attaining maximal rates of 172±17, 54±2.4, and 33±3.8 nmol K⁺/min/mg in KCl, KAc, or KH₂PO₄, respectively. NS11021 (50 nM) increased the maximal K⁺ uptake rate by 2.5-fold in the presence of KH₂PO₄ or KAc and increased mitochondrial volume, with little effect on Δ Ψ_m. In KCl, NS11021 increased K⁺ uptake by only 30% and did not increase volume. The effects of NS11021 on K⁺ uptake were inhibited by the K_Ca toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH₂PO₄, but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized Δ Ψ_m. A control compound lacking K_Ca activator properties did not increase K⁺ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K⁺ flux through mitoK_Ca mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained Δ Ψ_m.

Keywords: Mitochondrial volume; Membrane potential; Respiratory control ratio; Valinomycin; P/O ratio; K; _Ca; activator

Identification of the mitochondrial carrier that provides Yarrowia lipolytica with a fatty acid-induced and nucleotide-sensitive uncoupling protein-like activity by Luis A. Luévano-Martínez; Eva Moyano; Mario García de Lacoba; Eduardo Rial; Salvador Uribe-Carvajal (pp. 81-88).

Uncoupling proteins (UCPs) are mitochondrial carriers distributed throughout the eukaryotic kingdoms. While genes coding for UCPs have been identified in plants and animals, evidences for the presence of UCPs in fungi and protozoa are only functional. Here, it is reported that in the yeast Yarrowia lipolytica there is a fatty acid-promoted and GDP-sensitive uncoupling activity indicating the presence of a UCP. The uncoupling activity is higher in the stationary phase than in the mid-log growth phase. The in silico search on the Y. lipolytica genome led to the selection of two genes with the highest homology to the UCP family, XM_503525 and XM_500457. By phylogenetic analysis, XP_503525 was predicted to be an oxaloacetate carrier while XP_500457 would be a dicarboxylate carrier. Each of these two genes was cloned and heterologously expressed in Saccharomyces cerevisiae and the resulting phenotype was analyzed. The transport activity of the two gene products confirmed the phylogenetic predictions. In addition, only mitochondria isolated from yeasts expressing XP_503525 showed bioenergetic properties characteristic of a UCP: the proton conductance was increased by linoleic acid and inhibited by GDP. It is concluded that the XM_503525 gene from Y. lipolytica encodes for an oxaloacetate carrier although, remarkably, it also displays an uncoupling activity stimulated by fatty acids and inhibited by nucleotides.

Keywords: Uncoupling protein; UCP; Energy dissipation; Yarrowia lipolytica; Mitochondria; Heterologous expression; Yeast

Localization of Pcb antenna complexes in the photosynthetic prokaryote Prochlorothrix hollandica by Miroslava Herbstová; Radek Litvín; Zdenko Gardian; Josef Komenda; František Vácha (pp. 89-97).

The freshwater filamentous green oxyphotobacterium Prochlorothrix hollandica is an unusual oxygenic photoautotrophic cyanobacterium differing from most of the others by the presence of light-harvesting Pcb antenna binding both chlorophylls a and b and by the absence of phycobilins. The pigment–protein complexes of P. hollandica SAG 10.89 (CCAP 1490/1) were isolated from dodecylmaltoside solubilized thylakoid membranes on sucrose density gradient and characterized by biochemical, spectroscopic and immunoblotting methods. The Pcb antennae production is suppressed by high light conditions (>200 μmol photons m⁻² s⁻¹) in P. hollandica. PcbC protein was found either in higher oligomeric states or coupled to PS I (forming antenna rings around PS I). PcbA and PcbB are most probably only very loosely bound to photosystems; we assume that these pigment–protein complexes function as low light-induced mobile antennae. Further, we have detected α-carotene in substantial quantities in P. hollandica thylakoid membranes, indicating the presence of chloroplast-like carotenoid synthetic pathway which is not present in common cyanobacteria.

Keywords: Abbreviations; 2D SDS–PAGE; two-dimensional sodium dodecylsulfate–polyacrylamide gel electrophoresis; Cab; chlorophyll; a; /; b; binding; Car; carotenoid(s); Car/Chl; Car to Chl ratio; Chl; a; chlorophyll; a; Chl; a; /; b; Chl; a; to Chl; b; ratio; Chl; b; chlorophyll; b; Chl; chlorophyll(s); CN–PAGE; colorless native–polyacrylamide gel electrophoresis; CP 43; CP 47, core antennae of PS II; D1; D2, reaction center proteins of PS II; DM; n; -dodecyl-β-; d; -maltoside; DTT; dithiothreitol; F; _m; maximal fluorescence yield; F; _v; variable fluorescence yield; F; _v; /; F; _m; the ratio of variable to maximum fluorescence after dark adaptation, equivalent to the maximum quantum yield of photosystem II; HL; high light; HPLC; high-performance liquid chromatography; IsiA (CP 43′); iron stress-induced protein A also called CP 43′; kDa; kilodalton(s); LHC I; light-harvesting complex of PS I; LHC II; light-harvesting complex of PS II; LHC; light-harvesting complex; LL; low light; MES; 2-morpholinoethanesulphonic acid; MLL; moderate low light; Pcb; prochlorophyte chlorophyll; a; /; b; binding; Pheo; pheophytin; PMSF; phenylmethylsulphonyl fluoride; PS I; photosystem I; PS II; photosystem II; PS; photosystem; PVDF; polyvinylidene difluoride; SDS; sodium dodecylsulfate; SDS–PAGE; sodium dodecylsulfate–polyacrylamide gel electrophoresis; TM; thylakoid membrane(s); T; −; S; triplet-minus-singlet spectrum Prochlorothrix hollandica; Pcb antenna; Photosystem; α-Carotene; Oxyphotobacteria

Purification and characterization of photosystem I complex from Synechocystis sp. PCC 6803 by expressing histidine-tagged subunits by Hisako Kubota; Isamu Sakurai; Kenta Katayama; Naoki Mizusawa; Shunsuke Ohashi; Masami Kobayashi; Pengpeng Zhang; Eva-Mari Aro; Hajime Wada (pp. 98-105).

We generated Synechocystis sp. PCC 6803 strains, designated F-His and J-His, which express histidine-tagged PsaF and PsaJ subunits, respectively, for simple purification of the photosystem I (PSI) complex. Six histidine residues were genetically added to the C-terminus of the PsaF subunit in F-His cells and the N-terminus of the PsaJ subunit in J-His cells. The histidine residues introduced had no apparent effect on photoautotrophic growth of the cells or the activity of PSI and PSII in thylakoid membranes. PSI complexes could be simply purified from the F-His and J-His cells by Ni²⁺-affinity column chromatography. When thylakoid membranes corresponding to 20 mg chlorophyll were used, PSI complexes corresponding to about 7 mg chlorophyll could be purified in both strains. The purified PSI complexes could be separated into monomers and trimers by ultracentrifugation in glycerol density gradient and high activity was recorded for trimers isolated from the F-His and J-His strains. Blue-Native PAGE and SDS-PAGE analysis of monomers and trimers indicated the existence of two distinct monomers with different subunit compositions and no contamination of PSI with other complexes, such as PSII and Cyt b₆ f. Further analysis of proteins and lipids in the purified PSI indicated the presence of novel proteins in the monomers and about six lipid molecules per monomer unit in the trimers. These results demonstrate that active PSI complexes can be simply purified from the constructed strains and the strains are very useful tools for analysis of PSI.

Keywords: Abbreviations; BN; blue native; Chl; chlorophyll; Cm; chloramphenicol; Cm; ^R; chloramphenicol-resistant gene; DGDG; digalactosyldiacylglycerol; DM; n; -dodecyl β-; d; -maltoside; MGDG; monogalactosyldiacylglycerol; NDH; NADH dehydrogenase; PG; phosphatidylglycerol; PSI; photosystem I; PSII; photosystem II; SQDG; sulfoquinovosyldiacylglycerolCyanobacteria; Histidine tag; Photosystem I; Purification; Synechocystis; sp. PCC 6803

Effect of the P700 pre-oxidation and point mutations near A₀ on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii by Wojciech Giera; V.M. Ramesh; Andrew N. Webber; Ivo van Stokkum; Rienk van Grondelle; Krzysztof Gibasiewicz (pp. 106-112).

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700⁺). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A₀, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700⁺. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼7 ps and ∼25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A₀: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A₀. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700⁺. The same charge is proposed to be responsible for the fast A⁺A₀⁻→AA₀ charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A₀ axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A⁺A₀⁻.

Keywords: Abbreviations; A; ₀; primary acceptor; A; ₁; secondary acceptor; Chl; chlorophyll; LHCI; light-harvesting complex I; P; primary donor; PSI; Photosystem I; RC; reaction center; WT; wild typeTime-resolved fluorescence; Streak camera; Photosystem I; Chlamydomonas reinhardtii; Electron transfer cofactors; Chlorophyll; Photosynthesis

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BBA - Bioenergetics (v.1797, #1)