Energetic coupling between plastids and mitochondria drives CO2 assimilation in diatoms.

Diatoms are one of the most ecologically successful classes of photosynthetic marine eukaryotes in the contemporary oceans. Over the past 30 million years, they have helped to moderate Earth's climate by absorbing carbon dioxide from the atmosphere, sequestering it via the biological carbon pump and ultimately burying organic carbon in the lithosphere. The proportion of planetary primary production by diatoms in the modern oceans is roughly equivalent to that of terrestrial rainforests. In photosynthesis, the efficient conversion of carbon dioxide into organic matter requires a tight control of the ATP/NADPH ratio which, in other photosynthetic organisms, relies principally on a range of plastid-localized ATP generating processes. Here we show that diatoms regulate ATP/NADPH through extensive energetic exchanges between plastids and mitochondria. This interaction comprises the re-routing of reducing power generated in the plastid towards mitochondria and the import of mitochondrial ATP into the plastid, and is mandatory for optimized carbon fixation and growth. We propose that the process may have contributed to the ecological success of diatoms in the ocean.

Chlororespiration Controls Growth Under Intermittent Light.

Whereas photosynthetic function under steady-state light conditions has been well characterized, little is known about its changes that occur in response to light fluctuations. Chlororespiration, a simplified respiratory chain, is widespread across all photosynthetic lineages, but its role remains elusive. Here, we show that chlororespiration plays a crucial role in intermittent-light conditions in the green alga Chlamydomonas reinhardtii Chlororespiration, which is localized in thylakoid membranes together with the photosynthetic electron transfer chain, involves plastoquinone reduction and plastoquinol oxidation by a Plastid Terminal Oxidase (PTOX). We show that PTOX activity is critical for growth under intermittent light, with severe growth defects being observed in a mutant lacking PTOX2, the major plastoquinol oxidase. We demonstrate that the hampered growth results from a major change in the kinetics of redox relaxation of the photosynthetic electron transfer chain during the dark periods. This change, in turn, has a dramatic effect on the physiology of photosynthesis during the light periods, notably stimulating cyclic electron flow at the expense of the linear electron flow.

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean.

The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.

Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase.

We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 µs with [NO] = 200 µM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)⋅s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 µs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 µs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.

Estimation of the driving force for dioxygen formation in photosynthesis.

Photosynthetic water oxidation to molecular oxygen is carried out by photosystem II (PSII) over a reaction cycle involving four photochemical steps that drive the oxygen-evolving complex through five redox states Si (i = 0,…, 4). For understanding the catalytic strategy of biological water oxidation it is important to elucidate the energetic landscape of PSII and in particular that of the final S4 → S0 transition. In this short-lived chemical step the four oxidizing equivalents accumulated in the preceding photochemical events are used up to form molecular oxygen, two protons are released and at least one substrate water molecule binds to the Mn4CaO5 cluster. In this study we probed the probability to form S4 from S0 and O2 by incubating YD-less PSII in the S0 state for 2­3 days in the presence of (18)O2 and H2(16)O. The absence of any measurable (16,18)O2 formation by water-exchange in the S4 state suggests that the S4 state is hardly ever populated. On the basis of a detailed analysis we determined that the equilibrium constant K of the S4 → S0 transition is larger than 1.0 × 10(7) so that this step is highly exergonic. We argue that this finding is consistent with current knowledge of the energetics of the S0 to S4 reactions, and that the high exergonicity is required for the kinetic efficiency of PSII.

Corrigendum to "Influence of Histidine-198 of the D1 subunit on the properties of the primary electron donor, P680, of photosystem II in Thermosynechococcus elongatus".

Two mutants, D1-H198Q and D1-H198A, have been previously constructed in Thermosynechococcus elongatus with the aim at modifying the redox potential of the P680•+/P680 couple by changing the axial ligand of PD1, one the two chlorophylls of the Photosystem II primary electron donor [Sugiura et al., Biochim. Biophys. Acta 1777 (2008) 331-342]. However, after the publication of this work it was pointed out to us by Dr. Eberhard Schlodder (Technische Universität Berlin) that in both mutants the pheophytin band shift which is observed upon the reduction of QA was centered at 544nm instead of 547nm, clearly showing that the D1 protein corresponded to PsbA1 whereas the mutants were supposedly constructed in the psbA3 gene so that the conclusions in our previous paper were wrong. O2 evolving mutants have been therefore reconstructed and their analyze shows that they are now correct mutants which are suitable for further studies. Indeed, the D1-H198Q mutation downshifted by ≈3nm the P680•+/P680 difference absorption spectrum in the Soret region and increased the redox potential of the P680•+/P680 couple and the D1-H198A mutation decreased the redox potential of the P680•+/P680 couple all these effects being comparable to those which were observed in Synechocystis sp. PCC 6803 [Diner et al., Biochemistry 40 (2001) 9265-9281 and Merry et al. Biochemistry 37 (1998) 17,439-17,447]. We apologize for having presented wrong data and wrong conclusions in our earlier publication.

Nitric oxide-triggered remodeling of chloroplast bioenergetics and thylakoid proteins upon nitrogen starvation in Chlamydomonas reinhardtii.

Starving microalgae for nitrogen sources is commonly used as a biotechnological tool to boost storage of reduced carbon into starch granules or lipid droplets, but the accompanying changes in bioenergetics have been little studied so far. Here, we report that the selective depletion of Rubisco and cytochrome b6f complex that occurs when Chlamydomonas reinhardtii is starved for nitrogen in the presence of acetate and under normoxic conditions is accompanied by a marked increase in chlororespiratory enzymes, which converts the photosynthetic thylakoid membrane into an intracellular matrix for oxidative catabolism of reductants. Cytochrome b6f subunits and most proteins specifically involved in their biogenesis are selectively degraded, mainly by the FtsH and Clp chloroplast proteases. This regulated degradation pathway does not require light, active photosynthesis, or state transitions but is prevented when respiration is impaired or under phototrophic conditions. We provide genetic and pharmacological evidence that NO production from intracellular nitrite governs this degradation pathway: Addition of a NO scavenger and of two distinct NO producers decrease and increase, respectively, the rate of cytochrome b6f degradation; NO-sensitive fluorescence probes, visualized by confocal microscopy, demonstrate that nitrogen-starved cells produce NO only when the cytochrome b6f degradation pathway is activated.

The involvement of hydrogen-producing and ATP-dependent NADPH-consuming pathways in setting the redox poise in the chloroplast of Chlamydomonas reinhardtii in anoxia.

Photosynthetic microalgae are exposed to changing environmental conditions. In particular, microbes found in ponds or soils often face hypoxia or even anoxia, and this severely impacts their physiology. Chlamydomonas reinhardtii is one among such photosynthetic microorganisms recognized for its unusual wealth of fermentative pathways and the extensive remodeling of its metabolism upon the switch to anaerobic conditions. As regards the photosynthetic electron transfer, this remodeling encompasses a strong limitation of the electron flow downstream of photosystem I. Here, we further characterize the origin of this limitation. We show that it stems from the strong reducing pressure that builds up upon the onset of anoxia, and this pressure can be relieved either by the light-induced synthesis of ATP, which promotes the consumption of reducing equivalents, or by the progressive activation of the hydrogenase pathway, which provides an electron transfer pathway alternative to the CO2 fixation cycle.

An easily reversible structural change underlies mechanisms enabling desert crust cyanobacteria to survive desiccation.

Biological desert sand crusts are the foundation of desert ecosystems, stabilizing the sands and allowing colonization by higher order organisms. The first colonizers of the desert sands are cyanobacteria. Facing the harsh conditions of the desert, these organisms must withstand frequent desiccation-hydration cycles, combined with high light intensities. Here, we characterize structural and functional modifications to the photosynthetic apparatus that enable a cyanobacterium, Leptolyngbya sp., to thrive under these conditions. Using multiple in vivo spectroscopic and imaging techniques, we identified two complementary mechanisms for dissipating absorbed energy in the desiccated state. The first mechanism involves the reorganization of the phycobilisome antenna system, increasing excitonic coupling between antenna components. This provides better energy dissipation in the antenna rather than directed exciton transfer to the reaction center. The second mechanism is driven by constriction of the thylakoid lumen which limits diffusion of plastocyanin to P700. The accumulation of P700(+) not only prevents light-induced charge separation but also efficiently quenches excitation energy. These protection mechanisms employ existing components of the photosynthetic apparatus, forming two distinct functional modes. Small changes in the structure of the thylakoid membranes are sufficient for quenching of all absorbed energy in the desiccated state, protecting the photosynthetic apparatus from photoinhibitory damage. These changes can be easily reversed upon rehydration, returning the system to its high photosynthetic quantum efficiency.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Arabidopsis CURVATURE THYLAKOID1 proteins modify thylakoid architecture by inducing membrane curvature.

Chloroplasts of land plants characteristically contain grana, cylindrical stacks of thylakoid membranes. A granum consists of a core of appressed membranes, two stroma-exposed end membranes, and margins, which connect pairs of grana membranes at their lumenal sides. Multiple forces contribute to grana stacking, but it is not known how the extreme curvature at margins is generated and maintained. We report the identification of the CURVATURE THYLAKOID1 (CURT1) protein family, conserved in plants and cyanobacteria. The four Arabidopsis thaliana CURT1 proteins (CURT1A, B, C, and D) oligomerize and are highly enriched at grana margins. Grana architecture is correlated with the CURT1 protein level, ranging from flat lobe-like thylakoids with considerably fewer grana margins in plants without CURT1 proteins to an increased number of membrane layers (and margins) in grana at the expense of grana diameter in overexpressors of CURT1A. The endogenous CURT1 protein in the cyanobacterium Synechocystis sp PCC6803 can be partially replaced by its Arabidopsis counterpart, indicating that the function of CURT1 proteins is evolutionary conserved. In vitro, Arabidopsis CURT1A proteins oligomerize and induce tubulation of liposomes, implying that CURT1 proteins suffice to induce membrane curvature. We therefore propose that CURT1 proteins modify thylakoid architecture by inducing membrane curvature at grana margins.

Controlling electron transfer between the two cofactor chains of photosystem I by the redox state of one of their components.

Two functional electron transfer (ET) chains, related by a pseudo-C2 symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA(-) oxidation. This is the largest change of PhQA(-) oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700(+). This situation allows a second photochemical charge separation event to be initiated before PhQA(-) has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA(-) does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed.

Modification of the pheophytin redox potential in Thermosynechococcus elongatus Photosystem II with PsbA3 as D1.

In Photosystem II (PSII) of the cyanobacterium Thermosynechococcus elongatus, glutamate 130 in the high-light variant of the D1-subunit (PsbA3) was changed to glutamine in a strain lacking the two other genes for D1, psbA1 and psbA2. The resulting PSII (PsbA3/Glu130Gln) was compared with those from the "native" high-light (PsbA3-PSII) and low-light (PsbA1-PSII) variants, which differ by 21 amino acid including Glu130Gln. H-bonding from D1-Glu130Gln to the primary electron acceptor, PheophytinD1 (PheoD1), is known to affect the Em of the PheoD1/PheoD1(-) couple. The Gln130 mutation here had little effect on water splitting, charge accumulation and photosensitivity but did slow down S2QA(-) charge recombination and up-shift the thermoluminescence while increasing its yield. These changes were consistent with a ≈-30mV shift of the PheoD1/PheoD1(-)Em, similar to earlier single site-mutation results from other species and double the ≈-17mV shift seen for PsbA1-PSII versus PsbA3-PSII. This is attributed to the influence of the other 20 amino-acids that differ in PsbA3. A computational model for simulating S2QA(-) recombination matched the experimental trend: the S2QA(-) recombination rate in PsbA1-PSII differed only slightly from that in PsbA3-PSII, while in Glu130-PsbA3-PSII there was a more pronounced slowdown of the radical pair decay. The simulation predicted a major effect of the PheoD1/PheoD1(-) potential on (1)O2 yield (~60% in PsbA1-PSII, ~20% in PsbA3-PSII and ~7% in Gln130-PsbA3-PSII), reflecting differential sensitivities to high light.

The D1-173 amino acid is a structural determinant of the critical interaction between D1-Tyr161 (TyrZ) and D1-His190 in Photosystem II.

The main cofactors of Photosystem II (PSII) are borne by the D1 and D2 subunits. In the thermophilic cyanobacterium Thermosynechococcus elongatus, three psbA genes encoding D1 are found in the genome. Among the 344 residues constituting the mature form of D1, there are 21 substitutions between PsbA1 and PsbA3, 31 between PsbA1 and PsbA2, and 27 between PsbA2 and PsbA3. In a previous study (Sugiura et al., J. Biol. Chem. 287 (2012), 13336-13347) we found that the oxidation kinetics and spectroscopic properties of TyrZ were altered in PsbA2-PSII when compared to PsbA(1/3)-PSII. The comparison of the different amino acid sequences identified the residues Cys144 and Pro173 found in PsbA1 and PsbA3, as being substituted in PsbA2 by Pro144 and Met173, and thus possible candidates accounting for the changes in the geometry and/or the environment of the TyrZ/His190 phenol/imidizol motif. Indeed, these amino acids are located upstream of the α-helix bearing TyrZ and between the two α-helices bearing TyrZ and its hydrogen-bonded partner, D1/His190. Here, site-directed mutants of PSII, PsbA3/Pro173Met and PsbA2/Met173Pro, were analyzed using X- and W-band EPR and UV-visible time-resolved absorption spectroscopy. The Pro173Met substitution in PsbA2-PSII versus PsbA3-PSII is shown to be the main structural determinant of the previously described functional differences between PsbA2-PSII and PsbA3-PSII. In PsbA2-PSII and PsbA3/Pro173Met-PSII, we found that the oxidation of TyrZ by P680+● was specifically slowed during the transition between S-states associated with proton release. We thus propose that the increase of the electrostatic charge of the Mn4CaO5 cluster in the S2 and S3 states could weaken the strength of the H-bond interaction between TyrZ● and D1/His190 in PsbA2 versus PsbA3 and/or induce structural modification(s) of the water molecules network around TyrZ.

Proton gradient regulation 5-mediated cyclic electron flow under ATP- or redox-limited conditions: a study of ΔATpase pgr5 and ΔrbcL pgr5 mutants in the green alga Chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

Gordon research conference on photosynthesis: from evolution of fundamental mechanisms to radical re-engineering.

We provide here a News Report on the 2014 Gordon Research Conference on Photosynthesis, with the subtitle "From Evolution of Fundamental Mechanisms to Radical Re-Engineering." It was held at Mount Snow Resort, West Dover, Vermont, during August 10-15, 2014. After the formal sessions ended, four young scientists (Ute Ambruster of USA; Han Bao of USA; Nicoletta Liguori of the Netherlands; and Anat Shperberg-Avni of Israel) were recognized for their research; they each received a book from one of us (G) in memory of Colin A. Wraight (1945-2014), a brilliant and admired scientist who had been very active in the bioenergetics field in general and in past Gordon Conferences in particular, having chaired the 1988 Gordon Conference on Photosynthesis. (See an article on Wraight by one of us (Govindjee) at http://www.life.illinois.edu/plantbio/Features/ColinWraight/ColinWraight.html .).

Photosynthesis in Chondrus crispus: the contribution of energy spill-over in the regulation of excitonic flux.

Chondrus crispus is a species of red algae that grows on rocks from the middle intertidal into the subtidal zones of the North Atlantic coasts. As such, it has to cope with strongly variable abiotic conditions. Here we studied the response of the photosynthetic apparatus of this red alga to illumination. We found that, as previously described in the case of the unicellular alga Rhodella violacea (E. Delphin et al., Plant Physiol. 118 (1998) 103-113), a single multi-turnover saturating pulse of light is sufficient to induce a strong quenching of fluorescence. To elucidate the mechanisms underlying this fluorescence quenching, we combined room temperature and 77K fluorescence measurements with absorption spectroscopy to monitor the redox state of the different electron carriers in the chain. In addition, we studied the dependence of these various observables upon the excitation wavelength. This led us to identify energy spill-over from Photosystem II to Photosystem I rather than a qE-type non-photochemical quenching as the major source of fluorescence quenching that develops upon a series of 200ms pulses of saturating light results, in line with the conclusion of Ley and Butler (Biochim. Biophys. Acta 592 (1980) 349-363) from their studies of the unicellular red alga Porphyridium cruentum. In addition, we show that the onset of this spill-over is triggered by the reduction of the plastoquinone pool.

The requirement for carotenoids in the assembly and function of the photosynthetic complexes in Chlamydomonas reinhardtii.

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b6f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b6f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.

Modulation of the fluorescence yield in heliobacterial cells by induction of charge recombination in the photosynthetic reaction center.

Heliobacteria contain a very simple photosynthetic apparatus, consisting of a homodimeric type I reaction center (RC) without a peripheral antenna system and using the unique pigment bacteriochlorophyll (BChl) g. They are thought to use a light-driven cyclic electron transport pathway to pump protons, and thereby phosphorylate ADP, although some of the details of this cycle are yet to be worked out. We previously reported that the fluorescence emission from the heliobacterial RC in vivo was increased by exposure to actinic light, although this variable fluorescence phenomenon exhibited very different characteristics to that in oxygenic phototrophs (Collins et al. 2010). Here, we describe the underlying mechanism behind the variable fluorescence in heliobacterial cells. We find that the ability to stably photobleach P800, the primary donor of the RC, using brief flashes is inversely correlated to the variable fluorescence. Using pump-probe spectroscopy in the nanosecond timescale, we found that illumination of cells with bright light for a few seconds put them in a state in which a significant fraction of the RCs underwent charge recombination from P800 (+)A0 (-) with a time constant of ~20 ns. The fraction of RCs in the rapidly back-reacting state correlated very well with the variable fluorescence, indicating that nearly all of the increase in fluorescence could be explained by charge recombination of P800 (+)A0 (-), some of which regenerated the singlet excited state. This hypothesis was tested directly by time-resolved fluorescence studies in the ps and ns timescales. The major decay component in whole cells had a 20-ps decay time, representing trapping by the RC. Treatment of cells with dithionite resulted in the appearance of a ~18-ns decay component, which accounted for ~0.6 % of the decay, but was almost undetectable in the untreated cells. We conclude that strong illumination of heliobacterial cells can result in saturation of the electron acceptor pool, leading to reduction of the acceptor side of the RC and the creation of a back-reacting RC state that gives rise to delayed fluorescence.

Questioning the functional relevance of mitochondrial supercomplexes by time-resolved analysis of the respiratory chain.

Mitochondria are the powerhouses of eukaryotic cells as they feed metabolism with its major substrate. Oxidative-phosphorylation relies on the generation, by an electron/proton transfer chain, of an electrochemical transmembrane potential utilized to synthesize ATP. Although these fundamental principles are not a matter of debate, the emerging picture of the respiratory chain diverges from the linear and fluid scheme. Indeed, a growing number of pieces of evidence point to membrane compartments that possibly restrict the diffusion of electron carriers, and to supramolecular assembly of various complexes within various kinds of supercomplexes that modulate the thermodynamic and kinetic properties of the components of the chain. Here, we describe a method that allows the unprecedented time-resolved study of the respiratory chain in intact cells that is aimed at assessing these hypotheses. We show that, in yeast, cytochrome c is not trapped within supercomplexes and encounters no particular restriction to its diffusion which questions the functional relevance of these supramolecular edifices.