BBA - Bioenergetics (v.1858, #1)

In Memoriam (vi-x).

The purple Trp288Ala mutant of Synechocystis OCP persistently quenches phycobilisome fluorescence and tightly interacts with FRP by Nikolai N. Sluchanko; Konstantin E. Klementiev; Evgeny A. Shirshin; Georgy V. Tsoraev; Thomas Friedrich; Eugene G. Maksimov (1-11).
In Cyanobacteria, the Orange Carotenoid Protein (OCP) and Fluorescence Recovery Protein (FRP) are central to the photoprotective mechanism consisting in regulated quenching of phycobilisome (PBs) fluorescence. Due to a transient and flexible nature of the light-activated red quenching form, OCPR, which is obtained from the stable dark-adapted orange form, OCPO, by photoconversion, the detailed mechanism of photoprotection remains unclear. Here we demonstrate that our recently described W288A mutant of the Synechocystis OCP (hereinafter called OCPW288A) is a fully functional analogue of the OCPR form which is capable of constitutive PBs fluorescence quenching in vitro with no need of photoactivation. This PBs quenching effect is abolished in the presence of FRP, which interacts with OCPW288A with micromolar affinity and an apparent stoichiometry of 1:1, unexpectedly, implying dissociation of the FRP dimers. This establishes OCPW288A as a robust model system providing novel insights into the interplay between OCP and FRP to regulate photoprotection in cyanobacteria.Display Omitted
Keywords: Orange carotenoid protein; Synechocystis sp. PCC 6803; Echinenone; Size-exclusion chromatography; Protein-protein interaction;

Supercomplexes of plant photosystem I with cytochrome b6f, light-harvesting complex II and NDH by K.N. Sathish Yadav; Dmitry A. Semchonok; Lukáš Nosek; Roman Kouřil; Geoffrey Fucile; Egbert J. Boekema; Lutz A. Eichacker (12-20).
Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b6f (Cytb6f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb6f at the antenna side of PSI. The rectangular-shaped Cytb6f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb6f, may improve regulation of electron transport by the control of binding partners and distances in small domains.
Keywords: Photosystem I; Cytochrome b6f complex; NDH; Supercomplex; Electron microscopy;

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged.
Keywords: Mitochondria; Uncoupling proteins; Free fatty acids; Aldehydes; Purine nucleotides; Coenzyme Q;

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and Pi concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and Pi-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of Pi-dependent coupling can be observed also in the εCTD-less mutant, but the effects of Pi on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EFOF1 and increases responses to Pi. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/Pi binding, promoting ADP/Pi-dependent coupling.
Keywords: E. coli; ATP synthase; ε subunit; ADP binding; Activation; Uncoupling;

Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii by Jooyeon Jeong; Kwangryul Baek; Henning Kirst; Anastasios Melis; EonSeon Jin (45-55).
The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the “chloroplast signal recognition particle 54” gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.
Keywords: Chlorophyll antenna size; CpSRP54; LHC assembly; Photosynthesis; Productivity; TLA technology;

High photochemical trapping efficiency in Photosystem I from the red clade algae Chromera velia and Phaeodactylum tricornutum by Erica Belgio; Stefano Santabarbara; David Bína; Eliška Trsková; Miroslava Herbstová; Radek Kaňa; Giuseppe Zucchelli; Ondřej Prášil (56-63).
In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15–25 ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.Display Omitted
Keywords: Chromera velia; Phaeodactylum tricornutum; Red clade algae; Photosystem I; Fluorescence decay; Red spectral forms; Photochemical yield;

A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2 by Joris J. Snellenburg; Lucyna M. Wlodarczyk; Jan P. Dekker; Rienk van Grondelle; Ivo H.M. van Stokkum (64-72).
The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)− 1.Display Omitted
Keywords: Chlamydomonas reinhardtii; time-resolved fluorescence; target analysis; 77 K;

A variety of mitochondria-targeted small molecules have been invented to manipulate mitochondrial redox activities and improve function in certain disease states. 3-Hydroxypropyl-triphenylphosphonium-conjugated imidazole-substituted oleic acid (TPP-IOA) was developed as a specific inhibitor of cytochrome c peroxidase activity that inhibits apoptosis by preventing cardiolipin oxidation and cytochrome c release to the cytosol. Here we evaluate the effects of TPP-IOA on oxidative phosphorylation in isolated mitochondria and on mitochondrial function in live cells. We demonstrate that, at concentrations similar to those required to achieve inhibition of cytochrome c peroxidase activity, TPP-IOA perturbs oxidative phosphorylation in isolated mitochondria. In live SH-SY5Y cells, TPP-IOA partially collapsed mitochondrial membrane potential, caused extensive fragmentation of the mitochondrial network, and decreased apparent mitochondrial abundance within 3 h of exposure. Many cultured cell lines rely primarily on aerobic glycolysis, potentially making them less sensitive to small molecules disrupting oxidative phosphorylation. We therefore determined the anti-apoptotic efficacy of TPP-IOA in SH-SY5Y cells growing in glucose or in galactose, the latter of which increases reliance on oxidative phosphorylation for ATP supply. The anti-apoptotic activity of TPP-IOA that was observed in glucose media was not seen in galactose media. It therefore appears that, at concentrations required to inhibit cytochrome c peroxidase activity, TPP-IOA perturbs oxidative phosphorylation. In light of these data it is predicted that potential future therapeutic applications of TPP-IOA will be restricted to highly glycolytic cell types with limited reliance on oxidative phosphorylation.
Keywords: Oxidative phosphorylation; TPP-IOA; Apoptosis; Cytochrome c; Peroxidase; Triphenylphosphonium;

Investigation of the NADH/NAD+ ratio in Ralstonia eutropha using the fluorescence reporter protein Peredox by Vijay Tejwani; Franz-Josef Schmitt; Svea Wilkening; Ingo Zebger; Marius Horch; Oliver Lenz; Thomas Friedrich (86-94).
Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD+ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD+ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD+] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD+] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD+] ratios represents a novel and sensitive tool to determine the redox state of cells.
Keywords: Ralstonia eutropha; Fluorescence sensor protein; NADH:NAD+ ratio; Fluorescence lifetime; Decay-associated spectra; Hydrogenase;