BBA - Bioenergetics (v.1858, #5)

Lipid and carotenoid cooperation-driven adaptation to light and temperature stress in Synechocystis sp. PCC6803 by Tomas Zakar; Eva Herman; Sindhujaa Vajravel; Laszlo Kovacs; Jana Knoppová; Josef Komenda; Ildiko Domonkos; Mihaly Kis; Zoltan Gombos; Hajnalka Laczko-Dobos (337-350).
Polyunsaturated lipids are important components of photosynthetic membranes. Xanthophylls are the main photoprotective agents, can assist in protection against light stress, and are crucial in the recovery from photoinhibition. We generated the xanthophyll- and polyunsaturated lipid-deficient ROAD mutant of Synechocystis sp. PCC6803 (Synechocystis) in order to study the little-known cooperative effects of lipids and carotenoids (Cars). Electron microscopic investigations confirmed that in the absence of xanthophylls the S-layer of the cellular envelope is missing. In wild-type (WT) cells, as well as the xanthophyll-less (RO), polyunsaturated lipid-less (AD), and the newly constructed ROAD mutants the lipid and Car compositions were determined by MS and HPLC, respectively. We found that, relative to the WT, the lipid composition of the mutants was remodeled and the Car content changed accordingly. In the mutants the ratio of non-bilayer-forming (NBL) to bilayer-forming (BL) lipids was found considerably lower. Xanthophyll to β-carotene ratio increased in the AD mutant. In vitro and in vivo methods demonstrated that saturated, monounsaturated lipids and xanthophylls may stabilize the trimerization of Photosystem I (PSI). Fluorescence induction and oxygen-evolving activity measurements revealed increased light sensitivity of RO cells compared to those of the WT. ROAD showed a robust increase in light susceptibility and reduced recovery capability, especially at moderate low (ML) and moderate high (MH) temperatures, indicating a cooperative effect of xanthophylls and polyunsaturated lipids. We suggest that both lipid unsaturation and xanthophylls are required for providing the proper structure and functioning of the membrane environment that protects against light and temperature stress.Schematic figure of thylakoid membrane structure in Synechocystis wild-type (WT) and ROAD (xanthophyll- and polyunsaturated lipid-deficient) mutant cells.Display Omitted
Keywords: Lipid-carotenoid-protein interactions; Lipid remodeling; Xanthophylls; Photoinhibition; Temperature stress; Cyanobacteria;

Impact of copper ligand mutations on a cupredoxin with a green copper center by Magali Roger; Giuliano Sciara; Frédéric Biaso; Elisabeth Lojou; Xie Wang; Marielle Bauzan; Marie-Thérèse Giudici-Orticoni; Alejandro J. Vila; Marianne Ilbert (351-359).
Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.
Keywords: Copper; Cupredoxin; Green copper site; Metalloprotein; Metal center; Acidithiobacillus ferrooxidans;

Low-pH induced reversible reorganizations of chloroplast thylakoid membranes — As revealed by small-angle neutron scattering by Renáta Ünnep; Ottó Zsiros; Zsolt Hörcsik; Márton Markó; Anjana Jajoo; Joachim Kohlbrecher; Győző Garab; Gergely Nagy (360-365).
Energization of thylakoid membranes brings about the acidification of the lumenal aqueous phase, which activates important regulatory mechanisms. Earlier Jajoo and coworkers (2014 FEBS Lett. 588:970) have shown that low pH in isolated plant thylakoid membranes induces changes in the excitation energy distribution between the two photosystems. In order to elucidate the structural background of these changes, we used small-angle neutron scattering on thylakoid membranes exposed to low p2H (pD) and show that gradually lowering the p2H from 8.0 to 5.0 causes small but well discernible reversible diminishment of the periodic order and the lamellar repeat distance and an increased mosaicity — similar to the effects elicited by light-induced acidification of the lumen. Our data strongly suggest that thylakoids dynamically respond to the membrane energization and actively participate in different regulatory mechanisms.
Keywords: Chloroplast thylakoid membranes; Lamellar repeat distance; Low pH and p2H; Small-angle neutron scattering (SANS);

Searching for the low affinity ubiquinone binding site in cytochrome bo 3 from Escherichia coli by Sylvia K. Choi; Myat T. Lin; Hanlin Ouyang; Robert B. Gennis (366-370).
The cytochrome bo 3 ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo 3 contains two distinct ubiquinol binding sites: (i) a low affinity (QL) site which is the traditional substrate binding site; and (ii) a high affinity (QH) site where a “permanently” bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o3/CuB active site. Whereas the residues at the QH site are well defined, the location of the QL site remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305–314] identified a sequence motif G163EFX3GWX2Y173 as the likely QL site in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the QL substrate binding site.Display Omitted
Keywords: Ubiquinone; Mutagenesis; E. coli; Respiration; Cytochrome bo 3;

Multiple LHCII antennae can transfer energy efficiently to a single Photosystem I by Inge Bos; Kaitlyn M. Bland; Lijin Tian; Roberta Croce; Laurie K. Frankel; Herbert van Amerongen; Terry M. Bricker; Emilie Wientjes (371-378).
Photosystems I and II (PSI and PSII) work in series to drive oxygenic photosynthesis. The two photosystems have different absorption spectra, therefore changes in light quality can lead to imbalanced excitation of the photosystems and a loss in photosynthetic efficiency. In a short-term adaptation response termed state transitions, excitation energy is directed to the light-limited photosystem. In higher plants a special pool of LHCII antennae, which can be associated with either PSI or PSII, participates in these state transitions. It is known that one LHCII antenna can associate with the PsaH site of PSI. However, membrane fractions were recently isolated in which multiple LHCII antennae appear to transfer energy to PSI. We have used time-resolved fluorescence-streak camera measurements to investigate the energy transfer rates and efficiency in these membrane fractions. Our data show that energy transfer from LHCII to PSI is relatively slow. Nevertheless, the trapping efficiency in supercomplexes of PSI with ~ 2.4 LHCIIs attached is 94%. The absorption cross section of PSI can thus be increased with ~ 65% without having significant loss in quantum efficiency. Comparison of the fluorescence dynamics of PSI-LHCII complexes, isolated in a detergent or located in their native membrane environment, indicates that the environment influences the excitation energy transfer rates in these complexes. This demonstrates the importance of studying membrane protein complexes in their natural environment.
Keywords: Excitation energy transfer; State transitions; Light-harvesting complex; Time-resolved fluorescence;

Interaction between the photoprotective protein LHCSR3 and C2S2 Photosystem II supercomplex in Chlamydomonas reinhardtii by Dmitriy A. Semchonok; K.N. Sathish Yadav; Pengqi Xu; Bartlomiej Drop; Roberta Croce; Egbert J. Boekema (379-385).
Photosynthetic organisms can thermally dissipate excess of absorbed energy in high-light conditions in a process known as non-photochemical quenching (NPQ). In the green alga Chlamydomonas reinhardtii this process depends on the presence of the light-harvesting protein LHCSR3, which is only expressed in high light. LHCSR3 has been shown to act as a quencher when associated with the Photosystem II supercomplex and to respond to pH changes, but the mechanism of quenching has not been elucidated yet. In this work we have studied the interaction between LHCSR3 and Photosystem II C2S2 supercomplexes by single particle electron microscopy. It was found that LHCSR3 predominantly binds at three different positions and that the CP26 subunit and the LHCII trimer of C2S2 supercomplexes are involved in binding, while we could not find evidences for a direct association of LHCSR3 with the PSII core. At all three locations LHCSR3 is present almost exclusively as a dimer.
Keywords: Photosystem I; Chlamydomonas reinhardtii; LHCSR3; Electron; Microscopy;

Chloroplasts in vivo exposed to strong light export assimilates and excess reducing power to the cytoplasm for metabolic conversions and allocation to neighboring and distant organelles. The cytoplasmic streaming, being particularly fast in characean internodes, distributes the exported metabolites from brightly illuminated cell spots to light-limited regions, which is evident from the transient increase in chlorophyll fluorescence of shaded areas in response to illumination of distant cell regions situated upstream the liquid flow. It is not yet known whether long-distance communications between anchored chloroplasts are interfered by pH banding that commonly arises in characean internodes under the action of continuous or fluctuating light. In this study, microfluorometry, pH-microsensors, and local illumination were combined to examine long-distance transport and subsequent reentry of photosynthetic metabolites, including triose phosphates, into chloroplasts of cell regions producing external alkaline and acid bands. The lateral transmission of metabolic signals between distant chloroplasts was found to operate effectively in cell areas underlying acid zones but was almost fully blocked under alkaline zones. The rates of linear electron flow in chloroplasts of these regions were nearly equal under dim background light, but differed substantially at high light when availability of CO2, rather than irradiance, was the rate-limiting factor. Different productions of assimilates by chloroplasts underlying CO2-sufficient acid and CO2-deficient alkaline zones were a cause for contrasting manifestations of long-distance transport of photosynthetic metabolites. Nonuniform cytoplasmic pH in cells exhibiting pH bands might contribute to different activities of metabolic translocators under high and low pH zones.
Keywords: Long-distance communications; Photosynthetic electron transport; Cytoplasmic streaming; Proton flows;

Insights into proton translocation in cbb 3 oxidase from MD simulations by Catarina A. Carvalheda; Andrei V. Pisliakov (396-406).
Heme-copper oxidases are membrane protein complexes that catalyse the final step of the aerobic respiration, namely the reduction of oxygen to water. The energy released during catalysis is coupled to the active translocation of protons across the membrane, which contributes to the establishment of an electrochemical gradient that is used for ATP synthesis. The distinctive C-type (or cbb 3) cytochrome c oxidases, which are mostly present in proteobacteria, exhibit a number of unique structural and functional features, including high catalytic activity at low oxygen concentrations. At the moment, the functioning mechanism of C-type oxidases, in particular the proton transfer/pumping mechanism presumably via a single proton channel, is still poorly understood. In this work we used all-atom molecular dynamics simulations and continuum electrostatics calculations to obtain atomic-level insights into the hydration and dynamics of a cbb 3 oxidase. We provide the details of the water dynamics and proton transfer pathways for both the “chemical” and “pumped” protons, and show that formation of protonic connections is strongly affected by the protonation state of key residues, namely H243, E323 and H337.Display Omitted
Keywords: Molecular dynamics simulations; pK a calculations; Proton transfer; Water dynamics; Cytochrome c oxidase; Proton pump; Membrane protein;