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BBA - Bioenergetics (v.1807, #4)

Editorial Board (pp. i).

Characterization of photosystem II in transgenic tobacco plants with decreased iron superoxide dismutase by Yan Zhang; Shunhua Ding; Qingtao Lu; Zhipan Yang; Xiaogang Wen; Lixin Zhang; Congming Lu (pp. 391-403).

Iron superoxide dismutases (FeSODs) play an important role in preventing the oxidative damage associated with photosynthesis. To investigate the mechanisms of FeSOD in protection against photooxidative stress, we obtained transgenic tobacco ( Nicotiana tabacum) plants with severely decreased FeSOD by using a gene encoding tobacco chloroplastic FeSOD for the RNAi construct. Transgenic plants were highly sensitive to photooxidative stress and accumulated increased levels of O₂^•− under normal light conditions. Spectroscopic analysis and electron transport measurements showed that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed that there was a slow electron transfer between Q_A and Q_B and decreased redox potential of Q_B in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses showed that PSII protein accumulation was also decreased in transgenic plants. PSII photodamage and D1 protein degradation under high light treatment was increased in transgenic plants, whereas the PSII repair was not affected, indicating that the stability of the PSII complex was decreased in transgenic plants. The results in this study suggest that FeSOD plays an important role in maintaining PSII function by stabilizing PSII complexes in tobacco plants.► Transgenic tobacco plants with significant decreased FeSOD by RNAi were obtained. ► PSII activity was significantly reduced in transgenic plants. ► FeSOD plays an important role in maintaining PSII function by stabilizing PSII complex.

Keywords: Chlorophyll fluorescence; Iron superoxide dismutase; Photosystem II; Photoinhibition; Transformed tobacco (; Nicotiana tabacum; ); Thermoluminescence

Biochemical characterization of purified OmcS, a c-type cytochrome required for insoluble Fe(III) reduction in Geobacter sulfurreducens by Xinlei Qian; Tünde Mester; Leonor Morgado; Tsutomu Arakawa; Manju L. Sharma; Kengo Inoue; Crisjoe Joseph; Carlos A. Salgueiro; Michael J. Maroney; Derek R. Lovley (pp. 404-412).

Previous studies with Geobacter sulfurreducens have demonstrated that OmcS, an abundant c-type cytochrome that is only loosely bound to the outer surface, plays an important role in electron transfer to Fe(III) oxides as well as other extracellular electron acceptors. In order to further investigate the function of OmcS, it was purified from a strain that overproduces the protein. Purified OmcS had a molecular mass of 47015Da, and six low-spin bis-histidinyl hexacoordinated heme groups. Its midpoint redox potential was −212mV. A thermal stability analysis showed that the cooperative melting of purified OmcS occurs in the range of 65–82°C. Far UV circular dichroism spectroscopy indicated that the secondary structure of purified OmcS consists of about 10% α-helix and abundant disordered structures. Dithionite-reduced OmcS was able to transfer electrons to a variety of substrates of environmental importance including insoluble Fe(III) oxide, Mn(IV) oxide and humic substances. Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2s−¹) was observed as compared to that with Fe(III) citrate (2.9s−¹). The results, coupled with previous localization and gene deletion studies, suggest that OmcS is well-suited to play an important role in extracellular electron transfer.► OmcS has low spin, bis-his hexacoordinated heme groups. ► Midpoint redox potential of −212mV. ► It is able to reduce various soluble and insoluble metals such as iron, manganese, gold, uranium, and humic substances in vitro. ► Stopped flow analysis revealed that the reaction rate of OmcS oxidation has a hyperbolic dependence on the concentration of the studied substrates. ► A ten-fold faster reaction rate with anthraquinone-2,6-disulfonate (AQDS) (25.2s−¹) was observed as compared to that with Fe(III) citrate (2.9s−¹).

Keywords: Geobacter sulfurreducens; C; -type cytochrome; Iron reduction; Metal reduction; Humic substances

Exploration of the cytochrome c oxidase pathway puzzle and examination of the origin of elusive mutational effects by Suman Chakrabarty; Ida Namslauer; Peter Brzezinski; Arieh Warshel (pp. 413-426).

Gaining detailed understanding of the energetics of the proton-pumping process in cytochrome c oxidase (C cO) is a problem of great current interest. Despite promising mechanistic proposals, so far, a physically consistent model that would reproduce all the relevant barriers needed to create a working pump has not been presented. In addition, there are major problems in elucidating the origin of key mutational effects and in understanding the nature of the apparent p K_a values associated with the pH dependencies of specific proton transfer (PT) reactions in C cO. This work takes a key step in resolving the above problems, by considering mutations, such as the Asn139Asp replacement, that blocks proton pumping without affecting PT to the catalytic site. We first introduce a formulation that makes it possible to relate the apparent p K_a of Glu286 to different conformational states of this residue. We then use the new formulation along with the calculated p K_a values of Glu286 at these different conformations to reproduce the experimentally observed apparent p K_a of the residue. Next, we take the X-ray structures of the native and Asn139Asp mutant of the Paracoccus denitrificans C cO (N131D in this system) and reproduce for the first time the change in the primary PT pathways (and other key features) based on simulations that start with the observed structural changes. We also consider the competition between proton transport to the catalytic site and the pump site, as a function of the bulk pH, as well as the H/D isotope effect, and use this information to explore the relative height of the two barriers. The paper emphasizes the crucial role of energy-based considerations that include the PT process, and the delicate control of PT in C cO.► Effect of structural changes upon mutation in calculation of PT barriers in C cO. ► Modeling the relationship between conformational states and apparent p K_a of Glu286. ► Modeling pH dependent kinetics of PT in C cO based on competing pathways.

Keywords: Cytochrome; c; oxidase; Proton pump; Apparent pK; _a; Mutational effect; Energetics of proton transfer; Isotope effect

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis by Vamsi K. Moparthi; Brijesh Kumar; Cecilie Mathiesen; Hagerhall Cecilia Hägerhäll (pp. 427-436).

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na⁺/H⁺ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6–7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na⁺ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein , but expression of MrpA in the B. subtilis Δ mrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis Δ mrpA, but improved the growth of B. subtilis Δ mrpD only slightly. NuoN could restore the wild type properties of B. subtilis Δ mrpD, but had no effect on the Δ mrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na⁺ whereas MrpD transports H⁺ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na⁺ channel, NuoL, and two H⁺ channels, NuoM and NuoN, are present.Display Omitted► Protein complex formation of MrpABCDEFG is required for function only above pH 8. ► NuoL, NuoM and NuoN proteins from complex I could replace MrpA and MrpD in vivo. ► The MrpA and MrpD proteins are single ion transporters that together form an antiporter. ► Complex I probably contains one Na⁺ channel and two H⁺ channels.

Keywords: Na; ⁺; /H; ⁺; antiporter; Complex I; NuoL; NuoM; NuoN; Sha; Pha; Mnh

First solid-state NMR analysis of uniformly¹³C-enriched major light-harvesting complexes from Chlamydomonas reinhardtii and identification of protein and cofactor spin clusters by Anjali Pandit; Tomas Morosinotto; Michael Reus; Alfred R. Holzwarth; Roberto Bassi; Huub J.M. de Groot (pp. 437-443).

The light-harvesting complex II (LHCII) is the main component of the antenna system of plants and green algae and plays a major role in the capture of sun light for photosynthesis. The LHCII complexes have also been proposed to play a key role in the optimization of photosynthetic efficiency through the process of state 1–state 2 transitions and are involved in down-regulation of photosynthesis under excess light by energy dissipation through non-photochemical quenching (NPQ). We present here the first solid-state magic-angle spinning (MAS) NMR data of the major light-harvesting complex (LHCII) of Chlamydomonas reinhardtii, a eukaryotic green alga. We are able to identify nuclear spin clusters of the protein and of its associated chlorophyll pigments in¹³C–¹³C dipolar homonuclear correlation spectra on a uniformly¹³C-labeled sample. In particular, we were able to resolve several chlorophyll 13¹ carbon resonances that are sensitive to hydrogen bonding to the 13¹-keto carbonyl group. The data show that¹³C NMR signals of the pigments and protein sites are well resolved, thus paving the way to study possible structural reorganization processes involved in light-harvesting regulation through MAS solid-state NMR.► We performed MAS NMR on the major light-harvesting complex II of C. reinhardtii. ► We could resolve NMR signals of sites potentially involved in photoregulation. ► This opens perspectives to probe NPQ-related conformational changes by NMR.

Keywords: Photosynthetic light-harvesting; Non-photochemical quenching; lhc2 complex; Magic-angle spinning NMR

Impact of improved potassium accumulation on pH homeostasis, membrane potential adjustment and survival of Corynebacterium glutamicum by Ines Ochrombel; Lisa Ott; Kramer Reinhard Krämer; Andreas Burkovski; Kay Marin (pp. 444-450).

Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this study the uptake and the impact of the most abundant internal cation, potassium, were investigated in Actinobacteria, a group of high G+C Gram-positives with a number of prominent biotechnologically and medically important members. Genome analyses revealed a variety of different potassium uptake systems in this monophyletic group ranging from potassium channels common in virtually all Actinobacteria to different active carriers that were present predominantly in pathogenic members able to cope with various stress conditions. By applying Corynebacterium glutamicum as model system we provide experimental evidence that under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however, viability of C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB potassium transporter from the pathogen Corynebacterium jeikeium was heterologously expressed. We provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved maintenance of internal pH and membrane potential. The results indicate that the occurrence of active potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis and survival of bacterial cells under stress conditions.► Active potassium carriers are present in pathogenic Actinobacteria predominantly. ► Potassium uptake in the model system C. glutamicum is facilitated by a channel. ► The heterologous transporter KtrAB improves potassium accumulation. ► Thus pH homeostasis and membrane potential adjustment were improved. ► Thus survival under stress conditions was improved.

Keywords: Potassium transport; Potassium channel; Corynebacterium; pH homeostasis; Membrane potential adjustment

Electron transfer to the active site of the bacterial nitric oxide reductase is controlled by ligand binding to heme b₃ by Sarah J. Field; M. Dolores Roldan; Sophie J. Marritt; Julea N. Butt; David J. Richardson; Nicholas J. Watmough (pp. 451-457).

The active site of the bacterial nitric oxide reductase from Paracoccus denitrificans contains a dinuclear centre comprising heme b₃ and non heme iron (Fe_B). These metal centres are shown to be at isopotential with midpoint reduction potentials of E_m≈+80mV. The midpoint reduction potentials of the other two metal centres in the enzyme, heme c and heme b, are greater than the dinuclear centre suggesting that they act as an electron receiving/storage module. Reduction of the low-spin heme b causes structural changes at the dinuclear centre which allow access to substrate molecules. In the presence of the substrate analogue, CO, the midpoint reduction potential of heme b₃ is raised to a region similar to that of heme c and heme b. This leads us to suggest that reduction of the electron transfer hemes leads to an opening of the active site which allows substrate to bind and in turn raises the reduction potential of the active site such that electrons are only delivered to the active site following substrate binding.► The potentials of the active site metal centres of NOR are lower than the electron receiving hemes. ► Reduction of the electron receiving hemes opens the active site to ligands. ► CO raises the potential of the active site heme to allow internal electron transfer. ► The resulting compound reveals the EPR spectrum of the cryptic Fe_B centre.

Keywords: Carbon monoxide; Denitrification; Electron transfer; Hemes; Nitric oxide

Mitochondria–cytoskeleton interaction: Distribution of β-tubulins in cardiomyocytes and HL-1 cells by Rita Guzun; Minna Karu-Varikmaa; Marcela Gonzalez-Granillo; Andrey V. Kuznetsov; Lauriane Michel; Cécile Cottet-Rousselle; Saaremae Merle Saaremäe; Tuuli Kaambre; Madis Metsis; Michael Grimm; Charles Auffray; Valdur Saks (pp. 458-469).

Mitochondria–cytoskeleton interactions were analyzed in adult rat cardiomyocytes and in cancerous non-beating HL-1 cells of cardiac phenotype. We show that in adult cardiomyocytes βII-tubulin is associated with mitochondrial outer membrane (MOM). βI-tubulin demonstrates diffused intracellular distribution, βIII-tubulin is colocalized with Z-lines and βIV-tubulin forms microtubular network. HL-1 cells are characterized by the absence of βII-tubulin, by the presence of bundles of filamentous βIV-tubulin and diffusely distributed βI- and βIII-tubulins. Mitochondrial isoform of creatine kinase (MtCK), highly expressed in cardiomyocytes, is absent in HL-1 cells. Our results show that high apparent K_m for exogenous ADP in regulation of respiration and high expression of MtCK both correlate with the expression of βII-tubulin. The absence of βII-tubulin isotype in isolated mitochondria and in HL-1 cells results in increased apparent affinity of oxidative phosphorylation for exogenous ADP. This observation is consistent with the assumption that the binding of βII-tubulin to mitochondria limits ADP/ATP diffusion through voltage-dependent anion channel of MOM and thus shifts energy transfer via the phosphocreatine pathway. On the other hand, absence of both βII-tubulin and MtCK in HL-1 cells can be associated with their more glycolysis-dependent energy metabolism which is typical for cancer cells (Warburg effect).► βII-tubulin is attached to mitochondria and controls its function in cardiomyocytes. ► βII-tubulin and MitCK are absent in cancerous HL-1 cells of cardiac phenotype. ► βII, βIII, βIV and βI-tubulins prevent from fusion/fission of mitochondria. ► Mitochondrial interactosome remodelling determines mechanism of energy transfer.

Keywords: Cardiomyocytes; Creatine kinase; HL-1 cells; Mitochondrial interactosome; β-tubulin isotypes; Warburg effect

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BBA - Bioenergetics (v.1807, #4)