BBA - Bioenergetics (v.1604, #3)

The nuclear encoded subunits of complex I from bovine heart mitochondria by Judy Hirst; Joe Carroll; Ian M. Fearnley; Richard J. Shannon; John E. Walker (135-150).
NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, multi-subunit, membrane-bound assembly. Recently, the subunit compositions of complex I and three of its subcomplexes have been reevaluated comprehensively. The subunits were fractionated by three independent methods, each based on a different property of the subunits. Forty-six different subunits, with a combined molecular mass of 980 kDa, were identified. The three subcomplexes, Iα, Iβ and Iλ, correlate with parts of the membrane extrinsic and membrane-bound domains of the complex. Therefore, the partitioning of subunits amongst these subcomplexes has provided information about their arrangement within the L-shaped structure. The sequences of 45 subunits of complex I have been determined. Seven of them are encoded by mitochondrial DNA, and 38 are products of the nuclear genome, imported into the mitochondrion from the cytoplasm. Post-translational modifications of many of the nuclear encoded subunits of complex I have been identified. The seven mitochondrially encoded subunits, and seven of the nuclear encoded subunits, are homologues of the 14 subunits found in prokaryotic complexes I. They are considered to be sufficient for energy transduction by complex I, and they are known as the core subunits. The core subunits bind a flavin mononucleotide (FMN) at the active site for NADH oxidation, up to eight iron–sulfur clusters, and one or more ubiquinone molecules. The locations of some of the cofactors can be inferred from the sequences of the core subunits. The remaining 31 subunits of bovine complex I are the supernumerary subunits, which may be important either for the stability of the complex, or for its assembly. Sequence relationships suggest that some of them carry out reactions unrelated to the NADH:ubiquinone oxidoreductase activity of the complex.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Mitochondrion; Subunit composition; Electron-transport chain; Respiratory chain;

Over-reduced states of the Mn-cluster in cucumber leaves induced by dark-chilling treatment by Mieko Higuchi; Takumi Noguchi; Kintake Sonoike (151-158).
Oxygen evolution is inhibited when leaves of chilling-sensitive plants like cucumber are treated at 0 °C in the dark. The activity is restored by moderate illumination at room temperature. We examined the changes in the redox state of the Mn-cluster in cucumber leaves in the processes of dark-chilling inhibition and subsequent light-induced reactivation by means of thermoluminescence (TL). A TL B-band arising from S2QB charge recombination in PSII was observed upon single-flash illumination of untreated leaves, whereas four flashes were required to yield the B-band after dark-chilling treatment for 24 h. This three-step delay indicates that over-reduced states of the Mn-cluster such as the S−2 state were formed during the treatment. Fitting analysis of the flash-number dependence of the TL intensities showed that the Mn-cluster was more reduced with a longer period of the treatment and that S−3 was the lowest S-state detectable in the dark-chilled leaves. Measurements of the Mn content by atomic absorption spectroscopy showed that Mn atoms were gradually released from PSII during the dark-chilling treatment but re-bound to PSII by illumination at 30 °C. Thus, dark-chilling inhibition of oxygen evolution can be ascribed to the disintegration of the Mn-cluster due to its over-reduction. The observation of the S−3 state in the present in vivo system strongly suggests that S−3, which has been observed only by addition of exogenous reductants into in vitro preparations, is indeed a redox intermediate of the Mn-cluster in the processes of its disintegration and photoactivation.
Keywords: Mn-cluster; S-state; Thermoluminescence; Oxygen-evolving complex; Cucumber (Cucumis sativus L.); Dark-chilling;

The NADH:ubiquinone oxidoreductase of the mitochondrial respiratory chain is a large multisubunit complex in eukaryotes containing 30–40 different subunits. Analysis of this complex using blue-native gel electrophoresis coupled to tandem mass spectrometry (MS) has identified a series of 30 different proteins from the model dicot plant, Arabidopsis, and 24 different proteins from the model monocot plant, rice. These proteins have been linked back to genes from plant genome sequencing and comparison of this dataset made with predicted orthologs of complex I components in these plants. This analysis reveals that plants contain the series of 14 highly conserved complex I subunits found in other eukaryotic and related prokaryotic enzymes and a small set of 9 proteins widely found in eukaryotic complexes. A significant number of the proteins present in bovine complex I but absent from fungal complex I are also absent from plant complex I and are not encoded in plant genomes. A series of plant-specific nuclear-encoded complex I associated subunits were identified, including a series of ferripyochelin-binding protein-like subunits and a range of small proteins of unknown function. This represents a post-genomic and large-scale analysis of complex I composition in higher plants.
Keywords: Complex I; Plant mitochondria; Proteomics; Bioinformatics;

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation by O. Cunningham; A.M. McElligott; A.M. Carroll; E. Breen; C. Reguenga; M.E.M. Oliveira; J.E. Azevedo; R.K. Porter (170-179).
A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.
Keywords: Uncoupling protein; UCP 3; Peroxisome; Mitochondria; Antibody; Thyroid; Skeletal muscle;

The photosynthetic protist Euglena gracilis contains chloroplasts surrounded by three membranes which arise from secondary endosymbiosis. The genes petA and petD, encoding cytochrome f and subunit IV of the cytochrome bf complex, normally present in chloroplast genomes, are lacking from the chloroplast DNA (cpDNA) of E. gracilis. The bf complex of E. gracilis was isolated, and the identities of cytochrome f and subunit IV were established immunochemically, by heme-specific staining, and by Edman degradation. Based on N-terminal and conserved internal protein sequences, primers were designed and used for PCR gene amplification and cDNA sequencing. The complete sequence of the petA cDNA and the partial sequence of the petD cDNA from E. gracilis are described. Evidence is provided that in this protist, the petA and petD genes have migrated from the chloroplast to the nucleus. Both genes exhibit a typical nuclear codon usage, clearly distinct from the usage of chloroplast genes. The petA gene encodes an atypical cytochrome f, with a unique insertion of 62 residues not present in other f-type cytochromes. The petA gene also acquired a region that encodes a large tripartite chloroplast transit peptide (CTP), which is thought to allow the import of apocytochrome f through the three-membrane envelope of E. gracilis chloroplasts. This is the first description of petA and petD genes that are nucleus-localized.
Keywords: Chloroplast DNA; Euglena gracilis; Photosynthetic complex;

Bioenergetics (191-192).

Bioenergetics (193-194).