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BBA - Bioenergetics (v.1787, #2)
In Yarrowia lipolytica mitochondria, the alternative NADH dehydrogenase interacts specifically with the cytochrome complexes of the classic respiratory pathway by Sergio Guerrero-Castillo; Miriam Vázquez-Acevedo; Diego González-Halphen; Salvador Uribe-Carvajal (pp. 75-85).
In Yarrowia lipolytica, mitochondria contain a branched respiratory chain constituted by the classic complexes I, II, III and IV, plus an alternative external NADH dehydrogenase (NDH2e) and an alternative oxidase (AOX). The alternative enzymes are peripheral, single-subunit oxido-reductases that do not pump protons. Thus, the oxidation of NADH via NDH2e-ubiquinone-AOX would not contribute to the proton-motive force. The futile oxidation of NADH may be prevented if either NDH2e or AOX bind to the classic complexes, channelling electrons. By oxymetry, it was observed that the electrons from complex I reached both cytochrome oxidase and AOX. In contrast, NDH2e-derived electrons were specifically channelled/directed to the cytochrome complexes. In addition, the presence of respiratory supercomplexes plus the interaction of NDH2e with these complexes was evaluated using blue native PAGE, clear native PAGE, in-gel activities, immunoblotting, mass spectrometry, and N-terminal sequencing. NDH2e (but not the redirected matrix NDH2i from a mutant strain, Δnubm) was detected in association with the cytochromic pathway; this interaction seems to be strong, as it was not disrupted by laurylmaltoside. The association of NDH2e to complex IV was also suggested when both enzymes coeluted from an ion exchange chromatography column. In Y. lipolytica mitochondria the cytochrome complexes probably associate into supercomplexes; those were assigned as follows: I–III2, I–IV, I–III2–IV4, III2–IV, III2–IV2, IV2 and V2. The molecular masses of all the complexes and putative supercomplexes detected in Y. lipolytica were estimated by comparison with the bovine mitochondrial complexes. To our knowledge, this is the first evidence of supercomplex formation in Y. lipolytica mitochondria and also, the first description of a specific association between an alternative NADH dehydrogenase and the classic cytochrome pathway.
Keywords: Alternative NADH dehydrogenase; Cytochrome complex; Supercomplex; Yarrowia lipolytica; mitochondria
Purification, characterization and crystallization of menaquinol:fumarate oxidoreductase from the green filamentous photosynthetic bacterium Chloroflexus aurantiacus by Yueyong Xin; Yih-Kuang Lu; Raimund Fromme; Petra Fromme; Robert E. Blankenship ⁎ (pp. 86-96).
The integral membrane protein complex, menaquinol:fumarate oxidoreductase (mQFR) has been purified, identified and characterized from the thermophilic green filamentous anoxygenic photosynthetic bacterium Chloroflexus aurantiacus. The complex is composed of three subunits: a 74 kDa flavoprotein that contains a covalently bound flavin adenine dinucleotide, a 28 kDa iron-sulfur cluster-containing polypeptide, and a 27 kDa transmembrane polypeptide, which is also the binding site of two b-type hemes and two menaquinones. The purified complex has an apparent molecular mass of 260 kDa by blue-native PAGE, which is indicative of a native homodimeric form. The isolated complex is active in vitro in both fumarate reduction and succinate oxidation. It has been analyzed by visible absorption, redox titration, chemical analysis and EPR spectroscopy. In addition, phylogenetic analysis shows that the QFR of both C. aurantiacus and Chlorobium tepidum are most closely related to those found in the delta-proteobacteria. The purified enzyme was crystallized and X-ray diffraction data obtained up to 3.2 Å resolution.
Keywords: Abbreviations; mQFR; menaquinol fumarate oxidoreductase; SQR; succinate quinone oxidoreductase; FAD; flavin adenine dinucleotide; Em; redox midpoint potential; DDM; dodecyl maltoside; PMS; phenazine methosulfate; DCPIP; 2,6-dichloro-phenolindophenol; HQNO; heptyl-4-hydroxyquinoline N-oxide; Fp; FAD-containing subunit; Ip; Iron-sulfur-cluster containing subunit; Cp; Cytochrome containing membrane anchor subunitMenaquinol:fumarate oxidoreductase; Purification; Characterization; Crystallization; Electron transfer chain; Chloroflexus aurantiacus
The structure of genetically modified iron–sulfur cluster Fx in photosystem I as determined by X-ray absorption spectroscopy by Xiao-Min Gong; Yehoshua Hochman; Tal Lev; Grant Bunker; Chanoch Carmeli ⁎ (pp. 97-104).
Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe–4S] iron–sulfur cluster FX, located on the core subunits PsaA/B to iron–sulfur clusters FA/B on subunit PsaC. Structure function relations in the native and in the mutant ( psaB-C565S/D566E) of the cysteine ligand of FX cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of FX was determined in PS I lacking clusters FA/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe–4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe–S and Fe–Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe–O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to FX is discussed.
Keywords: Abbreviations; A; 0; primary electron acceptor in PS I; A; 1; secondary electron acceptor in PS I; Chl; chlorophyll; DCIP; 2,6-dichlorophenolindophenol; LAHG; light-activated heterotrophic growth; P700; primary electron donor of PS I; PS I; photosystem I; F; X; , F; A; and F; B; [4Fe–4S] clusters of PS I; XAS; X-ray absorption-spectroscopy; XANES; X-ray absorption-near edge structure; EXAFS; X-ray-extended fine structurePhotosystem I; Photosynthesis; Iron–sulfur cluster; X-ray absorption; Electron transfer; Subunit deletion; Site directed mutation; Transient absorption spectroscopy
Transcription of a “silent” cyanobacterial psbA gene is induced by microaerobic conditions by Cosmin Ionel Sicora ⁎; Felix M. Ho; Tiina Salminen; Stenbjörn Styring; Eva-Mari Aro ⁎ (pp. 105-112).
Cyanobacteria, contrary to higher plants, have a small psbA gene family encoding the reaction centre D1 protein subunit of photosystem II, the first macromolecular pigment-protein complex of the photosynthetic electron transport chain. Modulation of expression of multiple psbA genes in the family allows cyanobacteria to adapt to changing environmental conditions. To date, two different strategies for regulation of the psbA genes have emerged. One, characterized in Synechocystis PCC6803 and Gloeobacter violaceus PCC7421 involves the increased expression of one type of D1 protein to cope with the increased rate of damage. The other strategy, in Synechococcus PCC7942 and Anabaena PCC7120, is to replace the existing D1 with a new D1 form for the duration of the stress. However, most of the psbA gene families characterized to date contain also a divergent, apparently silent psbA gene of unknown function. This gene, present in Synechocystis, Anabaena and Thermosynechococcus elongatus BP-1 was not induced by any stress condition applied so far. Our data shows a reversible induction of the divergent psbA gene during the onset of argon-induced microaerobic conditions in Synechocystis, Anabaena and Thermosynechococcus elongatus. The unitary functional response of three unrelated cyanobacterial species, namely the induction of the expression of the divergent psbA gene as a reaction to the same environmental cue, indicates that these genes and the protein they encode are part of a specific cellular response to microaerobic conditions. There are no specific primary structure similarities between the different microaerobic inducible D1 forms, designated as D1′. Only three amino acid residues are consistently conserved in D1′. These modifications are: G80 to A, F158 to L and T286 to L. In silico mutation of the published D1 structure from Thermosynechococcus did not reveal major modifications. The point by point effects of the mutations on the local environment of the PSII structure are also discussed.
Keywords: Abreviations; Synechocystis; Synechocystis sp. PCC6803; Anabaena; Anabaena/Nostoc sp. PCC7120; Thermosynechococcus elongatus; Thermosynechococcus elongatus BP-1 psbA; Microaerobic effect; D1 protein; Cyanobacteria
Tuning of functional heme reduction potentials in Shewanella fumarate reductases by Miguel Pessanha; Emma L. Rothery; Caroline S. Miles; Graeme A. Reid; Stephen K. Chapman; Ricardo O. Louro; David L. Turner; Carlos A. Salgueiro ⁎; António V. Xavier (pp. 113-120).
The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.
Keywords: Abbreviations; fcc; 3; flavocytochrome; c; 3; Sffcc; 3; flavocytochrome; c; 3; from; S. frigidimarina; Sofcc; 3; flavocytochrome; c; 3; from; S. oneidensisRespiratory enzyme; Fumarate; Heme; NMR; Redox; Electrostatic interaction
Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga by Hideyuki Adachi; Yasufumi Umena; Isao Enami; Takahiro Henmi; Nobuo Kamiya; Jian-Ren Shen ⁎ (pp. 121-128).
Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P2221 with unit cell constants of a=146.8 Å, b=176.9 Å, and c=353.7 Å, and the other one had a space group of P212121 with unit cell constants of a=209.2 Å, b=237.5 Å, and c=299.8 Å. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 Å resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 Å resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.
Keywords: Abbreviations; Chl; chlorophyll; cyt; cytochrome; DDM; n-dodecyl-β-D-maltoside; NCS; non-crystallographic symmetry; OEC; oxygen-evolving complex; PSI; photosystems I; PSII; photosystems II; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresisPhotosystem II; Crystallization; Red algae; Cyanidium caldarium; Membrane protein; Dehydration
Antibiotics LL-Z1272 identified as novel inhibitors discriminating bacterial and mitochondrial quinol oxidases by Tatsushi Mogi ⁎; Hideaki Ui; Kazuro Shiomi; Satoshi Ōmura; Hideto Miyoshi; Kiyoshi Kita (pp. 129-133).
To counter antibiotic-resistant bacteria, we screened the Kitasato Institute for Life Sciences Chemical Library with bacterial quinol oxidase, which does not exist in the mitochondrial respiratory chain. We identified five prenylphenols, LL-Z1272β, γ, δ, ɛ and ζ, as new inhibitors for the Escherichia coli cytochrome bd. We found that these compounds also inhibited the E. coli bo-type ubiquinol oxidase and trypanosome alternative oxidase, although these three oxidases are structurally unrelated. LL-Z1272β and ɛ (dechlorinated derivatives) were more active against cytochrome bd while LL-Z1272γ, δ, and ζ (chlorinated derivatives) were potent inhibitors of cytochrome bo and trypanosome alternative oxidase. Thus prenylphenols are useful for the selective inhibition of quinol oxidases and for understanding the molecular mechanisms of respiratory quinol oxidases as a probe for the quinol oxidation site. Since quinol oxidases are absent from mammalian mitochondria, LL-Z1272β and δ, which are less toxic to human cells, could be used as lead compounds for development of novel chemotherapeutic agents against pathogenic bacteria and African trypanosomiasis.
Keywords: Quinol oxidase; Inhibitor; Natural antibiotic; Escherichia coli; Trypanosoma brucei; Alternative oxidase