BBA - Bioenergetics (v.1505, #2-3)
Proton transfer in Azotobacter vinelandii ferredoxin I: entatic Lys84 operates as elastic counterbalance for the proton-carrying Asp15 by Dmitry A. Cherepanov; Armen Y. Mulkidjanian (179-184).
In ferredoxin I from Azotobacter vinelandii, the reduction of a [3Fe-4S] iron-sulphur cluster is coupled with the protonation of the μ2S sulphur atom that is approx. 6 Å away from the protein boundary. The recent study of the site-specific mutants of ferredoxin I led to the conclusion that a particular surface aspartic residue (Asp15) is solely responsible for the proton transfer to the μ2S atom by ‘rapid penetrative excursions’ (K. Chen, J. Hirst, R. Camba, C.A. Bonagura, C.D. Stout, B.K. Burgess, F.A. Armstrong, Nature 405 (2000) 814–817). In the same paper it has been reported that the replacement of Asp15 by glutamate led to the blockage of the enzyme, although glutamate, with its longer and more flexible side chain, should apparently do even better as a mobile proton carrier than aspartate. We tackled this puzzling incompetence of Glu15 by molecular dynamics simulations. It was revealed that the conformational alterations of Asp15 are energetically balanced by the straining of the nearby Lys84 side chain in wild-type ferredoxin I but not in the Asp15→Glu mutant. Lys84 in ferredoxin I of A. vinelandii seems to represent the first case where the strained (entatic) conformation of a particular amino acid side chain could be directly identified in the ground state of an enzyme and assigned to a distinct mechanism of energy balance during the catalytic transition.
Keywords: Electron transfer; Proton transfer; Molecular dynamics; Enzyme catalysis; Ferredoxin I; Azotobacter vinelandii;
A novel scenario for the evolution of haem–copper oxygen reductases by Manuela M Pereira; Margarida Santana; Miguel Teixeira (185-208).
The increasing sequence information on oxygen reductases of the haem–copper superfamily, together with the available three-dimensional structures, allows a clear identification of their common, functionally important features. Taking into consideration both the overall amino acid sequences of the core subunits and key residues involved in proton transfer, a novel hypothesis for the molecular evolution of these enzymes is proposed. Three main families of oxygen reductases are identified on the basis of common features of the core subunits, constituting three lines of evolution: (i) type A (mitochondrial-like oxidases), (ii) type B (ba 3-like oxidases) and (iii) type C (cbb 3-type oxidases). The first group can be further divided into two subfamilies, according to the helix VI residues at the hydrophobic end of one of the proton pathways (the so-called D-channel): (i) type A1, comprising the enzymes with a glutamate residue in the motif –XGHPEV–, and (ii) type A2, enzymes having instead a tyrosine and a serine in the alternative motif –YSHPXV–. This second subfamily of oxidases is shown to be ancestor to the one containing the glutamate residue, which in the Bacteria domain is only present in oxidases from Gram-positive or purple bacteria. It is further proposed that the Archaea domain acquired terminal oxidases by gene transfer from the Gram-positive bacteria, implying that these enzymes were not present in the last common ancestor before the divergence between Archaea and Bacteria. In fact, most oxidases from archaea have a higher amino acid sequence identity and similarity with those from bacteria, mainly from the Gram-positive group, than with oxidases from other archaea. Finally, a possible relation between the dihaemic subunit (FixP) of the cbb 3 oxidases and subunit II of caa 3 oxidases is discussed. As the families of haem–copper oxidases can also be identified by their subunit II, a parallel evolution of subunits I and II is suggested.
Keywords: Haem–copper oxidase; Evolution; Oxygen;
Heterologous expression and properties of the γ-subunit of the Fe-only hydrogenase from Thermotoga maritima by Marc F.J.M. Verhagen; Thomas W. O’Rourke; Angeli Lal Menon; Michael W.W. Adams (209-219).
Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (αβγ) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (γ), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native γ-subunit has been hampered by interference from and the inability to separate intact γ-subunit from the other two subunits (α and β). To investigate the function and properties of the isolated γ-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact γ-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was −356 mV at pH 7.0 and 25°C, as measured by direct electrochemistry, and was pH dependent with a pK ox of 7.5 and a pK red of 7.7. The oxidized, recombinant γ-subunit was stable at 80°C under anaerobic conditions with a half-life greater than 24 h, as judged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant γ-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a K m of 5 μM and a V max of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated α-subunit were much less effective electron donors for the γ-subunit, with a V max of 0.01 U/mg and 0.1 U/mg, respectively.
Keywords: Thermotoga maritima HydC; Fe-only hydrogenase; Heterologous gene expression;
What controls the outer mitochondrial membrane permeability for ADP: facts for and against the role of oncotic pressure by Julius Liobikas; Dalia Marija Kopustinskiene; Adolfas Toleikis (220-225).
In our study 10% of bovine serum albumin was added to the physiological incubation medium to mimic the oncotic pressure of the cellular cytoplasm and to test for its effect on the respiration of isolated rat heart mitochondria, saponin- or saponin plus crude collagenase (type IV)-treated heart muscle fibers and saponin-treated rat quadriceps muscle fibers. Pyruvate and malate were used as substrates. We found that albumin slightly decreased the maximal ADP-stimulated respiration rate only for saponin-treated heart muscle fibers. The apparent K m ADP of oxidative phosphorylation increased significantly, by 70–100%, for isolated heart mitochondria, saponin plus collagenase-treated heart muscle fibers and for saponin-treated quadriceps muscle fibers but remained unchanged for saponin-treated heart muscle fibers. The saponin-treated heart muscle fibers were characterized by a very high control apparent K m ADP value (234±24 μM ADP) compared with other preparations (14–28 μM ADP). The results suggest that in vivo the oncotic pressure is not the relevant factor causing the low outer mitochondrial membrane permeability for ADP in cardiomyocytes, in contrast to quadriceps muscle cells. It is likely that the outer mitochondrial membrane-bound protein(s) which is supposed to remain in saponin-treated heart muscle fibers is responsible for this property of the membrane.
Keywords: Macromolecule; Rat heart mitochondria; Skinned heart fiber; Skinned skeletal muscle fiber; Oxidative phosphorylation; Kinetic constant;
Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro by Luca Tiano; Donatella Fedeli; Patrizia Ballarini; Giorgio Santoni; Giancarlo Falcioni (226-237).
Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes.
Keywords: Trout erythrocyte; Mitochondria; Ageing; Oxidative stress; Membrane potential; Antioxidant;
Site specific labeling of Rhodobacter sphaeroides reaction centers with dye probes for surface pH measurements by Szabolcs Osváth; Jonathan W Larson; Colin A Wraight (238-247).
Covalently bound pH sensitive dyes are an important tool for characterizing the proteolytic reactions of protein complexes that play key roles in biological energy transduction. Here we demonstrate the feasibility of this method for photosynthetic reaction centers (RCs) for the first time, by the highly selective attachment of two thiol reactive derivatives of fluorescein to the two H subunit cysteines of the photosynthetic RC from Rhodobacter sphaeroides R-26 The pK a shifts of the dyes upon binding to the protein and in response to high salt were measured, and interpreted based on the structure of the RC. 2-[(5-fluoresceinyl)aminocarbonyl]ethyl-methanethiosulfonate was attached to Cys H156 and fluorescein-5-maleimide to Cys H234. By following the absorption changes of bound fluorescein (500 nm), and those of the hydrophilic pH indicator 8-hydroxypyrene-1,3,6-tris-sulfonic acid (468 nm), the surface and bulk pH were monitored separately with less than 5% crosstalk. Flash-induced proton uptake and external calibrations by mixing with aliquots of acid were measured in different redox states of the RCs. The results indicate that the charge in the quinone acceptor complex after flash activation (primary quinone acceptor (QA)− or secondary quinone acceptor (QB)−) has no effect on the surface pH and potential in the vicinity of these two attachment sites, between pH 6.5 and 9. Application of the method to other surface locations is discussed.
Keywords: Photosynthetic reaction center; Proton transfer; pH dye; Surface probe;
Photosystem stoichiometry and state transitions in a mutant of the cyanobacterium Synechococcus sp. PCC 7002 lacking phycocyanin by Jindong Zhao; Gaozhong Shen; Donald A. Bryant (248-257).
Phycobilisomes (PBS) function as light-harvesting antenna complexes in cyanobacteria, red algae and cyanelles. They are composed of two substructures: the core and peripheral rods. Interposon mutagenesis of the cpcBA genes of Synechococcus sp. PCC 7002 resulted in a strain (PR6008) lacking phycocyanin and thus the ability to form peripheral rods. Difference absorption spectroscopy of whole cells showed that intact PBS cores were assembled in vivo in the cpcBA mutant strain PR6008. Fluorescence induction measurements demonstrated that the PBS cores are able to deliver absorbed light energy to photosystem (PS) II, and fluorescence induction transients in the presence of DCMU showed that PR6008 cells could perform a state 2 to state 1 transition with similar kinetics to that of the wild-type cells. Thus, PBS core assembly, light-harvesting functions and energy transfer to PS I were not dependent upon the assembly of the peripheral rods. The ratio of PS II:PS I in the PR6008 cells was significantly increased, nearly twice that of the wild-type cells, possibly a result of long-term adaptation to compensate for the reduced antenna size of PS II. However, the ratio of PBS cores:chlorophyll remained unchanged. This result indicates that approximately half of the PS II reaction centers in the PR6008 cells had no closely associated PBS cores.
Keywords: Cyanobacterium; Phycobilisome; Peripheral rod; State transition; Photosystem I; Photosystem II; Synechococcus sp.;
Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice by Urmo Braun; Kalju Paju; Margus Eimre; Evelin Seppet; Ehte Orlova; Lumme Kadaja; Sonata Trumbeckaite; Frank N. Gellerich; Stephan Zierz; Harald Jockusch; Enn K. Seppet (258-270).
The potential role of dystrophin-mediated control of systems integrating mitochondria with ATPases was assessed in muscle cells. Mitochondrial distribution and function in skinned cardiac and skeletal muscle fibers from dystrophin-deficient (MDX) and wild-type mice were compared. Laser confocal microscopy revealed disorganized mitochondrial arrays in m. gastrocnemius in MDX mice, whereas the other muscles appeared normal in this group. Irrespective of muscle type, the absence of dystrophin had no effect on the maximal capacity of oxidative phosphorylation, nor on coupling between oxidation and phosphorylation. However, in the myocardium and m. soleus, the coupling of mitochondrial creatine kinase to adenine nucleotide translocase was attenuated as evidenced by the decreased effect of creatine on the K m for ADP in the reactions of oxidative phosphorylation. In m. soleus, a low K m for ADP compared to the wild-type counterpart was found, which implies increased permeability for that nucleotide across the mitochondrial outer membrane. In normal cardiac fibers 35% of the ADP flux generated by ATPases was not accessible to the external pyruvate kinase-phosphoenolpyruvate system, which suggests the compartmentalized (direct) channeling of that fraction of ADP to mitochondria. Compared to control, the direct ADP transfer was increased in MDX ventricles. In conclusion, our data indicate that in slow-twitch muscle cells, the absence of dystrophin is associated with the rearrangement of the intracellular energy and feedback signal transfer systems between mitochondria and ATPases. As the mechanisms mediated by creatine kinases become ineffective, the role of diffusion of adenine nucleotides increases due to the higher permeability of the mitochondrial outer membrane for ADP and enhanced compartmentalization of ADP flux.
Keywords: Mitochondria; Adenosine 5′-triphosphatase; Muscle; Respiration; Regulation; Energy transfer;
Cold-induced changes in the energy coupling and the UCP3 level in rodent skeletal muscles by Ruben A. Simonyan; Maria Jimenez; Rolando B. Ceddia; Jean-Paul Giacobino; Patrick Muzzin; Vladimir P. Skulachev (271-279).
The mechanism of thermoregulatory uncoupling of respiration and phosphorylation in skeletal muscles has been studied. It is found that 24 h cold exposure results in (i) a 3-fold increase in the amount of UCP3 protein in rat skeletal muscle mitochondria, and (ii) pronounced lowering of the membrane potential in isolated rat or mouse skeletal muscle mitochondria. The decrease in membrane potential is reversed by adding bovine serum albumin. Cold exposure is also found to sensitize the membrane potential to the uncoupling action of added fatty acid (laurate). After laurate addition, the recoupling effects of GDP and carboxyatractylate decrease whereas that of albumin increases in mitochondria from cold-treated rats or mice. Changes similar to those induced by cold can be initiated by the in vivo addition of thyroxine. Cold exposure does not affect energy coupling in liver mitochondria. The possible involvement of UCP3 isoforms in nucleotide-sensitive and -insensitive uncoupling is discussed.
Keywords: Muscle mitochondria; Thermoregulatory uncoupling; Thyroxine; Uncoupling protein-3; Fatty acid;
Proton uptake associated with the reduction of the primary quinone QA influences the binding site of the secondary quinone QB in Rhodopseudomonas viridis photosynthetic reaction centers by Ulrich Zachariae; C.Roy D Lancaster (280-290).
Previously, two binding sites for the secondary quinone QB in the photosynthetic reaction center (RC) from Rhodopseudomonas viridis were identified by X-ray crystallography, a ‘proximal’ binding site close to the non-heme iron, and a ‘distal’ site, displaced by 4.2 Å along the path of the isoprenoid tail [C.R.D. Lancaster and H. Michel, Structure 5 (1997) 1339–1359]. The quinone ring planes in the two sites differ by roughly a 180° rotation around the isoprenoid tail. Here we present molecular dynamics simulations, which support the theory of a spontaneous transfer of QB between the distal site and the proximal site. In contrast to earlier computational studies on RCs, the molecular dynamics simulations of QB migration resulted in a proximal QB binding pattern identical to that of the crystallographic findings. Also, we demonstrate that the preference towards the proximal QB location is not necessarily attributed to reduction of QB to the semiquinone, but already to the preceding reduction of the primary quinone QA and resulting protonation changes in the protein. Energy mapping of the QB binding pocket indicates that the quinone ring rotation required for completion of the transfer between the two sites is improbable at the distal or proximal binding sites due to high potential barriers, but may be possible at a newly identified position near the distal binding site.
Keywords: Bioenergetics; Electron transfer; Membrane protein; Molecular dynamics; Protein electrostatics; Proton transfer; Blastochloris viridis;
Author Index (291-292).