BBA - Bioenergetics (v.1777, #2)
Editorial Board (ii).
Images of mitochondrial UCP 1 in mouse thymocytes using confocal microscopy by Alison E. Adams; Orla Hanrahan; Derek N. Nolan; H. Paul Voorheis; Padraic Fallon; Richard K. Porter (115-117).
The aim of this study was to demonstrate the constitutive expression of mitochondrial uncoupling protein 1 (UCP 1) in pure thymocytes using laser scanning confocal microscopic imagery. To that end we probed thymocytes from UCP 1 knock-out and wild-type mice. Mitochondrial location in thymocytes was determined using Mitotracker Red and the nucleus was labelled using Hoescht stain. We demonstrate that all cells investigated were thymocytes as determined by a monoclonal antibody specific for the thymocyte surface marker Thy 1 (CD90) pre-coupled to a fluorescent labelled (Alexa 448, green). Using a primary peptide antibody specific to UCP 1, and secondary fluorescently labelled (Alexa 647, magenta) antibody, we were able to demonstrate that UCP 1 is associated with mitochondria in thymocytes from UCP 1 wild-type mice but not thymocytes from UCP1-knock-out mice. These are the first images demonstrating the presence of UCP 1 in thymocyte mitochondria, in situ, and the first to clearly demonstrate UCP 1 expression in cells other than brown adipocytes. We conclude that mouse thymocytes contain UCP 1 in their mitochondria.
Keywords: Uncoupling protein-1; Thymus; Thymocytes; Mitochondria; UCP 1 knock-out mice; Confocal microscopy;
Uncoupling protein-2 accumulates rapidly in the inner mitochondrial membrane during mitochondrial reactive oxygen stress in macrophages by Tindaro M. Giardina; James H. Steer; Susan Z.Y. Lo; David A. Joyce (118-129).
Uncoupling protein-2 (UCP2) is a member of the inner mitochondrial membrane anion-carrier superfamily. Although mRNA for UCP2 is widely expressed, protein expression is detected in only a few cell types, including macrophages. UCP2 functions by an incompletely defined mechanism, to reduce reactive oxygen species production during mitochondrial electron transport. We observed that the abundance of UCP2 in macrophages increased rapidly in response to treatments (rotenone, antimycin A and diethyldithiocarbamate) that increased mitochondrial superoxide production, but not in response to superoxide produced outside the mitochondria or in response to H2O2. Increased UCP2 protein was not accompanied by increases in ucp2 gene expression or mRNA abundance, but was due to enhanced translational efficiency and possibly stabilization of UCP2 protein in the inner mitochondrial membrane. This was not dependent on mitochondrial membrane potential. These findings extend our understanding of the homeostatic function of UCP2 in regulating mitochondrial reactive oxygen production by identifying a feedback loop that senses mitochondrial reactive oxygen production and increases inner mitochondrial membrane UCP2 abundance and activity. Reactive oxygen species-induction of UCP2 may facilitate survival of macrophages and retention of function in widely variable tissue environments.
Keywords: Uncoupling protein-2; Macrophage; Superoxide; Reactive oxygen species; Mitochondria;
The psbA gene family responds differentially to light and UVB stress in Gloeobacter violaceus PCC 7421, a deeply divergent cyanobacterium by Cosmin I. Sicora; Christopher M. Brown; Otilia Cheregi; Imre Vass; Douglas A. Campbell (130-139).
Gloeobacter violaceus PCC 7421 is a slow-growing cyanobacterium which lacks thylakoid membranes, but whose five-membered psbA gene family encodes three isoform variants of the PsbA (D1) reaction center protein of Photosystem II. Under standard culture conditions Gloeobacter exhibits photosystem II electron transport, but several clear modifications in the redox potential of key cofactors bound by the PsbA protein are manifested in the flash-fluorescence characteristics. In other cyanobacteria dynamic expression of multiple psbA genes and turnover of PsbA isoforms is critical to counter excitation stress. We found that each of Gloeobacter's five psbA genes is expressed, with transcript abundances spanning 4.5 orders of magnitude. psbAI (glr2322) and psbAII (glr0779), encoding identical PsbA:2 form proteins, are constitutively expressed and dominate the psbA transcript pool under control conditions. psbAIII (gll3144) was strongly induced under photoinhibitory high irradiance stress, thereby contributing to a large increase in the psbA transcript pool that allowed cells to maintain their PsbA protein pools and then recover from irradiance stress, within one cellular generation. In contrast, under comparable photoinhibition provoked by UVB the cells were unable to maintain their psbA transcript and PsbA protein pools, and showed limited subsequent recovery. psbAIV (glr1706) and psbAV (glr2656), encoding two divergent PsbA isoforms, showed consistent trace expression but were never quantitatively significant contributors to the psbA transcript pool.
Keywords: Gloeobacter; D1 protein; Light; Photosystem II; psbA gene family; UV;
Access channels and methanol binding site to the CaMn4 cluster in Photosystem II based on solvent accessibility simulations, with implications for substrate water access by Felix M. Ho; Stenbjörn Styring (140-153).
Given the tightly packed environment of Photosystem II (PSII), channels are expected to exist within the protein to allow the movement of small molecules to and from the oxygen evolving centre. In this report, we calculate solvent contact surfaces from the PSII crystal structures to identify such access channels for methanol and water molecules. In a previous study of the effects of methanol on the EPR split S1-, S3-, and S0-signals [Su et al. (2006) Biochemistry 45, 7617–7627], we proposed that methanol binds to one and the same Mn ion in all S-states. We find here that while channels of methanol dimensions were able to make contact with the CaMn4 cluster, only 3Mn and 4Mn were accessible to methanol. Combining this observation with spectroscopic data in the literature, we propose that 3Mn is the ion to which methanol binds. Furthermore, by calculating solvent contact surfaces for water, we found analogous and more extensive water accessible channels within PSII. On the basis of their structure, orientation, and electrostatic properties, we propose functional assignments of these channels as passages for substrate water access to the CaMn4 cluster, and for the exit of O2 and H+ that are released during water oxidation. Finally, we discuss the possible existence of a gating mechanism for the control of substrate water access to the CaMn4 cluster, based on the observation of a gap within the channel system that is formed by Ca2+ and several mechanistically very significant residues in the vicinity of the cluster.
Keywords: Photosystem II; Channel; Solvent contact surface; Substrate water access; H+ exit pathway; O2 exit pathway;
Plastoquinol as a singlet oxygen scavenger in photosystem II by Jerzy Kruk; Achim Trebst (154-162).
It has been found that in Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to α-tocopherol. In relation to chlorophyll, after 18 h of growth under low light with the inhibitor, the content of α-tocopherol was 22.2 mol/1000 mol chlorophyll and that of total plastoquinone (oxidized and reduced) was 19 mol/1000 mol chlorophyll, while after 2 h of high-light stress the corresponding amounts dropped to 6.4 and 6.2 mol/1000 mol chlorophyll for α-tocopherol and total plastoquinone, respectively. The degradation of both prenyllipids was partially reversed by diphenylamine, a singlet oxygen scavenger. It was concluded that plastoquinol, as well as α-tocopherol is decomposed under high-light stress as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The levels of both α-tocopherol and of the reduced plastoquinone are not affected significantly in the absence of the inhibitor due to a high turnover rate of both prenyllipids, i.e., their degradation is compensated by fast biosynthesis. The calculated turnover rates under high-light conditions were twofold higher for total plastoquinone (0.23 nmol/h/ml of cell culture) than for α-tocopherol (0.11 nmol/h/ml). We have also found that the level of α-tocopherolquinone, an oxidation product of α-tocopherol, increases as the α-tocopherol is consumed. The same correlation was also observed for γ-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low-light growth conditions, the synthesis of plastoquinone-C, a hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. The presented data also suggest that the two plastoquinones may have different biosynthetic pathways in C. reinhardtii.
Keywords: Photosystem II; Plastoquinol; Plastoquinone; Pyrazolate; Singlet oxygen; Tocopherol; Tocopherolquinone;
Dual role for a bacteriophytochrome in the bioenergetic control of Rhodopsdeudomonas palustris: Enhancement of photosystem synthesis and limitation of respiration by Mila Kojadinovic; Aurélie Laugraud; Laurie Vuillet; Joël Fardoux; Laure Hannibal; Jean-Marc Adriano; Pierre Bouyer; Eric Giraud; André Verméglio (163-172).
In the purple photosynthetic bacterium Rhodopseudomonas palustris, far-red illumination induces photosystem synthesis via the action of the bacteriophytochrome RpBphP1. This bacteriophytochrome antagonizes the repressive effect of the transcriptional regulator PpsR2 under aerobic condition. We show here that, in addition to photosystem synthesis, far-red light induces a significant growth rate limitation, compared to cells grown in the dark, linked to a decrease in the respiratory activity. The phenotypes of mutants inactivated in RpBphP1 and PpsR2 show their involvement in this regulation. Based on enzymatic and transcriptional studies, a 30% decrease in the expression of the alpha-ketoglutarate dehydrogenase complex, a central enzyme of the Krebs cycle, is observed under far-red light. We propose that this decrease is responsible for the down-regulation of respiration in this condition. This regulation mechanism at the Krebs cycle level still allows the formation of the photosynthetic apparatus via the synthesis of key biosynthesis precursors but lowers the production of NADH, i.e. the respiratory activity. Overall, the dual action of RpBphP1 on the regulation of both the photosynthesis genes and the Krebs cycle allows a fine adaptation of bacteria to environmental conditions by enhancement of the most favorable bioenergetic process in the light, photosynthesis versus respiration.
Keywords: Bacteriophytochrome; Photosynthesis; Respiration; Alpha-ketoglutarate; Regulation; Rhodopseudomonas palustris;
Dopamine modulates mitochondrial function in viable SH-SY5Y cells possibly via its interaction with complex I: Relevance to dopamine pathology in schizophrenia by Hanit Brenner-Lavie; Ehud Klein; Rosa Zuk; Haifa Gazawi; Predrage Ljubuncic; Dorit Ben-Shachar (173-185).
Deleterious effects of dopamine (DA) involving mitochondrial dysfunction have an important role in DA-associated neuronal disorders, including schizophrenia and Parkinson's disease. DA detrimental effects have been attributed to its ability to be auto-oxidized to toxic reactive oxygen species. Since, unlike Parkinson's disease, schizophrenia does not involve neurodegenerative processes, we suggest a novel mechanism by which DA impairs mitochondrial function without affecting cell viability. DA significantly dissipated mitochondrial membrane potential (Δψ m) in SH-SY5Y cells. Bypassing complex I prevented the DA-induced depolarization. Moreover, DA inhibited complex I but not complex II activity in disrupted mitochondria, suggesting complex I participation in DA-induced mitochondrial dysfunction. We further demonstrated that intact mitochondria can accumulate DA in a saturated manner, with an apparent K m = 122.1 ± 28.6 nM and V max = 1.41 ± 0.15 pmol/mg protein/min, thereby enabling the interaction between DA and complex I. DA accumulation was an energy and Na+-dependent process. The pharmacological profile of mitochondrial DA uptake differed from that of other characterized DA transporters. Finally, relevance to schizophrenia is demonstrated by an abnormal interaction between DA and complex I in schizophrenic patients. These results suggest a non-lethal interaction between DA and mitochondria possibly via complex I, which can better explain DA-related pathological processes observed in non-degenerative disorders, such as schizophrenia.
Keywords: Dopamine; Mitochondria; Mitochondrial membrane potential; Complex I; Mitochondrial dopamine uptake; Schizophrenia;
Identification by time-resolved EPR of the peridinins directly involved in chlorophyll triplet quenching in the peridinin–chlorophyll a–protein from Amphidinium carterae by Marilena Di Valentin; Stefano Ceola; Enrico Salvadori; Giancarlo Agostini; Donatella Carbonera (186-195).
The mechanism of triplet–triplet energy transfer in the peridinin–chlorophyll–protein (PCP) from Amphidinium carterae was investigated by time-resolved EPR (TR-EPR). The approach exploits the concept of spin conservation during triplet–triplet energy transfer, which leads to spin polarization conservation in the observed TR-EPR spectra. The acceptor (peridinin) inherits the polarization of the donor (chlorophyll) in a way which depends on the relative geometrical arrangement of the donor–acceptor couple. Starting from the initially populated chlorophyll triplet state and taking the relative positions among Chls and peridinins from the X-ray structure of PCP, we calculated the expected triplet state polarization of any peridinin in the complex. Comparison with the experimental data allowed us to propose a path for triplet quenching in the protein. The peridinin–chlorophyll pair directly involved in the triplet–triplet energy transfer coincides with the one having the shortest center to center distance. A water molecule, which is coordinated to the central Mg atom of the Chl, is also placed in close contact with the peridinin. The results support the concept of localization of the triplet state mainly in one specific peridinin in each of the two pigment subclusters related by a pseudo C2 symmetry.
Keywords: PCP; Carotenoid; Triplet; TR-EPR;
Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation by Jiancong Xu; Gregory A. Voth (196-201).
The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a 3 (PRDa 3 ) for the first time. The results from these molecular dynamics simulations indicate that the PRDa 3 can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa 3 side chain isomerization and the proton translocation between Glu242 and the PRDa 3 site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa3 site.
Keywords: Cytochrome c oxidase; Multi-state empirical valence bond (MS-EVB); Free energy calculation; Proton pumping;
The polypeptides COX2A and COX2B are essential components of the mitochondrial cytochrome c oxidase of Toxoplasma gondii by Lorena Morales-Sainz; Adelma Escobar-Ramírez; Valentín Cruz-Torres; Adrián Reyes-Prieto; Miriam Vázquez-Acevedo; Reyna Lara-Martínez; Luis Felipe Jiménez-García; Diego González-Halphen (202-210).
Two genes encoding cytochrome c oxidase subunits, Cox2a and Cox2b, are present in the nuclear genomes of apicomplexan parasites and show sequence similarity to corresponding genes in chlorophycean algae. We explored the presence of COX2A and COX2B subunits in the cytochrome c oxidase of Toxoplasma gondii. Antibodies were raised against a synthetic peptide containing a 14-residue fragment of the COX2A polypeptide and against a hexa-histidine-tagged recombinant COX2B protein. Two distinct immunochemical stainings localized the COX2A and COX2B proteins in the parasite's mitochondria. A mitochondria-enriched fraction exhibited cyanide-sensitive oxygen uptake in the presence of succinate. T. gondii mitochondria were solubilized and subjected to Blue Native Electrophoresis followed by second dimension electrophoresis. Selected protein spots from the 2D gels were subjected to mass spectrometry analysis and polypeptides of mitochondrial complexes III, IV and V were identified. Subunits COX2A and COX2B were detected immunochemically and found to co-migrate with complex IV; therefore, they are subunits of the parasite's cytochrome c oxidase. The apparent molecular mass of the T. gondii mature COX2A subunit differs from that of the chlorophycean alga Polytomella sp. The data suggest that during its biogenesis, the mitochondrial targeting sequence of the apicomplexan COX2A precursor protein may be processed differently than the one from its algal counterpart.
Keywords: Toxoplasma gondii; Cytochrome c oxidase; COX2A subunit; COX2B subunit; Fragmented cox2 gene; Apicomplexan parasite;
Differential efficacy of inhibition of mitochondrial and bacterial cytochrome bc 1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin by Frederik A.J. Rotsaert; Martina G. Ding; Bernard L. Trumpower (211-219).
We have compared the efficacy of inhibition of the cytochrome bc 1 complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC50 ∼ 10 nM, while the IC50 for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc 1 complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc 1 complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.
Keywords: bc 1 complex; Center N; Inhibitor; Yeast; Bovine; Paracoccus denitrificans; Ilicicolin H; Funiculosin;
Spectroscopic study on the communication between a heme a 3 propionate, Asp399 and the binuclear center of cytochrome c oxidase from Paracoccus denitrificans by Petra Hellwig; Andreas Böhm; Ute Pfitzner; Werner Mäntele; Bernd Ludwig (220-226).
The proton pumping mechanism of cytochrome c oxidase on a molecular level is highly disputed. Recently theoretical calculations and real time electron transfer measurements indicated the involvement of residues in the vicinity of the ring A propionate of heme a 3, including Asp399 and the CuB ligands His 325, 326. In this study we probed the interaction of Asp399 with the binuclear center and characterize the protonation state of its side chain. Redox induced FTIR difference spectra of mutations at the site in direct comparison to wild type, indicate that below pH 5 Asp 399 displays signals typical for the deprotonation of the acidic residue with reduction of the enzyme. Interestingly at a pH higher than 5, no contributions from Asp 399 are evident. In order to probe the interaction of the site with the binuclear center we followed the rebinding of CO by infrared spectroscopy for mutations on residue Asp399 to Glu, Asn and Leu. Previously different CO conformers have been identified for bacterial cytochrome c oxidases, and its pH dependent behaviour discussed to be relevant for catalysis. Interestingly we observe the lack of this pH dependency and a strong influence on the observable conformers for all mutants studied here, clearly suggesting a communication of the site with the heme-copper center and the nearby histidine residues.
Keywords: Cytochrom c oxidase; FTIR spectroscopy; Heme propionates; CO conformers;
Interaction of transmembrane helices in ATP synthase subunit a in solution as revealed by spin label difference NMR by Oleg Y. Dmitriev; Karen H. Freedman; Joseph Hermolin; Robert H. Fillingame (227-237).
Subunit a in the membrane traversing F0 sector of Escherichia coli ATP synthase is known to fold with five transmembrane helices (TMHs) with residue 218 in TMH IV packing close to residue 248 in TMH V. In this study, we have introduced a spin label probe at Cys residues substituted at positions 222 or 223 and measured the effects on the Trp ɛNH indole NMR signals of the seven Trp residues in the protein. The protein was purified and NMR experiments were carried out in a chloroform–methanol–H2O (4:4:1) solvent mixture. The spin label at positions 222 or 223 proved to broaden the signals of W231, W232, W235 and W241 located at the periplasmic ends of TMH IV and TMH V and the connecting loop between these helices. The broadening of W241 would require that the loop residues fold back on themselves in a hairpin-like structure much like it is predicted to fold in the native membrane. Placement of the spin label probe at several other positions also proved to have broadening effects on some of these Trp residues and provided additional constraints on folding of TMH IV and TMH V. The effects of the 223 probes on backbone amide resonances of subunit a were also measured by an HNCO experiment and the results are consistent with the two helices folding back on themselves in this solvent mixture. When Cys and Trp were substituted at residues 206 and 254 at the cytoplasmic ends of TMHs IV and V respectively, the W254 resonance was not broadened by the spin label at position 206. We conclude that the helices fold back on themselves in this solvent system and then pack at an angle such that the cytoplasmic ends of the polypeptide backbone are significantly displaced from each other.
Keywords: ATP synthase subunit a; Proton transport; Transmembrane helices; Solution NMR; Spin label; Membrane protein structure;