BBA - Bioenergetics (v.1797, #2)
Editorial Board (i).
Mitochondrial DNA mutations and human disease by Helen A.L. Tuppen; Emma L. Blakely; Douglass M. Turnbull; Robert W. Taylor (113-128).
Mitochondrial disorders are a group of clinically heterogeneous diseases, commonly defined by a lack of cellular energy due to oxidative phosphorylation (OXPHOS) defects. Since the identification of the first human pathological mitochondrial DNA (mtDNA) mutations in 1988, significant efforts have been spent in cataloguing the vast array of causative genetic defects of these disorders. Currently, more than 250 pathogenic mtDNA mutations have been identified. An ever-increasing number of nuclear DNA mutations are also being reported as the majority of proteins involved in mitochondrial metabolism and maintenance are nuclear-encoded. Understanding the phenotypic diversity and elucidating the molecular mechanisms at the basis of these diseases has however proved challenging. Progress has been hampered by the peculiar features of mitochondrial genetics, an inability to manipulate the mitochondrial genome, and difficulties in obtaining suitable models of disease. In this review, we will first outline the unique features of mitochondrial genetics before detailing the diseases and their genetic causes, focusing specifically on primary mtDNA genetic defects. The functional consequences of mtDNA mutations that have been characterised to date will also be discussed, along with current and potential future diagnostic and therapeutic advances.
Keywords: mtDNA; Mitochondrial disease; Molecular mechanism; Diagnosis; Treatment;
Quantum chemistry as a tool in bioenergetics by Margareta R.A. Blomberg; Per E.M. Siegbahn (129-142).
Recent developments of quantum chemical methods have made it possible to tackle crucial questions in bioenergetics. The most important systems, cytochrome c oxidase in cellular respiration and photosystem II (PSII) in photosynthesis will here be used as examples to illustrate the power of the quantum chemical tools. One main contribution from quantum chemistry is to put mechanistic suggestions onto an energy scale. Accordingly, free energy profiles can be constructed both for reduction of molecular oxygen in cytochrome c oxidase and water oxidation in PSII, including O–O bond cleavage and formation, and also proton pumping in cytochrome c oxidase. For the construction of the energy diagrams, the computational results sometimes have to be combined with experimental information, such as reduction potentials and rate constants for individual steps in the reactions.
Keywords: Quantum chemistry; Cytochrome c oxidase; Photosynthesis; Reaction mechanisms; Proton pumping;
Alteration of mitochondrial oxidative phosphorylation in aged skeletal muscle involves modification of adenine nucleotide translocator by Gilles Gouspillou; Isabelle Bourdel-Marchasson; Richard Rouland; Guillaume Calmettes; Jean-Michel Franconi; Véronique Deschodt-Arsac; Philippe Diolez (143-151).
The process of skeletal muscle aging is characterized by a progressive loss of muscle mass and functionality. The underlying mechanisms are highly complex and remain unclear. This study was designed to further investigate the consequences of aging on mitochondrial oxidative phosphorylation in rat gastrocnemius muscle, by comparing young (6 months) and aged (21 months) rats. Maximal oxidative phosphorylation capacity was clearly reduced in older rats, while mitochondrial efficiency was unaffected. Inner membrane properties were unaffected in aged rats since proton leak kinetics were identical to young rats. Application of top-down control analysis revealed a dysfunction of the phosphorylation module in older rats, responsible for a dysregulation of oxidative phosphorylation under low activities close to in vivo ATP turnover. This dysregulation is responsible for an impaired mitochondrial response toward changes in cellular ATP demand, leading to a decreased membrane potential which may in turn affect ROS production and ion homeostasis. Based on our data, we propose that modification of ANT properties with aging could partly explain these mitochondrial dysfunctions.
Keywords: Mitochondrial oxidative phosphorylation; Top-down control analysis; Skeletal muscle; Adenine nucleotide translocator; Aging;
Characterization of two different acyl carrier proteins in complex I from Yarrowia lipolytica by Krzysztof Dobrynin; Albina Abdrakhmanova; Sebastian Richers; Carola Hunte; Stefan Kerscher; Ulrich Brandt (152-159).
Acyl carrier proteins of mitochondria (ACPMs) are small (∼ 10 kDa) acidic proteins that are homologous to the corresponding central components of prokaryotic fatty acid synthase complexes. Genomic deletions of the two genes ACPM1 and ACPM2 in the strictly aerobic yeast Yarrowia lipolytica resulted in strains that were not viable or retained only trace amounts of assembled mitochondrial complex I, respectively. This suggested different functions for the two proteins that despite high similarity could not be complemented by the respective other homolog still expressed in the deletion strains. Remarkably, the same phenotypes were observed if just the conserved serine carrying the phosphopantethein moiety was exchanged with alanine. Although this suggested a functional link to the lipid metabolism of mitochondria, no changes in the lipid composition of the organelles were found. Proteomic analysis revealed that both ACPMs were tightly bound to purified mitochondrial complex I. Western blot analysis revealed that the affinity tagged ACPM1 and ACPM2 proteins were exclusively detectable in mitochondrial membranes but not in the mitochondrial matrix as reported for other organisms. Hence we conclude that the ACPMs can serve all their possible functions in mitochondrial lipid metabolism and complex I assembly and stabilization as subunits bound to complex I.
Keywords: Complex I; Yarrowia lipolytica; Mitochondrial acyl carrier protein; ACPM; Assembly defect;
Purification and characterization of a stable oxygen-evolving Photosystem II complex from a marine centric diatom, Chaetoceros gracilis by Ryo Nagao; Tatsuya Tomo; Eri Noguchi; Saori Nakajima; Takehiro Suzuki; Akinori Okumura; Yasuhiro Kashino; Mamoru Mimuro; Masahiko Ikeuchi; Isao Enami (160-166).
Oxygen-evolving Photosystem II particles (crude PSII) retaining a high oxygen-evolving activity have been prepared from a marine centric diatom, Chaetoceros gracilis (Nagao et al., 2007). The crude PSII, however, contained a large amount of fucoxanthin chlorophyll a/c-binding proteins (FCP). In this study, a purified PSII complex which was deprived of major components of FCP was isolated by one step of anion exchange chromatography from the crude PSII treated with Triton X-100. The purified PSII was still associated with the five extrinsic proteins of PsbO, PsbQ', PsbV, Psb31 and PsbU, and showed a high oxygen-evolving activity of 2135 μmol O2 (mg Chl a)− 1 h− 1 in the presence of phenyl-p-benzoquinone which was virtually independent of the addition of CaCl2. This activity is more than 2.5-fold higher than the activity of the crude PSII. The activity was completely inhibited by 3-(3,4)-dichlorophenyl-(1,1)-dimethylurea (DCMU). The purified PSII contained 42 molecules of Chl a, 2 molecules of diadinoxanthin and 2 molecules of Chl c on the basis of two molecules of pheophytin a, and showed typical absorption and fluorescence spectra similar to those of purified PSIIs from the other organisms. In this study, we also found that the crude PSII was significantly labile, as a significant inactivation of oxygen evolution, chlorophyll bleaching and degradation of PSII subunits were observed during incubation at 25 °C in the dark. In contrast, these inactivation, bleaching and degradation were scarcely detected in the purified PSII. Thus, we succeeded for the first time in preparation of a stable PSII from diatom cells.
Keywords: Oxygen evolution; Photosystem II; Extrinsic protein; Diatom; Chaetoceros gracilis;
Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport by Torrie T. Jones; Gregory J. Brewer (167-176).
Respiratory enzyme complex dysfunction is mechanistically involved in mitochondrial failure leading to neurodegenerative disease, but the pathway is unclear. Here, age-related differences in mitochondrial respiration were measured in both whole and permeabilized neurons from 9-month and 24-month adult rat cortex cultured in common conditions. After permeabilization, respiration increased in both ages of neurons with excess substrates. To dissect specific deficiencies in the respiratory chain, inhibitors for each respiratory chain complex were used to isolate their contributions. Relative to neurons from 9-month rats, in neurons isolated from 24-month rats, complexes I, III, and IV were more sensitive to selective inhibition. Flux control point analysis identified complex I in neurons isolated from 24-month rats as the most sensitive to endogenous substrate availability. The greatest age-related deficit in flux capacity occurred at complex IV with a 29% decrease in neurons isolated from 24-month rats relative to those from 9-month rats. The deficits in complexes I and III may contribute to a redox shift in the quinone pool within the electron transport chain, further extending these age-related deficits. Together these changes could lead to an age-related catastrophic decline in energy production and neuronal death.
Keywords: Oxidative phosphorylation; Aging; Mitochondria; Coenzyme Q; NADH; Rotenone;
The onset of NPQ and ΔμH + upon illumination of tobacco plants studied through the influence of mitochondrial electron transport by Pierre Cardol; Rosine De Paepe; Fabrice Franck; Giorgio Forti; Giovanni Finazzi (177-188).
The relationship between the development of photoprotective mechanisms (non-photochemical quenching, NPQ), the generation of the electrochemical proton gradient in the chloroplast and the capacity to assimilate CO2 was studied in tobacco dark-adapted leaves at the onset of illumination with low light. These conditions induce the generation of a transient NPQ, which relaxes in the light in parallel with the activation of the Calvin cycle. Wild-type plants were compared with a CMSII mitochondrial mutant, which lacks the respiratory complex I and shows a delayed activation of photosynthesis. In the mutant, a slower onset of photosynthesis was mirrored by a decreased capacity to develop NPQ. This correlates with a reduced efficiency to reroute electrons at the PSI reducing side towards cyclic electron flow around PSI and/or other alternative acceptor pools, and with a smaller ability to generate a proton motive force in the light. Altogether, these data illustrate the tight relationship existing between the capacity to evacuate excess electrons accumulated in the intersystem carriers and the capacity to dissipate excess photons during a dark to light transition. These data also underline the essential role of respiration in modulating the photoprotective response in dark-adapted leaves, by poising the cellular redox state.
Keywords: Non-photochemical quenching; Respiration; Photosynthesis; Cyclic electron flow; Electrochemical proton gradient;
Impact of mitochondriotropic quercetin derivatives on mitochondria by Lucia Biasutto; Nicola Sassi; Andrea Mattarei; Ester Marotta; Paola Cattelan; Antonio Toninello; Spiridione Garbisa; Mario Zoratti; Cristina Paradisi (189-196).
Mitochondria-targeted polyphenols are being developed with the intent to intervene on the levels of reactive oxygen species (ROS) in mitochondria. Polyphenols being more than just anti-oxidants, the interaction of these derivatives with the organelles needs to be characterised. We have studied the effects of two quercetin derivatives, 3-(4-O-triphenylphosphoniumbutyl)quercetin iodide (Q3BTPI) and its tetracetylated analogue (QTA3BTPI), on the inner membrane aspecific permeability, transmembrane voltage difference and respiration of isolated rat liver mitochondria. While the effects of low concentrations were too small to be reliably defined, when used in the 5–20 μM range these compounds acted as inducers of the mitochondrial permeability transition (MPT), an effect due to pro-oxidant activity. Furthermore, Q3BTPI behaved as an uncoupler of isolated mitochondria, causing depolarisation and stimulating oxygen consumption. When applied to tetramethylrhodamine methyl ester (TMRM)-loaded HepG2 or Jurkat cells uptake of the compounds was predictably associated with a loss of TMRM fluorescence, but there was no indication of MPT induction. A production of superoxide could be detected in some cells upon prolonged incubation of MitoSOX®-loaded cells with QTA3BTPI. The overall effects of these model mitochondriotropic polyphenols may thus differ considerably depending on whether their hydroxyls are protected or not and on the experimental system. In vivo assays will be needed for a definitive assessment of their bioactivities.
Keywords: Mitochondrial permeability transition; Mitochondria-targeted polyphenol; Reactive oxygen species; Transmembrane potential; Uncoupling;
Patients with Leber hereditary optic neuropathy fail to compensate impaired oxidative phosphorylation by Alex Korsten; Irenaeus F.M. de Coo; Liesbeth Spruijt; L. Elly A. de Wit; Hubert J.M. Smeets; Wim Sluiter (197-203).
Ninety-five percent of Leber hereditary optic neuropathy (LHON) patients carry a mutation in one out of three mtDNA-encoded ND subunits of complex I. Penetrance is reduced and more male than female carriers are affected. To assess if a consistent biochemical phenotype is associated with LHON expression, complex I- and complex II-dependent adenosine triphosphate synthesis rates (CI-ATP, CII-ATP) were determined in digitonin-permeabilized peripheral blood mononuclear cells (PBMCs) of thirteen healthy controls and for each primary mutation of a minimum of three unrelated patients and of three unrelated carriers with normal vision and were normalized per mitochondrion (citrate synthase activity) or per cell (protein content). We found that in mitochondria, CI-ATP and CII-ATP were impaired irrespective of the primary LHON mutation and clinical expression. An increase in mitochondrial density per cell compensated for the dysfunctional mitochondria in LHON carriers but was insufficient to result in a normal biochemical phenotype in early-onset LHON patients.
Keywords: Biochemical phenotype; Leber hereditary optic neuropathy; Mitochondrial density; Oxidative phosphorylation; Peripheral blood mononuclear cell;
Incorporation of the chlorophyll d-binding light-harvesting protein from Acaryochloris marina and its localization within the photosynthetic apparatus of Synechocystis sp. PCC6803 by Daping Yang; Yang Qing; Chen Min (204-211).
The gene encoding a chlorophyll d-binding light-harvesting protein, pcbA from Acaryochloris marina (now called as accessory Chlorophyll Binding Protein CBPII) marked with a His-tag was transformed into the genome of Synechocystis PCC6803. Protein gel electrophoresis and western blotting confirmed that this foreign chlorophyll d-binding protein CBPII was expressed and integrated into the thylakoid membrane and bound with chlorophyll a, the only type of chlorophyll present in Synechocystis PCC 6803. Native electrophoresis suggested that CBPII interacts with photosystem II of Synechocystis PCC 6803. Surprisingly, spectral analyses showed that the phycobiliproteins were suppressed in the transformed Synechocystis pcbA +, with a lower ratio of phycobilins to chlorophyll a. These results suggest that there are competitive interactions between the external antenna system of phycobiliproteins and the integral antenna system of chlorophyll-bound protein complexes.
Keywords: Antenna; Chlorophyll; Light-harvesting; Membrane protein; Photosynthesis; Transformation;
Functional analysis of Photosystem I light-harvesting complexes (Lhca) gene products of Chlamydomonas reinhardtii by Milena Mozzo; Manuela Mantelli; Francesca Passarini; Stefano Caffarri; Roberta Croce; Roberto Bassi (212-221).
The outer antenna system of Chlamydomonas reinhardtii Photosystem I is composed of nine gene products, but due to difficulty in purification their individual properties are not known. In this work, the functional properties of the nine Lhca antennas of Chlamydomonas, have been investigated upon expression of the apoproteins in bacteria and refolding in vitro of the pigment–protein complexes. It is shown that all Lhca complexes have a red-shifted fluorescence emission as compared to the antenna complexes of Photosystem II, similar to Lhca from higher plants, but less red-shifted. Three complexes, namely Lhca2, Lhca4 and Lhca9, exhibit emission maxima above 707 nm and all carry an asparagine as ligand for Chl 603. The comparison of the protein sequences and the biochemical/spectroscopic properties of the refolded Chlamydomonas complexes with those of the well-characterized Arabidopsis thaliana Lhcas shows that all the Chlamydomonas complexes have a chromophore organization similar to that of A. thaliana antennas, particularly to Lhca2, despite low sequence identity. All the major biochemical and spectroscopic properties of the Lhca complexes have been conserved through the evolution, including those involved in “red forms” absorption. It has been proposed that in Chlamydomonas PSI antenna size and polypeptide composition can be modulated in vivo depending on growth conditions, at variance as compared to higher plants. Thus, the different properties of the individual Lhca complexes can be functional to adapt the architecture of the PSI–LHCI supercomplex to different environmental conditions.
Keywords: Photosynthesis; Green alga; Chlamydomonas reinhardtii; Light-harvesting complexes; Fluorescence;
Structural characterization of a family of cytochromes c 7 involved in Fe(III) respiration by Geobacter sulfurreducens by P.R. Pokkuluri; Y.Y. Londer; X. Yang; N.E.C. Duke; J. Erickson; V. Orshonsky; G. Johnson; M. Schiffer (222-232).
Periplasmic cytochromes c 7 are important in electron transfer pathway(s) in Fe(III) respiration by Geobacter sulfurreducens. The genome of G. sulfurreducens encodes a family of five 10-kDa, three-heme cytochromes c 7. The sequence identity between the five proteins (designated PpcA, PpcB, PpcC, PpcD, and PpcE) varies between 45% and 77%. Here, we report the high-resolution structures of PpcC, PpcD, and PpcE determined by X-ray diffraction. This new information made it possible to compare the sequences and structures of the entire family. The triheme cores are largely conserved but are not identical. We observed changes, due to different crystal packing, in the relative positions of the hemes between two molecules in the crystal. The overall protein fold of the cytochromes is similar. The structure of PpcD differs most from that of the other homologs, which is not obvious from the sequence comparisons of the family. Interestingly, PpcD is the only cytochrome c 7 within the family that has higher abundance when G. sulfurreducens is grown on insoluble Fe(III) oxide compared to ferric citrate. The structures have the highest degree of conservation around “heme IV”; the protein surface around this heme is positively charged in all of the proteins, and therefore all cytochromes c 7 could interact with similar molecules involving this region. The structures and surface characteristics of the proteins near the other two hemes, “heme I” and “heme III”, differ within the family. The above observations suggest that each of the five cytochromes c 7 could interact with its own redox partner via an interface involving the regions of heme I and/or heme III; this provides a possible rationalization for the existence of five similar proteins in G. sulfurreducens.
Keywords: Cytochrome c 7 structure; Heme-puckering; Multiheme cytochrome; Electron transfer; Fe(III) reduction; Geobacter sulfurreducens;
3,5-diiodo-L-thyronine upregulates rat-liver mitochondrial FoF1-ATP synthase by GA-binding protein/nuclear respiratory factor-2 by Roberto Mangiullo; Antonio Gnoni; Fabrizio Damiano; Luisa Siculella; Franco Zanotti; Sergio Papa; Gabriele V. Gnoni (233-240).
Besides triiodothyronine (T3), 3,5-diiodo-L-thyronine (T2) has been reported to affect mitochondrial bioenergetic parameters. T2 effects have been considered as independent of protein synthesis. Here, we investigated the effect of in vivo chronic T2 administration to hypothyroid rats on liver mitochondrial FoF1-ATP synthase activity and expression. T2 increased state 4 and state 3 oxygen consumption and raised ATP synthesis and hydrolysis, which were reduced in hypothyroid rats. Immunoblotting analysis showed that T2 up-regulated the expression of several subunits (α, β, FoI-PVP and OSCP) of the ATP synthase. The observed increase of β-subunit mRNA accumulation suggested a T2-mediated nuclear effect. Then, the molecular basis underlying T2 effects was investigated. Our results support the notion that the β-subunit of ATP synthase is indirectly regulated by T2 through, at least in part, the activation of the transcription factor GA-binding protein/nuclear respiratory factor-2. These findings provide new insights into the T2 role on bioenergetic mechanisms.
Keywords: 3,5-diiodothyronine; FoF1-ATP synthase; Nuclear respiratory factor-2; Oxidative phosphorylation; Rat-liver mitochondria;
Carotenoid-triggered energy dissipation in phycobilisomes of Synechocystis sp. PCC 6803 diverts excitation away from reaction centers of both photosystems by Marina G. Rakhimberdieva; Irina V. Elanskaya; Wim F.J. Vermaas; Navassard V. Karapetyan (241-249).
Cyanobacteria are capable of using dissipation of phycobilisome-absorbed energy into heat as part of their photoprotective strategy. Non-photochemical quenching in cyanobacteria cells is triggered by absorption of blue-green light by the carotenoid-binding protein, and involves quenching of phycobilisome fluorescence. In this study, we find direct evidence that the quenching is accompanied by a considerable reduction of energy flow to the photosystems. We present light saturation curves of photosystems’ activity in quenched and non-quenched states in the cyanobacterium Synechocystis sp. PCC 6803. In the quenched state, the quantum efficiency of light absorbed by phycobilisomes drops by about 30–40% for both photoreactions—P700 photooxidation in the photosystem II-less strain and photosystem II fluorescence induction in the photosystem I-less strain of Synechocystis. A similar decrease of the excitation pressure on both photosystems leads us to believe that the core–membrane linker allophycocyanin APC-LCM is at or beyond the point of non-photochemical quenching. We analyze 77 K fluorescence spectra and suggest that the quenching center is formed at the level of the short-wavelength allophycocyanin trimers. It seems that both chlorophyll and APC-LCM may dissipate excess energy via uphill energy transfer at physiological temperatures, but neither of the two is at the heart of the carotenoid-binding protein-dependent non-photochemical quenching mechanism.
Keywords: Allophycocyanin; Cyanobacteria; Energy dissipation; Fluorescence quenching; Photosystem;
A quantum mechanics/molecular mechanics study of the tyrosine residue, TyrD, of Photosystem II by Richard Hart; Patrick J. O'Malley (250-254).
QM/MM calculations have been used to monitor the oxidation of the D2-Tyr160, TyrD, residue involved in redox reactions in Photosystem II. The results indicate that in the reduced form the residue is involved in hydrogen bond donation via its phenolic head group to the τ-nitrogen of the neighboring D2-His189 residue. Oxidation to form the radical is accompanied by spontaneous transfer of the phenolic hydrogen to the τ-nitrogen of D2-His189 leading to the formation of a tyrosyl-imidazolium ion complex. Deprotonation of the imidazolium ion leads to the formation of a tyrosyl-imidazole neutral hydrogen-bonded complex. Comparison of calculated and experimental hyperfine coupling tensors and g-tensors suggests that the neutral imidazole complex is formed at physiological temperatures while the imidazolium complex may be stabilized at cryogenic temperatures.
Keywords: Photosystem II; Oxygen Evolving Complex; Tyrosine radical; B3LYP; Electron Transfer;
Active proton leak in mitochondria: A new way to regulate substrate oxidation by Arnaud Mourier; Anne Devin; Michel Rigoulet (255-261).
The main function of mitochondria is energy transduction, from substrate oxidation to the free energy of ATP synthesis, through oxidative phosphorylation. For physiological reasons, the degree of coupling between these two processes must be modulated in order to adapt redox potential and ATP turnover to cellular needs. Such a modulation leads to energy wastage. To this day, two energy wastage mechanisms have been described: the membrane passive proton conductance (proton leak) and the decrease in the coupling efficiency between electrons transfer and proton extrusion at the proton pumps level (redox or proton slipping). In this paper, we describe a new energy wastage mechanism of interest. We show that in isolated yeast mitochondria, the membrane proton conductance is strictly dependent on the external dehydrogenases activity. An increase in their activity leads to an increase in the membrane proton conductance. This proton permeability is independent of the respiratory chain and ATP synthase proton pumps. We propose to name this new mechanism “active proton leak.” Such a mechanism could allow a wide modulation of substrate oxidation in response to cellular redox constraints.
Keywords: Mitochondria; Energy wastage; Proton leak; Dehydrogenase; Yeast;
Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein–protein interaction surface by Susana Frago; Isaias Lans; José A. Navarro; Manuel Hervás; Dale E. Edmondson; Miguel A. De la Rosa; Carlos Gómez-Moreno; Stephen G. Mayhew; Milagros Medina (262-271).
Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP+ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.
Keywords: Flavodoxin; Reduction potential; FMN analogues; Photosystem I; Protein-protein interaction; Electron transfer; Ferredoxin-NADP+ reductase;
Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography by Natalya V. Dudkina; Gert T. Oostergetel; Dagmar Lewejohann; Hans-Peter Braun; Egbert J. Boekema (272-277).
The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at ∼ 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F1 headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.
Keywords: ATP synthase; Electron tomography; Mitochondria; Polytomella;
Structural and functional studies on Ycf12 (Psb30) and PsbZ-deletion mutants from a thermophilic cyanobacterium by Kenji Takasaka; Masako Iwai; Yasufumi Umena; Keisuke Kawakami; Yukari Ohmori; Masahiko Ikeuchi; Yuichiro Takahashi; Nobuo Kamiya; Jian-Ren Shen (278-284).
Ycf12 (Psb30) and PsbZ are two low molecular weight subunits of photosystem II (PSII), with one and two trans-membrane helices, respectively. In order to study the functions of these two subunits from a structural point of view, we constructed deletion mutants lacking either Ycf12 or PsbZ from Thermosynechococcus elongatus, and purified, crystallized and analyzed the structure of PSII dimer from the two mutants. Our results showed that Ycf12 is located in the periphery of PSII, close to PsbK, PsbZ and PsbJ, and corresponded to the unassigned helix X1 reported previously, in agreement with the recent structure at 2.9 Å resolution (A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nat. Struct. Mol. Biol. 16 (2009) 334–342). On the other hand, crystals of PsbZ-deleted PSII showed a remarkably different unit cell constants from those of wild-type PSII, indicating a role of PsbZ in the interactions between PSII dimers within the crystal. This is the first example for a different arrangement of PSII dimers within the cyanobacterial PSII crystals. PSII dimers had a lower oxygen-evolving activity from both mutants than that from the wild type. In consistent with this, the relative content of PSII in the thylakoid membranes was lower in the two mutants than that in the wild type. These results suggested that deletion of both subunits affected the PSII activity, thereby destabilized PSII, leading to a decrease in the PSII content in vivo. While PsbZ was present in PSII purified from the Ycf12-deletion mutant, Ycf12 was present in crude PSII but absent in the finally purified PSII from the PsbZ-deletion mutant, indicating a preferential, stabilizing role of PsbZ for the binding of Ycf12 to PSII. These results were discussed in terms of the PSII crystal structure currently available.
Keywords: Photosystem II; Mutant; Crystal structure; Ycf12; PsbZ; Oxygen evolution;
Regulation of vascular smooth muscle cell bioenergetic function by protein glutathiolation by Bradford G. Hill; Ashlee N. Higdon; Brian P. Dranka; Victor M. Darley-Usmar (285-295).
Protein thiolation by glutathione is a reversible and regulated post-translational modification that is increased in response to oxidants and nitric oxide. Because many mitochondrial enzymes contain critical thiol residues, it has been hypothesized that thiolation reactions regulate cell metabolism and survival. However, it has been difficult to differentiate the biological effects due to protein thiolation from other oxidative protein modifications. In this study, we used diamide to titrate protein glutathiolation and examined its impact on glycolysis, mitochondrial function, and cell death in rat aortic smooth muscle cells. Treatment of cells with diamide increased protein glutathiolation in a concentration-dependent manner and had comparably little effect on protein–protein disulfide formation. Diamide increased mitochondrial proton leak and decreased ATP-linked mitochondrial oxygen consumption and cellular bioenergetic reserve capacity. Concentrations of diamide above 200 μM promoted acute bioenergetic failure and caused cell death, whereas lower concentrations of diamide led to a prolonged increase in glycolytic flux and were not associated with loss of cell viability. Depletion of glutathione using buthionine sulfoximine had no effect on basal protein thiolation or cellular bioenergetics but decreased diamide-induced protein glutathiolation and sensitized the cells to bioenergetic dysfunction and death. The effects of diamide on cell metabolism and viability were fully reversible upon addition of dithiothreitol. These data suggest that protein thiolation modulates key metabolic processes in both the mitochondria and cytosol.
Keywords: Mitochondria; Oxidative stress; Glutathionylation; Glycolysis; Extracellular flux; Reserve capacity;
Visualizing changes in electron distribution in coupled chains of cytochrome bc 1 by modifying barrier for electron transfer between the FeS cluster and heme c 1 by Ewelina Cieluch; Krzysztof Pietryga; Marcin Sarewicz; Artur Osyczka (296-303).
Cytochrome c 1 of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc 1 by modifying heme c 1 redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c 1 which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c 1 back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c 1 and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c 1 without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc 1 revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc 1 exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites.
Keywords: Electron transfer; Cytochrome bc 1; Complex III; Q cycle; Redox midpoint potential; Kinetic model;
Inhibition of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F by carbon monoxide: An FTIR and EPR spectroscopic study by Maria-Eirini Pandelia; Hideaki Ogata; Leslie J. Currell; Marco Flores; Wolfgang Lubitz (304-313).
X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628–11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni–SIa reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni–SCO and Ni–SCOred. At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni–R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni–SCO state results in Ni–SIa that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol−1. In addition, formation of a paramagnetic CO-inhibited state (Ni–CO) was observed that results from the interaction of carbon monoxide with the Ni–L state. It is proposed that the nickel in Ni–CO is in a formal monovalent state (Ni1+).
Keywords: [NiFe] hydrogenase; Desulfovibrio vulgaris; Carbon monoxide inhibition; Spectro-electrochemistry; Rapid-scan FTIR; EPR;
Mitochondrial Complex I decrease is responsible for bioenergetic dysfunction in K-ras transformed cells by Alessandra Baracca; Ferdinando Chiaradonna; Gianluca Sgarbi; Giancarlo Solaini; Lilia Alberghina; Giorgio Lenaz (314-323).
Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.
Keywords: K-ras; Cancer; Mitochondria; Respiration; ATP synthesis; Complex I;
UCP1 ectopically expressed in murine muscle displays native function and mitigates mitochondrial superoxide production by Susanne Keipert; Susanne Klaus; Gerhard Heldmaier; Martin Jastroch (324-330).
Mitochondrial uncoupling in skeletal muscle has raised a major interest as a therapeutic target for treatment of obesity, insulin sensitivity, and age-related disease. These physiological effects could be demonstrated in several mouse models ectopically expressing uncoupling protein 1 (UCP1). Here, we investigated whether UCP1 expressed under the control of the human skeletal actin (HSA) promoter in mouse skeletal muscle can be regulated, and whether it affects mitochondrial superoxide production. We show that the skeletal muscle UCP1 can be fully inhibited by a purine nucleotide (GDP) and reactivated by fatty acids (palmitate). During mitochondrial resting state (State 4), mitochondrial superoxide production is about 76% lower in transgenic mice. We suggest that this reduction is due to uncoupling activity as the administration of GDP restores superoxide production to wildtype levels. Our study confirms native behaviour of UCP1 in skeletal muscle and demonstrates beneficial effects on prevention of mitochondrial reactive oxygen species production which may reduce age-related deleterious processes.
Keywords: Uncoupling protein; Mitochondrial respiration; Superoxide production; Skeletal muscle; Proton conductance;