BBA - Bioenergetics (v.1807, #11)
Editorial Board (i).
Cardiac mitochondria in heart failure: Normal cardiolipin profile and increased threonine phosphorylation of complex IV by Mariana Rosca; Paul Minkler; Charles L. Hoppel (1373-1382).
Mitochondrial dysfunction is a major contributor in heart failure (HF). We investigated whether the decrease in respirasome organization reported by us previously in cardiac mitochondria in HF is due to changes in the phospholipids of the mitochondrial inner membrane or modifications of the subunits of the electron transport chain (ETC) complexes. The contents of the main phospholipid species, including cardiolipin, as well as the molecular species of cardiolipin were unchanged in cardiac mitochondria in HF. Oxidized cardiolipin molecular species were not observed. In heart mitochondria isolated from HF, complex IV not incorporated into respirasomes exhibits increased threonine phosphorylation. Since HF is associated with increased adrenergic drive to cardiomyocytes, this increased protein phosphorylation might be explained by the involvement of cAMP-activated protein kinase. Does the preservation of cAMP-induced phosphorylation changes of mitochondrial proteins or the addition of exogenous cAMP have similar effects on oxidative phosphorylation? The usage of phosphatase inhibitors revealed a specific decrease in complex I-supported respiration with glutamate. In saponin-permeabilized cardiac fibers, pre-incubation with cAMP decreases oxidative phosphorylation due to a defect localized at complex IV of the ETC inter alia. We propose that phosphorylation of specific complex IV subunits decreases oxidative phosphorylation either by limiting the incorporation of complex IV in supercomplexes or by decreasing supercomplex stability.► Heart failure causes no changes in phospholipid composition of cardiac mitochondria. ► Complex IV not assembled into respirasomes shows higher threonine phosphorylation. ► Complex IV phosphorylation may attenuate OXPHOS via respirasome assembly/stability.
Keywords: Mitochondria; Supercomplex; Oxidative phosphorylation; Cardiolipin; Protein threonine phosphorylation;
The acidic domain of cytochrome c 1 in Paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc 1 complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c 552 by Michela Castellani; Jeffrey Havens; Thomas Kleinschroth; Francis Millett; Bill Durham; Francesco Malatesta; Bernd Ludwig (1383-1389).
The cytochrome bc 1 complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c 1 subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c 552.► The role of a unique acidic domain in the c 1 subunit of complex III is addressed. ► In a deletion variant, neither kCAT nor KM are changed. ► Laser flash photolysis indicates same substrate interaction in the variant complex.
Keywords: Electron transfer; Cytochrome bc 1 complex; Complex III; Cytochrome bc 1 Δac complex; Laser flash photolysis; Steady state kinetic;
Mitochondrial NADH:ubiquinone oxidoreductase (complex I) in eukaryotes: A highly conserved subunit composition highlighted by mining of protein databases by Pierre Cardol (1390-1397).
Complex I (NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain. Compared to its bacterial counterpart which encompasses 14–17 subunits, mitochondrial complex I has almost tripled its subunit composition during evolution of eukaryotes, by recruitment of so-called accessory subunits, part of them being specific to distinct evolutionary lineages. The increasing availability of numerous broadly sampled eukaryotic genomes now enables the reconstruction of the evolutionary history of this large protein complex. Here, a combination of profile-based sequence comparisons and basic structural properties analyses at the protein level enabled to pinpoint homology relationships between complex I subunits from fungi, mammals or green plants, previously identified as "lineage-specific" subunits. In addition, homologs of at least 40 mammalian complex I subunits are present in representatives of all major eukaryote assemblages, half of them having not been investigated so far (Excavates, Chromalveolates, Amoebozoa). This analysis revealed that complex I was subject to a phenomenal increase in size that predated the diversification of extant eukaryotes, followed by very few lineage-specific additions/losses of subunits. The implications of this subunit conservation for studies of complex I are discussed.► More than 40 complex I subunit homologs are identified in all eukaryote assemblages. ► These subunits were massively recruited at the stem branch of all extant eukaryotes. ► Very few proteins remain candidates for the specificity in lineages or species. ► A conserved enzyme supports the study of complex I in alternative model organisms.
Keywords: Mitochondrial NADH:ubiquinone oxidoreductase; Profile-based search; Eukaryote evolution; Database mining; Complex I subunit composition;
The cytochrome bd respiratory oxygen reductases by Vitaliy B. Borisov; Robert B. Gennis; James Hemp; Michael I. Verkhovsky (1398-1413).
Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme–copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.► Physiological functions and genetics of cytochrome bd terminal oxidases reviewed. ► Structural and catalytic properties of cytochromes bd discussed. ► Phylogenetic analysis of cytochromes bd and their homologues presented.
Keywords: Metabolism; Molecular bioenergetics; Oxidoreduction; Bacterial physiology; Microbe; Disease;
[Fe4S4]- and [Fe3S4]-cluster formation in synthetic peptides by Alessandra Hoppe; Maria-Eirini Pandelia; Wolfgang Gärtner; Wolfgang Lubitz (1414-1422).
[Fe4S4]- and [Fe3S4]-clusters are ubiquitous iron–sulfur motifs in biological systems. The [Fe3S4] composition is, however, of much lower natural abundance than the more typical [Fe4S4]-clusters. In the present study formation of [Fe3S4]-clusters has been examined using chemically synthesized model peptides consisting of 33 amino acids (maquettes). Maquettes are effective synthetic analogs for metal–ion binding sites, allowing for a facile modification of the primary coordination sphere of iron–sulfur clusters. Maquettes have been designed following the [FeS]-cluster-binding motif of dimethyl sulfoxide reductase subunit B (DmsB) from Escherichia coli that carries a [Fe4S4]-cluster, but incorporates a [Fe3S4]-cluster instead upon mutation of one of the coordinating cysteines. The time-dependent formation of iron–sulfur clusters and the effects of exchanging selected amino acids in the model peptides, known to regulate the [Fe3S4] to [Fe4S4] ratio in the DmsB protein, were monitored by UV/Vis- and EPR-spectroscopy. Exchange of cysteines within the conserved CxxCxxC motif has a much stronger effect on cluster formation and stoichiometry than the exchange of a coordinating external cysteine. Amino acid exchange in the binding motif shows a dependence of the cluster stoichiometry on the amino acid side chain. Formation of [Fe3S4]-clusters in maquettes is less favorable compared to native proteins. The [Fe3S4] moiety appears to be a rather transient species towards the more stable (final) incorporation of a [Fe4S4]-cluster. Results are best described by an assembly mechanism that considers a successive coordination of the iron atoms by the peptide, rather than incorporation of an already pre-formed mercaptoethanol-coordinated [Fe4S4]-cluster.► Formation of [Fe3S4] and [Fe4S4]-clusters using chemically synthesized peptides. ► [Fe3] to [Fe4] stoichiometry depends on the aminoacid exchange within binding motif. ► Assembly mechanism considering successive coordination of the Fe ions by the peptide.
Keywords: Iron–sulfur clusters; Maquette; Peptide synthesis; Cluster assembly; EPR spectroscopy;
Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700 by Eberhard Schlodder; Martin Hussels; Marianne Çetin; Navassard V. Karapetyan; Marc Brecht (1423-1431).
Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700+. However, the efficiency of the fluorescence quenching by oxidized P700+ was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5 K: ≅ 719 nm; emission maximum at 5 K: ≅ 740 nm) was found to be strongly quenched by P700+ similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760 nm at 5 K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques.► Quenching by P700+. ► Red/reddest antenna states. ► Reassignment of absorption bands.
Keywords: Photosystem I; Fluorescence quenching; Single molecule spectroscopy; Long-wavelength antenna chlorophyll; Arthrospira platensis; Thermosynechococcus elongatus;
SDH mutations in cancer by Chiara Bardella; Patrick J. Pollard; Ian Tomlinson (1432-1443).
The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis.► Germline loss-of-function mutations in any of the SDH genes cause HPGL/PCC syndrome. ► Germline loss-of-function mutations in SDHAF2 gene cause HPGL/PCC syndrome. ► SDH deficiency induces pseudohypoxia. ► Succinate accumulation might inhibits α-ketoglutarate-dependent dioxygenases.
Keywords: Succinate dehydrogenase; Mitochondrial tumor suppressor genes; Hereditary paraganglioma-phaechromocytoma syndrome;
The medium reorganization energy for the charge transfer reactions in proteins by Lev I. Krishtalik (1444-1456).
A low static dielectric permittivity of proteins causes the low reorganization energies for the charge transfer reactions inside them. This reorganization energy does not depend on the pre-existing intraprotein electric field. The charge transferred inside the protein interacts with its aqueous surroundings; for many globular proteins, the effect of this surroundings on the reorganization energy is comparable with the effect of reorganization of the protein itself while for the charge transfer in the middle of membrane the aqueous phase plays a minor role. Reorganization energy depends strongly on the system considered, and hence there is no sense to speak on the “protein reorganization energy” as some permanent characteristic parameter. We employed a simple algorithm for calculation of the medium reorganization energy using the numerical solution of the Poisson–Boltzmann equation. Namely, the reaction field energy was computed in two versions — all media having optical dielectric permittivity, and all the media with the static one; the difference of these two quantities gives the reorganization energy. We have calculated reorganization energies for electron transfer in cytochrome c, various ammine-ruthenated cytochromes c, azurin, ferredoxin, cytochrome c oxidase, complex of methylamine dehydrogenase with amicyanin, and for proton transfer in α-chymotrypsin. It is shown that calculation of the medium reorganization energy can be a useful tool in analysis of the mechanisms of the charge transfer reactions in proteins.► Reorganization energy does not depend on the intraprotein permanent electric field. ► “Protein reorganization energy” is not a constant typical of all proteins. ► We propose a simple algorithm for calculation of the medium reorganization energy. ► We demonstrated the usefulness of this algorithm for 8 charge transfer reactions. ► We quantified the effect of reorganization of the protein's aqueous surroundings.
Keywords: Protein reorganization energy; Cytochrome c; Azurin; Ferredoxin; Cytochrome c oxidase; Interprotein electron transfer;
Photochemical characterization of a novel fungal rhodopsin from Phaeosphaeria nodorum by Ying Fan; Peter Solomon; Richard P. Oliver; Leonid S. Brown (1457-1466).
Eukaryotic microbial rhodopsins are widespread bacteriorhodopsin-like proteins found in many lower eukaryotic groups including fungi. Many fungi contain multiple rhodopsins, some significantly diverged from the original bacteriorhodopsin template. Although few fungal rhodopsins have been studied biophysically, both fast-cycling light-driven proton pumps and slow-cycling photosensors have been found. The purpose of this study was to characterize photochemically a new subgroup of fungal rhodopsins, the so-called auxiliary group. The study used the two known rhodopsin genes from the fungal wheat pathogen, Phaeosphaeria nodorum. One of the genes is a member of the auxiliary group while the other is highly similar to previously characterized proton-pumping Leptosphaeria rhodopsin. Auxiliary rhodopsin genes from a range of species form a distinct group with a unique primary structure and are located in carotenoid biosynthesis gene cluster. Amino acid conservation pattern suggests that auxiliary rhodopsins retain the transmembrane core of bacteriorhodopsins, including all residues important for proton transport, but have unique polar intramembrane residues. Spectroscopic characterization of the two yeast-expressed Phaeosphaeria rhodopsins showed many similarities: absorption spectra, conformation of the retinal chromophore, fast photocycling, and carboxylic acid protonation changes. It is likely that both Phaeosphaeria rhodopsins are proton-pumping, at least in vitro. We suggest that auxiliary rhodopsins have separated from their ancestors fairly recently and have acquired the ability to interact with as yet unidentified transducers, performing a photosensory function without changing their spectral properties and basic photochemistry.► For the first time, member of a new group of fungal rhodopsins was characterized. ► Bioinformatic analysis suggests its carotenoid-related photosensory function. ► Two distinct rhodopsins of Phaeosphaeria have similar absorption maxima. ► Both rhodopsins have photochemical properties indicative of proton pumping.
Keywords: Photosensory transduction; Membrane proteins; Fungal rhodopsins; Retinal proteins; Photochemical cycle; Proton pumping;
The stereochemistry of chlorophyll-c 3 from the haptophyte Emiliania huxleyi: The (132 R)-enantiomers of chlorophylls-c are exclusively selected as the photosynthetically active pigments in chromophyte algae by Tadashi Mizoguchi; Yuki Kimura; Taichi Yoshitomi; Hitoshi Tamiaki (1467-1473).
Chlorophyll(Chl)-c pigments in algae, diatoms and some prokaryotes are characterized by the fully conjugated porphyrin π-system as well as the acrylate residue at the 17-position. The precise structural characterization of Chl-c 3 from the haptophyte Emiliania huxleyi was performed. The conformations of the π-conjugated peripheral substituents, the 3-/8-vinyl, 7-methoxycarbonyl and 17-acrylate moieties were evaluated, in a solution, using nuclear Overhauser enhancement correlations and molecular modeling calculations. The rotation of the 17-acrylate residue was considerably restricted, whereas the other three substituents readily rotated at ambient temperature. Moreover, the stereochemistry at the 132-position was determined by combination of chiral high-performance liquid chromatography (HPLC) with circular dichroism (CD) spectroscopy. Compared with the CD spectra of the structurally related, synthetic (132 R)- and (132 S)-protochlorophyllide(PChlide)-a, naturally occurring Chl-c 3 had exclusively the (132 R)-configuration. To elucidate this natural selection of a single enantiomer, we analyzed the three major Chl-c pigments (Chl-c 1, c 2 and c 3) in four phylogenetically distinct classes of Chl-c containing algae, i.e., heterokontophyta, dinophyta, cryptophyta and haptophyta using chiral HPLC. All the photosynthetic organisms contained only the (132 R)-enantiomerically pure Chls-c, and lacked the corresponding enantiomeric (132 S)-forms. Additionally, Chl-c 2 was found in all the organisms as the common Chl-c. These results throw a light on the biosynthesis as well as photosynthetic function of Chl-c pigments: Chl-c 2 is derived from 8-vinyl-PChlide-a by dehydrogenation of the 17-propionate to acrylate residues as generally proposed, and the (132 R)-enantiomers of Chls-c function as photosynthetically active, light-harvesting pigments together with the principal Chl-a and carotenoids.► The stereochemistry of chlorophyll(Chl)-c 3 from a haptophyte was characterized. ► Chiral HPLC and CD spectra showed natural Chl-c 3 to be exclusively (132 R)-enantiomer. ► Four phylogenetically distinct classes of algae containing Chls-c were also examined. ► All the photosynthetic organisms contained only (132 R)-enantiomerically pure Chls-c.
Keywords: Chlorophyll-c; Chiral HPLC; Enantiomer; Chromophyte algae; Light-harvesting complexes;
Characteristics of the turnover of uncoupling protein 3 by the ubiquitin proteasome system in isolated mitochondria by Shona A. Mookerjee; Martin D. Brand (1474-1481).
Uncoupling protein 3 (UCP3) is implicated in mild uncoupling and the regulation of mitochondrial ROS production. We previously showed that UCP3 turns over rapidly in C2C12 myoblasts, with a half-life of 0.5–4 h, and that turnover can be reconstituted in vitro. We show here that rapid degradation of UCP3 in vitro in isolated brown adipose tissue mitochondria required the 26S proteasome, ubiquitin, ATP, succinate to generate a high membrane potential, and a pH of 7.4 or less. Ubiquitin containing lysine-48 was both necessary and sufficient to support UCP3 degradation, implying a requirement for polyubiquitylation at this residue. The 20S proteasome did not support degradation. UCP3 degradation was prevented by simultaneously blocking matrix ATP generation and import, showing that ATP in the mitochondrial matrix was required. Degradation did not appear to require a transmembrane pH gradient, but was very sensitive to membrane potential: degradation was halved when membrane potential decreased 10–20 mV from its resting value, and was not significant below about 120 mV. We propose that matrix ATP and a high membrane potential are needed for UCP3 to be polyubiquitylated through lysine-48 of ubiquitin and exported to the cytosolic 26S proteasome, where it is de-ubiquitylated and degraded.► The rapid turnover of UCP3 was characterized in vitro. ► UCP3 degradation requires matrix ATP and a high membrane potential. ► K48-linked polyubiquitylation is necessary and sufficient to promote UCP3 degradation. ► UCP3 degradation is highly sensitive to small changes in membrane potential.
Keywords: Uncoupling protein; UCP3; Mitochondrion; In vitro; Ubiquitin; 26S proteasome;
The complex of cytochrome c and cytochrome c peroxidase: The end of the road? by Alexander N. Volkov; Peter Nicholls; Jonathan A.R. Worrall (1482-1503).
Cytochrome c (Cc) and cytochrome c peroxidase (CcP) form a physiological complex in the inter-membrane space of yeast mitochondria, where CcP reduces hydrogen peroxide to water using the electrons provided by ferrous Cc. The Cc–CcP system has been a popular choice of study of interprotein biological electron transfer (ET) and in understanding dynamics within a protein–protein complex. In this review we have charted seven decades of research beginning with the discovery of CcP and leading to the latest functional and structural work, which has clarified the mechanism of the intermolecular ET, addressed the putative functional role of a low-affinity binding site, and identified lowly-populated intermediates on the energy landscape of complex formation. Despite the remarkable attention bestowed on this complex, a number of outstanding issues remain to be settled on the way to a complete understanding of Cc–CcP interaction.Display Omitted►We review the protein complex between cytochrome c and cytochrome c peroxidase. ►X-ray crystallographic studies of the 1:1 complex are highlighted. ►The ‘encounter state’ of the complex determined by NMR is illustrated. ►The electron transfer mechanisms for the 1:1 and 2:1 complexes are discussed.
Keywords: Cytochrome c; Cytochrome c peroxidase; Protein complex; Electron transfer; NMR spectroscopy; X-ray crystallography;