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BBA - Bioenergetics (v.1710, #2-3)
Commentary on: ‘Old and new data, new issues: The mitochondrial Δ ψ’ by H. Tedeschi by David G. Nicholls (pp. 63-65).
A recent review [H.Tedeschi, Old and New Data, New Issues: the Mitochondrial Δ ψ, Biochim. Biophys. Acta, Bioenerg. (2005)] has questioned the validity of experiments performed more than 30 years ago that provided the first quantitative estimates of the protonmotive force generated by mitochondria. This commentary explains that the review author's confusion stems from ignoring the data in these papers on the trans-membrane pH gradient.
Keywords: Mitochondria; Chemiosmotic theory; Membrane potential; Protonmotive force
Reply to David Nicholls' response by Henry Tedeschi (pp. 66-66).
All evidence presently available shows that there is no metabolically dependent electric potential (Δ Ψ) across the mitochondrial inner membrane. The data supporting this statement are documented in our review article and in the present communication. The evidence purported to show a Δ Ψ results from a miscalculation or the mistaken assumption that the distribution of lipophilic cations is in response to a Δ Ψ.
Keywords: Mitochondria; Mitochondrial membrane potential; Oxidative-phosphorylation; Chemiosmosis
Application of classical molecular dynamics for evaluation of proton transfer mechanism on a protein by Ran Friedman; Esther Nachliel; Menachem Gutman (pp. 67-77).
Proton transfer reactions on surfaces are prevalent in biology, chemistry and physics. In the present study, we employed classical Molecular Dynamics simulations to search for the presence of transient configurations that enable proton transfer, or proton sharing, between adjacent carboxylate groups on the protein surface. The results demonstrate that, during random fluctuations of the residues on the surface, there are repeated situations in which nearby carboxylates either share a common proton through a hydrogen bond, or are connected by a few water molecules that form conducting networks. These networks do not extend out of the common Coulomb cage of the participating residues and the lifetimes of the bridged structures are sufficiently long to allow passage of a proton between the carboxylates. The detection of domains capable of supporting a rapid proton transfer on a protein supports the notion that clusters of carboxylates are the operative elements of proton collecting antennae, as in bacteriorhodopsin, cytochrome c oxidase or the photosynthetic reaction center.
Keywords: Abbreviations; CPMD; Car-Parrinello Molecular Dynmics; MD; Molecular Dynamics; PDB; Protein Data Bank; RMSD; Root Mean Square DeviationMolecular dynamics; Proton transfer; Protein surface
Mitochondrial dysfunction associated with cardiac ischemia/reperfusion can be attenuated by oxygen tension control. Role of oxygen-free radicals and cardiolipin by G. Petrosillo; N. Di Venosa; F.M. Ruggiero; M. Pistolese; D. D'Agostino; E. Tiravanti; T. Fiore; G. Paradies (pp. 78-86).
Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocites. Mitochondrial respiration is an important source of ROS generation and hence a potential contributor to cardiac reperfusion injury. Appropriate treatment strategy could be particularly useful to limit this ROS generation and associated mitochondrial dysfunction. In the present study, we examined the effect of lowering the oxygen tension, at the onset of the reperfusion, on various parameters of mitochondrial bioenergetics in rat heart tissue. After isolation of mitochondria from control, ischemic, normoxic and hypoxic reperfused rat heart, various bioenergetic parameters were evaluated such as rates of mitochondrial oxygen consumption, complex I and complex III activity, H2O2 production and in addition, the degree of lipid peroxidation, cardiolipin content and cardiolipin oxidation. We found that normoxic reperfusion significantly altered all these mitochondrial parameters, while hypoxic reperfusion had a protective effect attenuating these alterations. This effect appears to be due, at least in part, to a reduction of mitochondrial ROS generation with subsequent preservation of cardiolipin integrity, protection of mitochondrial function and improvement of post-ischemic hemodynamic function of the heart.
Keywords: Mitochondrial respiration; Complexes I and III activity; ROS generation; Cardiolipin oxidation; Rat heart hypoxic reperfusion
Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes by Stefan Dröse; Alexander Galkin; Ulrich Brandt (pp. 87-95).
The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of ΔpH and Δ ψ. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-β-d-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,ΔpH and Δ ψ, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.
Keywords: Abbreviations; ACMA; 9-amino-6-chloro-2-methoxyacridine; DBQ; n; -decylubiquinone; LM; n; -lauryl-β-; d; -maltoside; ETH 157; N; ,; N; ′-dibenzyl-; N; ,; N; ′-diphenyl-1,2-phenylenedioxydiacetamide; FCCP; carbonyl-cyanide-; p; -trifluoro-methoxy-phenylhydrazone; HAR; hexaammineruthenium(III)-chloride; OG; n; -octyl-β-; d; -glucopyranoside; Q-1; 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone; TX-100; Triton X-100Mitochondria; Complex I; Reconstitution; H; +; -pumping; Yarrowia lipolytica
The mitochondrial channel VDAC has a cation-selective open state by Evgeny Pavlov; Sergey M. Grigoriev; Laurent M. Dejean; Chaninah L. Zweihorn; Carmen A. Mannella; Kathleen W. Kinnally (pp. 96-102).
The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance “open� state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.
Keywords: Abbreviations; E; rev; equilibrium reversal potential; IMS; intermembrane space; TOM; translocase outer membrane; VDAC; voltage dependent anion-selective channelVDAC; Mitochondrial porin; Ion selectivity; Mitochondria; Patch clamp; Gating
The interaction of 5′-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates by Sung-Kun Kim; Afroza Rahman; Jeremy T. Mason; Masakazu Hirasawa; Richard C. Conover; Michael K. Johnson; Myroslawa Miginiac-Maslow; Eliane Keryer; David B. Knaff; Thomas Leustek (pp. 103-112).
APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation–reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/ C. reinhardtii thioredoxin h1 adduct is −280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe–4S] center by either APS or the competitive inhibitor 5′-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe–4S] cluster.
Keywords: Abbreviations; APS; 5′-adenylylsulfate; BsAPR; Bacillus subtilis; APS reductase; CNBr; cyanogen bromide; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); DTT; dithiothreitol; GRX; glutaredoxin; IPTG; isopropyl-β-; d; -thiogalactopyranoside; MALDI-TOF; Matrix-Assisted Laser Desorption Ionization-Time Of Flight; PaAPR; Pseudomonas aeruginosa; APS reductase; PAGE; polyacrylamide gel electrophoresis; RR; resonance Raman; SDS; sodium dodecylsulfate; TRX; thioredoxin; TRX C35A; the C35A mono-cysteine variant of; Escherichia coli; thioredoxin; TRX; h; 1C39S; the C39S mono-cysteine variant of; Chlamydomonas reinhardtii; thioredoxin; h; 1; WT; wild type Pseudomonas aeruginosa; APS reductase; Thioredoxin; Iron–sulfur cluster; Disulfide-linked protein complex
Physiological role of rhodoquinone in Euglena gracilis mitochondria by Norma A. Castro-Guerrero; Ricardo Jasso-Chávez; Rafael Moreno-Sánchez (pp. 113-121).
Rhodoquinone (RQ) participates in fumarate reduction under anaerobiosis in some bacteria and some primitive eukaryotes. Euglena gracilis, a facultative anaerobic protist, also possesses significant rhodoquinone-9 (RQ9) content. Growth under low oxygen concentration induced a decrease in cytochromes and ubiquinone-9 (UQ9) content, while RQ9 and fumarate reductase (FR) activity increased. However, in cells cultured under aerobic conditions, a relatively high RQ9 content was also attained together with significant FR activity. In addition, RQ9 purified from E. gracilis mitochondria was able to trigger the activities of cytochrome bc1 complex, bc1-like alternative component and alternative oxidase, although with lower efficiency (higher Km, lower Vm) than UQ9. Moreover, purified E. gracilis mitochondrial NAD+-independentd-lactate dehydrogenase (d-iLDH) showed preference for RQ9 as electron acceptor, whereasl-iLDH and succinate dehydrogenase preferred UQ9. These results indicated a physiological role for RQ9 under aerobiosis and microaerophilia in E. gracilis mitochondria, in which RQ9 mediates electron transfer betweend-iLDH and other respiratory chain components, including FR.
Keywords: Microaerophilia; Rhodoquinone redox state; Fumarate reductase; Pyridine nucleotide-independent; d; -lactate dehydrogenase; Cytochrome; bc; 1; complex; Alternative oxidase