Oxidative Phosphorylation

Modular kinetic analysis reveals differences in Cd 2+ and Cu 2+ ion-induced impairment of oxidative phosphorylation in liver Jolita Ciapaite 1 , Zita Nauciene 1,2 , Rasa Baniene 2 , Marijke J. Wagner 3 , Klaas Krab 3 and Vida Mildaziene 1 1 Centre of Environmental Research, Faculty of Natural Sciences, Vytautas Magnus University, Kaunas, Lithuania 2 Institute for Biomedical Research, Kaunas Medical University, Lithuania 3 Department of Molecular Cell Physiology, Institute for Molecular Cell Biology, VU University, Amsterdam, The Netherlands Many pollutants, even at low effective concentrations, can harm living organisms by weakening their ability to cope with long-term environmental challenges. At excess amounts, the heavy metals cadmium and copper are toxic and carcinogenic [1]. The ability of cadmium and copper to accumulate in the bones, liver and kid- neys determines their toxicity. Their deleterious effects can be ameliorated to some extent by binding to metallothionein [2]. Cellular dysfunction induced by cadmium and copper is thought to involve alterations Keywords cadmium and copper; lipid peroxidation; metabolic control analysis; modular kinetic analysis; oxidative phosphorylation Correspondence J. Ciapaite, Centre of Environmental Research, Faculty of Natural Sciences, Vytautas Magnus University, Vileikos 8, LT-44404 Kaunas, Lithuania Fax: +370 37 327916 Tel: +370 37 455193 E-mail: jolita.ciapaite@falw.vu.nl (Received 29 January 2009, revised 18 April 2009, accepted 5 May 2009) doi:10.1111/j.1742-4658.2009.07084.x Impaired mitochondrial function contributes to copper- and cadmium- induced cellular dysfunction. In this study, we used modular kinetic analy- sis and metabolic control analysis to assess how Cd 2+ and Cu 2+ ions affect the kinetics and control of oxidative phosphorylation in isolated rat liver mitochondria. For the analysis, the system was modularized in two ways: (a) respiratory chain, phosphorylation and proton leak; and (b) coen- zyme Q reduction and oxidation, with the membrane potential (Dw) and fraction of reduced coenzyme Q as the connecting intermediate, respec- tively. Modular kinetic analysis results indicate that both Cd 2+ and Cu 2+ ions inhibited the respiratory chain downstream of coenzyme Q. Moreover, Cu 2+ , but not Cd 2+ ions stimulated proton leak kinetics at high Dw val- ues. Further analysis showed that this difference can be explained by Cu 2+ ion-induced production of reactive oxygen species and membrane lipid peroxidation. In agreement with modular kinetic analysis data, metabolic control analysis showed that Cd 2+ and Cu 2+ ions increased control of the respiratory and phosphorylation flux by the respiratory chain module (mainly because of an increase in the control exerted by cytochrome bc 1 and cytochrome c oxidase), decreased control by the phosphorylation module and increased negative control of the phosphorylation flux by the proton leak module. In summary, we showed that there is a subtle differ- ence in the mode of action of Cd 2+ and Cu 2+ ions on the mitochondrial function, which is related to the ability of Cu 2+ ions to induce reactive oxygen species production and lipid peroxidation. Abbreviations C J P i , flux control coefficient, quantifying the control of phosphorylation flux J P by module i; C J R i , flux control coefficient, quantifying the control of respiratory flux J R by module i; CoQ, coenzyme Q; COX, cytochrome c oxidase; DCPIP, 2,6-dichlorophenolindophenol; J L, proton leak flux; Oxidative Phosphorylation Oxidative Phosphorylation Bởi: OpenStaxCollege You have just read about two pathways in glucose catabolism—glycolysis and the citric acid cycle—that generate ATP Most of the ATP generated during the aerobic catabolism of glucose, however, is not generated directly from these pathways Rather, it is derived from a process that begins with moving electrons through a series of electron transporters that undergo redox reactions This causes hydrogen ions to accumulate within the matrix space Therefore, a concentration gradient forms in which hydrogen ions diffuse out of the matrix space by passing through ATP synthase The current of hydrogen ions powers the catalytic action of ATP synthase, which phosphorylates ADP, producing ATP Electron Transport Chain The electron transport chain ([link]) is the last component of aerobic respiration and is the only part of glucose metabolism that uses atmospheric oxygen Oxygen continuously diffuses into plants; in animals, it enters the body through the respiratory system Electron transport is a series of redox reactions that resemble a relay race or bucket brigade in that electrons are passed rapidly from one component to the next, to the endpoint of the chain where the electrons reduce molecular oxygen, producing water There are four complexes composed of proteins, labeled I through IV in [link], and the aggregation of these four complexes, together with associated mobile, accessory electron carriers, is called the electron transport chain The electron transport chain is present in multiple copies in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes 1/8 Oxidative Phosphorylation The electron transport chain is a series of electron transporters embedded in the inner mitochondrial membrane that shuttles electrons from NADH and FADH2 to molecular oxygen In the process, protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water Complex I To start, two electrons are carried to the first complex aboard NADH This complex, labeled I, is composed of flavin mononucleotide (FMN) and an iron-sulfur (Fe-S)containing protein FMN, which is derived from vitamin B2, also called riboflavin, is one of several prosthetic groups or co-factors in the electron transport chain A prosthetic group is a non-protein molecule required for the activity of a protein Prosthetic groups are organic or inorganic, non-peptide molecules bound to a protein that facilitate its function; prosthetic groups include co-enzymes, which are the prosthetic groups of enzymes The enzyme in complex I is NADH dehydrogenase and is a very large protein, containing 45 amino acid chains Complex I can pump four hydrogen ions across the membrane from the matrix into the intermembrane space, and it is in this way that the hydrogen ion gradient is established and maintained between the two compartments separated by the inner mitochondrial membrane Q and Complex II Complex II directly receives FADH2, which does not pass through complex I The compound connecting the first and second complexes to the third is ubiquinone (Q) The Q molecule is lipid soluble and freely moves through the hydrophobic core of the membrane Once it is reduced, (QH2), ubiquinone delivers its electrons to the next complex in the electron transport chain Q receives the electrons derived from NADH from complex I and the electrons derived from FADH2 from complex II, including succinate dehydrogenase This enzyme and FADH2 form a small complex that delivers electrons directly to the electron transport chain, bypassing the first complex Since 2/8 Oxidative Phosphorylation these electrons bypass and thus not energize the proton pump in the first complex, fewer ATP molecules are made from the FADH2 electrons The number of ATP molecules ultimately obtained is directly proportional to the number of protons pumped across the inner mitochondrial membrane Complex III The third complex is composed of cytochrome b, another Fe-S protein, Rieske center (2Fe-2S center), and cytochrome c proteins; this complex is also called cytochrome oxidoreductase Cytochrome proteins have a prosthetic group of heme The heme molecule is similar to the heme in hemoglobin, but it carries electrons, not oxygen As a result, the iron ion at its core is reduced and oxidized as it passes the electrons, fluctuating between different oxidation states: Fe++ (reduced) and Fe+++ (oxidized) The heme molecules in the cytochromes have slightly different characteristics due to the effects of the different proteins binding them, giving slightly different characteristics to each complex Complex III pumps protons through the membrane and passes its electrons to cytochrome c for transport to the fourth complex of proteins and enzymes (cytochrome c is the acceptor of electrons from Q; however, whereas Q carries pairs of electrons, cytochrome c can accept only one at a time) Complex IV The fourth ...RESEARC H Open Access Patients with chronic fatigue syndrome performed worse than controls in a controlled repeated exercise study despite a normal oxidative phosphorylation capacity Ruud CW Vermeulen 1* , Ruud M Kurk 1 , Frans C Visser 1 , Wim Sluiter 2 , Hans R Scholte 3 Abstract Background: The aim of this study was to investigate the possibility that a decreased mitochondrial ATP synthesis causes muscular and mental fatigue and plays a role in the pathophysiology of the chronic fatigue syndrome (CFS/ME). Methods: Female patients (n = 15) and controls (n = 15) performed a cardiopulmonary exercise test (CPET) by cycling at a continuously increased work rate till maximal exertion. The CPET was repeated 24 h later. Before the tests, blood was taken for the isolation of peripheral blood mononuclear cells (PBMC), which were processed in a special way to preserve their oxidative phosphorylation, which was tested later in the presence of ADP and phosphate in permeabilized cells with glutamate, malate and malonate plus or minus the complex I inhibitor rotenone, and succinate with rotenone plus or minus the complex II inhibitor malonate in order to measure the ATP production via Complex I and II, respectively. Plasma CK was determined as a surrogate measure of a decreased oxidative phosphorylation in muscle, since the previous finding that in a group of patients with external ophthalmoplegia the oxygen consumption by isolated muscle mitochondria correlated negatively with plasma creatine kinase, 24 h after exercise. Results: At both exercise tests the patients reached the anaerobic threshold and the maximal exercise at a much lower oxygen consumption than the controls and this worsened in the second test. This implies an increase of lactate, the product of anaerobic glycolysis, and a decrease of the mitochondrial ATP production in the patients. In the past this was also found in patients with defects in the mitochondrial oxidative phosphorylation. However the oxidative phosphorylation in PBMC was similar in CFS/ME patients and controls. The plasma creatine kinase levels before and 24 h after exercise were low in patients and controls, suggesting normality of the muscular mitochondrial oxidative phosphorylation. Conclusion: The decrease in mitochondrial ATP synthesis in the CFS/ME patients is not caused by a defect in the enzyme complexes catalyzing oxidative phosphorylation, but in another factor. Trial registration: Clinical trials registration number: NL16031.040.07. Background Chronic fatigue syndrome/myalgic encephalopathy (CFS/ ME) as a syndrome was defined in consensus meetings by Fukuda et al [1]. Fatigue was the major criterion in the definition. It was described as suddenly o ccurring, not explained, not caused by exercise, insufficiently relieved by rest and causing a major reduction in physical capa- city. The additional symptoms of the syndrome were headache, pain in muscles and joints, unrefreshing sleep, postexertional malaise, sore throat, painful lymph glands and insuf ficien t concentration. The combination of fati- gue and four or more of the additional symptoms lasting at least for 6 months, suf ficed for the diagnosis of CFS/ ME. The main exclusion c rite rion for CFS/ME, was the * Correspondence: rv@cvscentrum.nl 1 CFS/ME and Pain Research Center Amsterdam, 1 CHAPTER 4: THE CITRIC ACID AND OXIDATIVE PHOSPHORYLATION INTERNATIONAL UNIVERSITY SCHOOL OF BIOTECHNOLOGY BIOCHEMISTRY 2 Learning objectives 1. To understand the intermediates in CAC 2. The ATPs produce in CAC 3. The CO2 is released in CAC 4. The electrons are transferred in the electron transport chain 5. The oxidative phosphorylation 3 Content Citric acid cycle Introduction Cellular location Catabolism Anabolism & catabolism Sources of acetyl-CoA Fatty acid –aminoacid- monosaccharides 4 Content Reactants & products Cyclical reaction pathway Fate of acetyl CoA carbon regulation: inhibition Energetics Anaerobic Anaplerotic reactions Oxidative phoshorylation Introduction Mitochondrial anatomy Shuttle system Introduction to the transport chain Comlex I- comlex II- comlex III and Cytochrome C- comlex IV 5 Citric Acid Cycle INTRODUCTION The citric acid cycle is a central metabolic pathway that completes the oxidative degradation of fatty acids, amino acids, and monosaccharides. During aerobic catabolism, these biomolecules are broken down to smaller molecules that ultimately contribute to a cell’s energetic or molecular needs. 6 FIG. 01: Citric acid cycle is the central metabolic pathway 7 INTRODUCTION Early metabolic steps, including glycolysis and the activity of the pyruvate dehydrogenase complex, yield a two-carbon fragment called an acetyl group, which is linked to a large cofactor known as coenzyme A (or CoA). It is during the citric acid cycle that acetyl-CoA is oxidized to the waste product, carbon dioxide, along with the reduction of the cofactors NAD+ and ubiquinone. 8 FIG. 02: Early catabolic pathway 9 FIG. 03: Citric acid cycle is the central metabolic pathway 10 INTRODUCTION The citric acid cycle serves two main purposes: 1.To increase the cell’s ATP-producing potential by generating a reduced electron carriers such as NADH and reduced ubiquinone; and 2.To provide the cell with a variety of metabolic precursors. [...]... carriers and their role in coupling the citric acid cycle to downstream reactions that produce ATP; Describe the amphibolic character of the citric acid cycle; and Understand the reactions that replenish citric acid cycle intermediates 13 CELLULAR LOCATION Both prokaryotic and eukaryotic cells use the citric acid cycle to help meet their energetic and molecular needs In respiring prokaryotes, the citric acid. ..FIG 04: Main purposes of CAC 11 INTRODUCTION Be able to describe the sources of acetyl groups that enter the citric acid cycle; Trace the conversion of substrates to products through each of the citric acid cycle’s eight reactions and understand how flux through the cycle is regulated; Understand the energetic output of the citric acid cycle; 12 INTRODUCTION Describe the role of the reduced... and degrading molecules are considered amphibolic Amphi is a Greek prefix meaning both 23 FIG 10: The CAC is amphibolic 24 SOURCES OF ACETYL-CoA 25 The skeleton drawings of the monosaccharide glucose, the fatty acid palmitic acid, Int. J. Med. Sci. 2008, 5 143 International Journal of Medical Sciences ISSN 1449-1907 www.medsci.org 2008 5(3):143-151 © Ivyspring International Publisher. All rights reserved Research Paper OXIDATIVE PHOSPHORYLATION: Kinetic and Thermodynamic Correlation between Electron Flow, Proton Translocation, Oxygen Consumption and ATP Synthesis under Close to In Vivo Concentrations of Oxygen Baltazar D. Reynafarje 1 and Jorge Ferreira 2 1. Johns Hopkins University School of Medicine, Department of Biological Chemistry, Baltimore, Maryland 21205, USA. 2. Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 70000 Santiago-7, Chile. Correspondence to: Jorge Ferreira, Programa de Farmacología Molecular y Clínica, Facultad de Medicina, Universidad de Chile, Independencia 1027, Casilla 70000 Santiago-7, Chile. E-mail: jferreir@med.uchile.cl, Fax: +56 2 735 5580, Tel: +56 2 978 6069. Received: 2008.04.15; Accepted: 2008.06.05; Published: 2008.06.09 For the fist time the mitochondrial process of oxidative phosphorylation has been studied by determining the extent and initial rates of electron flow, H + translocation, O 2 uptake and ATP synthesis under close to in vivo concentrations of oxygen. The following novel results were obtained. 1) The real rates of O 2 uptake and ATP synthesis are orders of magnitude higher than those observed under state-3 metabolic conditions. 2) The phosphorylative process of ATP synthesis is neither kinetically nor thermodynamically related to the respiratory process of H + ejection. 3) The ATP/O stoichiometry is not constant but varies depending on all, the redox potential ( Δ E h ), the degree of reduction of the membrane and the relative concentrations of O 2 , ADP, and protein. 4) The free energy of electron flow is not only used for the enzymatic binding and release of substrates and products but fundamentally for the actual synthesis of ATP from ADP and Pi. 5) The concentration of ADP that produces half-maximal responses of ATP synthesis (EC 50 ) is not constant but varies depending on both Δ E h and O 2 concentration. 6) The process of ATP synthesis exhibits strong positive catalytic cooperativity with a Hill coefficient, n, of ~3.0. It is concluded that the most important factor in determining the extent and rates of ATP synthesis is not the level of ADP or the proton gradient but the concentration of O 2 and the state of reduction and/or protonation of the membrane. Key words: Energy transduction, proton gradient, free energy of electron flow and ATP synthesis Introduction The central and most important aspect of the mitochondrial process of energy transduction in aerobic organisms is the mechanism by which the free energy of respiration is transformed into the chemical of ATP. Since the formulation of the chemiosmotic hypothesis [1], it is firmly believed that the processes of electron flow, H + ejection, O 2 uptake and ATP synthesis are always kinetically and thermodynamically related. Thus, it is common practice to evaluate the number of molecules of ATP formed per atom of oxygen consumed by simply evaluating the H + /O ratio [2], or by solely determining the amount of O 2 consumed under state-3 metabolic conditions [3]. In this context, it is also stated that (a) “electrons do not flow from fuel molecules to O 2 unless ATP needs to be synthesized” [4], and (b) the respiratory energy of electron flow is only used to bind ADP and Pi and to release the spontaneously formed ATP from the catalytic sites of the synthase [5-8]. It is also asserted that the control of electron flux by O 2 is minimal and that in a way not specified the phosphorylative process of ATP synthesis controls the flow of electrons through the mitochondrial respiratory chain [9]. We provide here evidence that the process of ATP synthesis does not depend on the vectorial ejection of H + and the Genome Biology 2005, 6:R11 comment reviews reports deposited research refereed research interactions information Open Access 2005Tripoliet al.Volume 6, Issue 2, Article R11 Research Comparison of the oxidative phosphorylation (OXPHOS) nuclear genes in the genomes of Drosophila melanogaster, Drosophila pseudoobscura and Anopheles gambiae Gaetano Tripoli * , Domenica D'Elia † , Paolo Barsanti * and Corrado Caggese * Addresses: * University of Bari, DAPEG Section of Genetics, via Amendola 165/A, 70126 Bari, Italy. † CNR, Institute of Biomedical Technology, Section of Bari, via Amendola 122/D, 70126 Bari, Italy. Correspondence: Corrado Caggese. E-mail: caggese@biologia.uniba.it © 2005 Tripoli et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Evolution of oxidative phosphorylation genes in Diptera<p>An analysis of nuclear-encoded oxidative phosphorylation genes in <it>Drosophila</it> and <it>Anopheles</it> reveals that pairs of duplicated genes have strikingly different expression patterns.</p> Abstract Background: In eukaryotic cells, oxidative phosphorylation (OXPHOS) uses the products of both nuclear and mitochondrial genes to generate cellular ATP. Interspecies comparative analysis of these genes, which appear to be under strong functional constraints, may shed light on the evolutionary mechanisms that act on a set of genes correlated by function and subcellular localization of their products. Results: We have identified and annotated the Drosophila melanogaster, D. pseudoobscura and Anopheles gambiae orthologs of 78 nuclear genes encoding mitochondrial proteins involved in oxidative phosphorylation by a comparative analysis of their genomic sequences and organization. We have also identified 47 genes in these three dipteran species each of which shares significant sequence homology with one of the above-mentioned OXPHOS orthologs, and which are likely to have originated by duplication during evolution. Gene structure and intron length are essentially conserved in the three species, although gain or loss of introns is common in A. gambiae. In most tissues of D. melanogaster and A. gambiae the expression level of the duplicate gene is much lower than that of the original gene, and in D. melanogaster at least, its expression is almost always strongly testis-biased, in contrast to the soma-biased expression of the parent gene. Conclusions: Quickly achieving an expression pattern different from the parent genes may be required for new OXPHOS gene duplicates to be maintained in the genome. This may be a general evolutionary mechanism for originating phenotypic changes that could lead to species differentiation. Background The accessibility of whole-genome sequence data for several organisms, together with the development of efficient compu- ter-based search tools, has revolutionized modern biology, allowing in-depth comparative analysis of genomes [1-4]. In many cases, comparisons among species at various levels of divergence have helped to define protein-coding genes, rec- ognize nonfunctional genes, and find regulatory sequences and other functional elements in the genome. When applied to a set of genes correlated by function and/or subcellular Published: 31 January 2005 Genome Biology 2005, 6:R11 Received: 24 September 2004 Revised: 8 December 2004 Accepted: 7 January 2005 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2005/6/2/R11 R11.2 Genome Biology 2005, Volume 6, Issue 2, Article R11 Tripoli et al. http://genomebiology.com/2005/6/2/R11 Genome Biology 2005, 6:R11 localization of their products, intra- and interspecies compar- ative analyses can be especially ... of sunlight in the process of photophosphorylation Recall that the production of ATP using the process of chemiosmosis in mitochondria is called oxidative phosphorylation The overall result of... electrons directly to the electron transport chain, bypassing the first complex Since 2/8 Oxidative Phosphorylation these electrons bypass and thus not energize the proton pump in the first complex,... without the aid of ion channels Similarly, hydrogen ions in the matrix space can only 3/8 Oxidative Phosphorylation pass through the inner mitochondrial membrane through an integral membrane