scholarly journals A Fraction of Yeast Cu,Zn-Superoxide Dismutase and Its Metallochaperone, CCS, Localize to the Intermembrane Space of Mitochondria

2001 ◽  
Vol 276 (41) ◽  
pp. 38084-38089 ◽  
Author(s):  
Lori A. Sturtz ◽  
Kerstin Diekert ◽  
Laran T. Jensen ◽  
Roland Lill ◽  
Valeria Cizewski Culotta
Keyword(s):  
Superoxide Dismutase ◽  
Intermembrane Space

scholarly journals Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration

2017 ◽  
Vol 37 (20) ◽  
Author(s):  
Courtney J. Banks ◽  
Nathan W. Rodriguez ◽  
Kyle R. Gashler ◽  
Rushika R. Pandya ◽  
Jeffrey B. Mortenson ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Cell Survival ◽  
Metabolic Regulation ◽  
Posttranslational Regulation ◽  
Signaling Proteins ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Defective Mutant ◽  
Respiratory Complex I ◽  
Survival Signaling

ABSTRACT In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival.

scholarly journals Absence of the iron-containing superoxide dismutase in mitochondria from mustard (Brassica campestris)

1981 ◽  
Vol 195 (1) ◽  
pp. 229-233 ◽  
Author(s):  
M L Salin ◽  
S M Bridges
Keyword(s):  
Superoxide Dismutase ◽  
Brassica Campestris ◽  
Polyacrylamide Gel ◽  
Superoxide Dismutases ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Mature Leaves ◽  
Etiolated Seedlings

Mitochondria were isolated from mature leaves as well as etiolated seedlings of Brassica campestris (mustard), a eukaryote previously shown to possess the iron-containing isoenzyme of superoxide dismutase. On the basis of KCN- and H2O2-sensitivity, and on polyacrylamide-gel analysis, only the cuprozinc and mangano superoxide dismutases were found in mitochondria. The iron-containing enzyme was absent. The mangano enzyme was found in the mitochondrial matrix, whereas the cuprozinc enzyme appeared to be localized in the intermembrane space.

scholarly journals Mitochondrial Protein Oxidation in Yeast Mutants Lacking Manganese-(MnSOD) or Copper- and Zinc-containing Superoxide Dismutase (CuZnSOD)

2004 ◽  
Vol 279 (50) ◽  
pp. 51817-51827 ◽  
Author(s):  
Kristin M. O'Brien ◽  
Reinhard Dirmeier ◽  
Marcella Engle ◽  
Robert O. Poyton
Keyword(s):  
Oxidative Stress ◽  
Superoxide Dismutase ◽  
Stationary Phases ◽  
Mitochondrial Protein ◽  
Protein Carbonylation ◽  
Intermembrane Space ◽  
Flight Mass Spectrometry ◽  
Specific Proteins ◽  
Mitochondrial Intermembrane Space ◽  
Yeast Mutants

Saccharomyces cerevisiaeexpresses two forms of superoxide dismutase (SOD): MnSOD, encoded bySOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded bySOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type orsodmutant strains. Moreover, mitochondrial protein carbonylation levels insod1,sod2, andsod1sod2mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither MnSOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly insod1andsod1sod2mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated insod1andsod2mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in asod1mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in asod2mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that MnSOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.

Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space

2001 ◽  
Vol 353 (2) ◽  
pp. 411-416 ◽  
Author(s):  
Derick HAN ◽  
Everett WILLIAMS ◽  
Enrique CADENAS
Keyword(s):  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Respiratory Chain ◽  
Superoxide Anion ◽  
Complex Iii ◽  
Spin Adduct ◽  
Intermembrane Space ◽  
Epr Signal ◽  
Mitochondrial Respiratory

It has been generally accepted that superoxide anion generated by the mitochondrial respiratory transport chain are vectorially released into the mitochondrial matrix, where they are converted to hydrogen peroxide through the catalytic action of Mn-superoxide dismutase. Release of superoxide anion into the intermembrane space is a controversial topic, partly unresolved by the reaction of superoxide anion with cytochrome c, which faces the intermembrane space and is present in this compartment at a high concentration. This study was aimed at assessing the topological site(s) of release of superoxide anion during respiratory chain activity. To address this issue, mitoplasts were prepared from isolated mitochondria by digitonin treatment to remove portions of the outer membrane along with portions of cytochrome c. EPR analysis in conjunction with spin traps of antimycin-supplemented mitoplasts revealed the formation of a spin adduct of superoxide anion. The EPR signal was (i) abrogated by superoxide dismutase, (ii) decreased competitively by exogenous ferricytochrome c and (iii) broadened by the membrane-impermeable spin-broadening agent chromium trioxalate. These results confirm the production and release of superoxide anion towards the cytosolic side of the inner mitochondrial membrane. In addition, co-treatment of mitoplasts with myxothiazol and antimycin A, resulting in an inhibition of the oxidation of ubiquinol to ubisemiquinone, abolished the EPR signal, thus suggesting that ubisemiquinone autoxidation at the outer site of the complex-III ubiquinone pool is a pathway for superoxide anion formation and subsequent release into the intermembrane space. The generation of superoxide anion towards the intermembrane space requires consideration of the mitochondrial steady-state values for superoxide anion and hydrogen peroxide, the decay pathways of these oxidants in this compartment and the implications of these processes for cytosolic events.

scholarly journals Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

2007 ◽  
Vol 405 (1) ◽  
pp. 173-179 ◽  
Author(s):  
Pedro Iñarrea ◽  
Hadi Moini ◽  
Derick Han ◽  
Daniel Rettori ◽  
Ignacio Aguiló ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Thioredoxin Reductase ◽  
Oxidative Modification ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
Cytochrome C Release ◽  
Mitochondrial Energy Metabolism ◽  
Mitochondrial Functions ◽  
The Impact ◽  
Exogenous H2o2

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 μM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

scholarly journals Cmc1p Is a Conserved Mitochondrial Twin CX9C Protein Involved in Cytochrome c Oxidase Biogenesis

2008 ◽  
Vol 28 (13) ◽  
pp. 4354-4364 ◽  
Author(s):  
Darryl Horn ◽  
Hassan Al-Ali ◽  
Antoni Barrientos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Superoxide Dismutase ◽  
Cytochrome C Oxidase ◽  
Mitochondrial Enzymes ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Copper Chaperones ◽  
Copper Trafficking ◽  
Mitochondrial Inner Membrane ◽  
Copper Incorporation

ABSTRACT Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.

scholarly journals Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa

1980 ◽  
Vol 187 (2) ◽  
pp. 321-328 ◽  
Author(s):  
Landis E. A. Henry ◽  
Richard Cammack ◽  
Jean-Paul Schwitzguebel ◽  
John M. Palmer ◽  
David O. Hall
Keyword(s):  
Superoxide Dismutase ◽  
Neurospora Crassa ◽  
Analytical Ultracentrifugation ◽  
Intracellular Localization ◽  
Total Activity ◽  
Superoxide Dismutases ◽  
Intermembrane Space ◽  
Matrix Space ◽  
Isolation And Characterization ◽  
High Yields

1. Neurospora crassa was found to contain two distinct superoxide dismutases. 2. Most of the activity is associated with the cytosolic fraction and was shown to be the Cu/Zn-containing form of the protein. 3. Mitochondria isolated from Neurospora crassa showed two distinct superoxide dismutases: a cyanide-sensitive Cu/Zn-containing protein and a cyanide-insensitive form which probably contains manganese. 4. Localization experiments, using selective marker enzymes and digitonin fractionation, indicated that the cyanide-sensitive form is localized in the intermembrane space, whereas the cyanide-insensitive form is confined to the mitochondrial matrix space. 5. The cytosolic Cu/Zn-containing superoxide dismutase was isolated in high yields and extensively characterized by using e.p.r. spectroscopy, isoelectric focusing and analytical ultracentrifugation. 6. E.p.r. spectroscopy was used to monitor changes in the copper environment of the native protein after the addition of a number of potential inhibitors and after high-pH treatment. 7. Both of the cyanide-sensitive Cu/Zn-containing enzymes (cytosolic and mitochondrial) appeared to have identical properties which in turn were different from the cyanide-insensitive enzyme. 8. It is probable that the cyanide-insensitive enzyme was not previously detected, owing to its low amount (less than 10% of the total activity), greater lability than the cyanide-sensitive enzyme and the necessity of obtaining a mitochondrial-enriched fraction before its isolation.

scholarly journals Redox activation of mitochondrial intermembrane space Cu,Zn-superoxide dismutase

2005 ◽  
Vol 387 (1) ◽  
pp. 203-209 ◽  
Author(s):  
Pedro IÑARREA ◽  
Hadi MOINI ◽  
Daniel RETTORI ◽  
Derick HAN ◽  
Jesús MARTÍNEZ ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Outer Membrane ◽  
Alkylating Agents ◽  
Liver Mitochondria ◽  
Oxidative Modification ◽  
Mitochondrial Outer Membrane ◽  
Rat Liver Mitochondria ◽  
Intermembrane Space ◽  
H2o2 Treatment ◽  
Mitochondrial Intermembrane Space

The localization of Cu,Zn-superoxide dismutase in the mitochondrial intermembrane space suggests a functional relationship with superoxide anion (O2•−) released into this compartment. The present study was aimed at examining the functionality of Cu,Zn-superoxide dismutase and elucidating the molecular basis for its activation in the intermembrane space. Intact rat liver mitochondria neither scavenged nor dismutated externally generated O2•−, unless the mitochondrial outer membrane was disrupted selectively by digitonin. The activation of the intermembrane space Cu,Zn-superoxide dismutase following the disruption of mitochondrial outer membrane was largely inhibited by bacitracin, an inhibitor of protein disulphide-isomerase. Thiol alkylating agents, such as N-methylmaleimide or iodoacetamide, decreased intermembrane space Cu,Zn-superoxide dismutase activation during, but not after, disruption of the outer membrane. This inhibitory effect was overcome by exposing mitochondria to low micromolar concentrations of H2O2 before disruption of the outer membrane in the presence of the alkylating agents. Moreover, H2O2 treatment alone enabled intact mitochondria to scavenge externally generated O2•−. These findings suggest that intermembrane space Cu,Zn-superoxide dismutase is inactive in intact mitochondria and that an oxidative modification of its critical thiol groups is necessary for its activation.

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