Metals in Redox Biology
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Transcript of Metals in Redox Biology
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Metals in Redox Biology
Annelie Mollbrink, Charlotte Lindfors, Anna Joe and Caitlin McAtee
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Metals involved in hydroxyl radical formation OH ͘
• Iron (Fe)• Copper (Cu)• Chromium (Cr)• Vanadium (V)
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The Fenton Reaction
1) Fe2+ + H2O2 Fe3+ + OH + OH ͘ -
Ferrous iron catalyzes the formation of hydroxyl radicals from hydrogen peroxidase
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The Iron Catalyzed Haber-Weiss Reaction
• O2- reduces the iron salt:
• The Fenton reaction:
• Net = the Haber-Weiss reaction:
• Fe3+ + O2- Fe2
+ + O2
• Fe2+ + H2O2 Fe3+ + OH + OH ͘ -
• O2- + H2O2 O2 + OH + OH- ͘
Iron salt as catalyst
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Non Transition Metalscan also induce oxidative stress
• Lead (Pb)• Arsenic (As)
• Indirect?• GSH-levels?• Impaired defense
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How do mammalian cells import/export metals?
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Proteins involved in iron transport
• Heme carrier protein 1 (HCP1)
• Divalent metal transporter 1 (DMT1)
• Duodenal cytochrome b (Dcytb) – ferrireductase, reduces ferric Fe3+ to ferrous Fe2+
• Ferroportin (FPN) – iron exporter
• Hephestin – ferroxidase, oxidase Fe2+ to Fe3+
• Ferritin
• Hemosiderin
• Transferrin
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Iron exporterFerroportin
(FPN)Iron-regulated transporter 1
(IREG1)Metal transport protein 1
(MTP1)
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Iron exporterFerroportin
(FPN)Iron-regulated transporter 1
(IREG1)Metal transport protein 1
(MTP1)
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Iron exporterFerroportin
(FPN)Iron-regulated transporter 1
(IREG1)Metal transport protein 1
(MTP1)
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Iron exporterFerroportin
(FPN)Iron-regulated transporter 1
(IREG1)Metal transport protein 1
(MTP1)
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Iron absorption by the enterocyte
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Iron transport in the hepatocyte
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How is metal content regulated in the mitochondria?
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Metal ion pools within mitochondria
• Iron, Copper, Zinc• Two pools of iron • Bioavailable
– Iron pool expanded in yeast lacking Mtm1, Grx5, Ssq1 Sod2 inactivation
• Less bioavailable– High accumulation of mitochondrial iron in cells lacking Yfh1 no Sod2
inactivation– Iron is insoluble Fe (III)
• Factors controlling distribution of iron into these two pools are unknown
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How are different metal cofactors incorporated into metalloenzymes?
Metalloenzymes: enzymes that have a tightly bound metal ion
Metal ions are normally incorporated into the enzymes during enzyme synthesis
-Directly incorporated into their cognate sites on proteins: copper ions
-Become part of prosthetic groups, cofactors or complexes prior to insertion of theses moieties into target proteins: molybdenum cofactor (MOCO), Fe–S clusters, heme group
Hausinger et al., ASM News (2001)
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molybdenum cofactor
Johnson et al., J. Biol. Chem.,1980Kisker et al., 1997 Cell
-Moco is labile and oxygen-sensitive
-cofactor is deeply buried within theholo-enzyme
-Molybdenum cofactor insertion is a tightly controlled process that involves specific interactions between the proteins that promote cofactor delivery, enzyme-specific chaperones, and the apoenzyme.
Nitrate reductase
Enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion.
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MOLECLUAR CHAPERONES FOR FE-S CLUSTER ASSEMBLY
-Isc pathway : contains HscA and HscB proteins homologues of the DnaJ and DnaK molecular chaperones.
-This interaction is enhanced by HscB, which can bind to both IscU and HscA, leading to a strong enhancement of the intrinsic HscA ATPase activity.
-HscA binds to a conserved stretch of amino acids (LPPVK) in IscU. The LPPVK motif is located near a highly conserved Cys (Cys106) residue in IscU, so IscU binding to HscAB and subsequent ATP hydrolysis might alter the interaction of this cysteine with clusters on IscU.
FE-S CLUSTER
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Heme group
Kranz et al., 1998 Molecular Microbiology
There are three systems that deliver the heme group to the apoprotein. maintain in the reduced state both the iron atom in the heme molecule and cysteine residues on the protein.
R. capsulatus Cytochrome C2+ heme
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Copper
Metallochaperones: a shuttle protein for delivering copper Cox17: delivers copper to cytochrome oxydase in mitochondria Ccs: to cytosolic superoxide dismutase Atx1: to multicopper oxidase in Golgi
Copper trafficking pathway in euk.
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Metalloproteins:Aconitases
http://employees.csbsju.edu/hjakubowski/classes/ch112/pathways-charts/tca1.gif
• Converts Citrate to Isocitrate
• Senses:• Oxidative
Stress• Iron Starvation
References:J. Green and M.S. Paget. Nature Reviews Microbiology 2 954-966 (2004).Y. Tang and J.R. Guest. Microbiology 145 3069-3079 (1999).K.K. Singh et. Al. Molecular Cancer 5:14 (2006).X.J. Chen et. Al. Science 307 714-717 (2005).
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Aconitase Function
• Fe-S clusters• High Iron:– [4Fe-4S] clusters– Clusters are catalysts
• Low Iron/Oxidative Stress:– [3Fe-4S] clusters– Clusters
disassembled, Catalytic activity lost
• Binding to mRNA can stabilize transcript or inhibit translation
J. Green and M.S. Paget. Nature Reviews Microbiology 2 954-966 (2004).
Apo-aconitase
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Aconitases
• E. coli– AcnA: Stress-induced stationary-phase enzyme• 53% identical to human iron regulatory protein 1
– AcnB: Citric acid cycle enzyme (exponential phase)• More sensitive to oxidative stress/Fe starvation• 15-17% identical to AcnA
• Mammalian– M-Aconitase: mitochondrial
• Yeast– Aco1p: Shown to play a role in mitochondrial DNA
stability
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Aconitase mRNA Binding Activity
• Aconitases bind specifically to acn 3’ UTRs
• A5 and B5: AcnA KD≈8 µM, AcnB KD≈1.3 µM
• Ox stress: Activity down ~60%, but protein exp. Increases 3-4 fold
Citric Acid Cycle Gene Iron-regulated Bacterioferritin Gene
S: Unliganded SepharoseAs: AcnA-SepharoseBs: AcnB-SepharoseT: Total Unfractionated RNAM: Standard Markers
Lanes 1 and 4: No proteinLane 2: 12 µg apo-AcnALane 3: 24 µg apo-AcnALane 5: 3 µg apo-AcnBLane 6: 5 µg apo-AcnB
Y. Tang and J.R. Guest. Microbiology 145 3069-3079 (1999).
UTRs that were synthesized in vitro by T3 RNA pol + primers
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Aconitase in Prostate Cancer
• Normal Prostate Cells: Citrate Producing• Malignant Prostate Cells: Citrate
Oxidizing• Immunohistochemistry shows levels of
m-Aconitase are similar in all prostate tissues
• Accumulation of zinc in normal prostate cells could be inhibiting m-Aconitase
BPH: Benign Prostatic HyperplasiaPIN: Prostatic Intraepithelial Neoplastic Foci
NC
K.K. Singh et. Al. Molecular Cancer 5:14 (2006).
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Thank You