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Metals in Redox Biology

Metals in Redox Biology. Annelie Mollbrink, Charlotte Lindfors, Anna Joe and Caitlin McAtee. Metals involved in hydroxyl radical formation ͘OH. Iron (Fe) Copper (Cu) Chromium ( Cr ) Vanadium (V) . The Fenton Reaction. Fe 2+ + H 2 O 2  Fe 3+ + ͘OH + OH -

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Metals in Redox Biology

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  1. Metals in RedoxBiology Annelie Mollbrink, Charlotte Lindfors, Anna Joe and CaitlinMcAtee

  2. Metalsinvolved in hydroxylradical formation ͘OH • Iron (Fe) • Copper (Cu) • Chromium (Cr) • Vanadium (V)

  3. The Fenton Reaction • Fe2+ + H2O2 Fe3+ + ͘OH + OH- Ferrousironcatalyzes the formation of hydroxylradicals from hydrogen peroxidase

  4. 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

  5. Non TransitionMetalscanalsoinduceoxidative stress • Lead (Pb) • Arsenic (As) • Indirect? • GSH-levels? • Impaireddefense

  6. How do mammalian cells import/export metals?

  7. 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

  8. Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) • Metal transport protein 1 (MTP1)

  9. Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) • Metal transport protein 1 (MTP1)

  10. Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) • Metal transport protein 1 (MTP1)

  11. Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) • Metal transport protein 1 (MTP1)

  12. Iron absorption by the enterocyte

  13. Iron transport in the hepatocyte

  14. How is metal content regulated in the mitochondria?

  15. 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

  16. 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)

  17. molybdenum cofactor Enzyme-specific chaperonesplay a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion. Johnson et al., J. Biol. Chem.,1980 Kiskeret al., 1997 Cell Nitrate reductase -Mocois labile and oxygen-sensitive -cofactor is deeply buried within the holo-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.

  18. FE-S CLUSTER MOLECLUAR CHAPERONES FOR FE-S CLUSTER ASSEMBLY -Iscpathway : contains HscA and HscBproteins 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 HscAATPaseactivity. -HscAbinds 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 IscUbinding to HscAB and subsequent ATP hydrolysis might alter the interaction of this cysteine with clusters on IscU.

  19. Heme group There are three systemsthat 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 Kranzet al., 1998 Molecular Microbiology

  20. Copper Metallochaperones: a shuttle protein for delivering copper Cox17: delivers copper to cytochromeoxydase in mitochondria Ccs: to cytosolic superoxide dismutase Atx1: to multicopperoxidase in Golgi Copper trafficking pathway in euk.

  21. Metalloproteins:Aconitases • Converts Citrate to Isocitrate • Senses: • Oxidative Stress • Iron Starvation References: J. Green and M.S. Paget. Nature Reviews Microbiology2 954-966 (2004). Y. Tang and J.R. Guest. Microbiology145 3069-3079 (1999). K.K. Singh et. Al. Molecular Cancer5:14 (2006). X.J. Chen et. Al. Science307 714-717 (2005). http://employees.csbsju.edu/hjakubowski/classes/ch112/pathways-charts/tca1.gif

  22. 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 Apo-aconitase • Binding to mRNA can stabilize transcript or inhibit translation J. Green and M.S. Paget. Nature Reviews Microbiology2 954-966 (2004).

  23. 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

  24. Aconitase mRNA Binding Activity Citric Acid Cycle Gene Iron-regulated Bacterioferritin Gene • 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 S: UnligandedSepharose As: AcnA-Sepharose Bs: AcnB-Sepharose T: Total Unfractionated RNA M: Standard Markers UTRs that were synthesized in vitro by T3 RNA pol + primers Lanes 1 and 4: No protein Lane 2: 12 µg apo-AcnA Lane 3: 24 µg apo-AcnA Lane 5: 3 µg apo-AcnB Lane 6: 5 µg apo-AcnB Y. Tang and J.R. Guest. Microbiology145 3069-3079 (1999).

  25. 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 NC BPH: Benign Prostatic Hyperplasia PIN: Prostatic Intraepithelial Neoplastic Foci K.K. Singh et. Al. Molecular Cancer5:14 (2006).

  26. Thank You

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