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Metals in Redox Biology Annelie Mollbrink, Charlotte Lindfors, Anna Joe and Caitlin McAtee Metals involved in hydroxyl radical formation ͘OH • • • • Iron Copper Chromium Vanadium (Fe) (Cu) (Cr) (V) The Fenton Reaction 1) Fe2+ + H2O2 Fe3+ + ͘OH + OH- Ferrous iron catalyzes the formation of hydroxyl radicals from hydrogen peroxidase The Iron Catalyzed Haber-Weiss Reaction • O2- reduces the iron salt: • Fe3+ + O2- Fe2+ + O2 • The Fenton reaction: • Fe2+ + H2O2 Fe3+ + ͘OH + OH- • Net = the Haber-Weiss reaction: • O2- + H2O2 O2 + ͘OH + OHIron salt as catalyst Non Transition Metals can also induce oxidative stress • Lead (Pb) • Arsenic (As) • Indirect? • GSH-levels? • Impaired defense How do mammalian cells import/export metals? 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 Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) Metal transport protein 1 (MTP1) Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) Metal transport protein 1 (MTP1) Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) Metal transport protein 1 (MTP1) Iron exporter Ferroportin (FPN) Iron-regulated transporter 1 (IREG1) Metal transport protein 1 (MTP1) Iron absorption by the enterocyte Iron transport in the hepatocyte How is metal content regulated in the mitochondria? 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 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) molybdenum cofactor Enzyme-specific chaperones play a central role in the biogenesis of multisubunit molybdoenzymes by coordinating subunits assembly and molybdenum cofactor insertion. Johnson et al., J. Biol. Chem.,1980 Kisker et al., 1997 Cell Nitrate reductase -Moco is 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. FE-S CLUSTER 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. Heme group 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 Kranz et al., 1998 Molecular Microbiology 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. Metalloproteins: Aconitases • 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). http://employees.csbsju.edu/hjakubowski/classes/ch112/pathways-charts/tca1.gif 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 Microbiology 2 954-966 (2004). 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 Aconitase mRNA Binding Activity Citric Acid Cycle Gene Iron-regulated Bacterioferritin Gene S: Unliganded Sepharose 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. Microbiology 145 3069-3079 (1999). • 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 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 Cancer 5:14 (2006). Thank You