Transcript Metals in Redox Biology - University of Nebraska–Lincoln

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