Copper Metabolism

1
Copper Metabolism

• Copper is an essential trace metal that is
  required for
• protection against radicals
• electron transport chain
• pigmentation
• neuropeptide peptide hormone processing
• collagen maturation

However, free Cu ions are highly toxic to both
eukaryotic and prokaryotic cells because of their
ability to generate reactive oxygen species
Copper levels are controlled in cells primarily
by metal responsive transcription factors that
regulate the expression levels of metal chelating
proteins called metallothioneins
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Targets of Copper Transport
The protein targets for Cu are located primarily
in the extracellular matrix or plasma
However, two of the most important targets
(cytochrome oxidase and superoxide dismutase) are
intracellular enzymes
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Copper content of human blood
Human plasma contains about 100 µg of Cu per 100
ml It is distributed in various proteins and low
molecular weight complexes
Under some circumstances as much as 90 of copper
transport is carried out by ceruloplasmin
4
Normal copper balance
About 60 of each mg of Cu taken in our diet is
absorbed The same amount is typically lost on a
daily basis Some fraction of the total Cu being
distributed is also reabsorbed
5
Copper Distribution in Cells
Copper typically binds to transport proteins (P1)
for cellular uptake Once in the cells Cu can be
shuttled to target proteins (P2 P4) either in
the cytosol or the mitochondria Cu binding to
other proteins (P3) is used for transport to
other cells or to metallothionein (MT) for
excretion
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Pathways of Copper distribution
Dietary intake and excretion of Cu is greater
than that of Fe even though there is much less Cu
in the human body Most of the Cu resides in
tissues, with only small amounts in blood, liver
and kidneys
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Copper Uptake species comparison
Different species can use different approaches to
control copper uptake, with different systems in
place to respond to either low copper or high
copper availability
Different metal responsive transcription factors
(Mac1, Ace1, MTF-1) bind to metal responsive
elements (MRE) in DNA regulatory regions The
binding occurs by means of zinc-finger DNA
binding domains Binding controls expression of
either copper import proteins (at low Cu levels)
or metallothioneins (at high Cu levels)
B.B.A. 1763, 737 (2006)
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Copper Uptake in Yeast
high affinity transporter
low affinity transporter
metallothioneins
copper chaperones
superoxide dismutase
ATPase
The mitochondria has its own copper delivery
systems
B.B.A. 1763, 759 (2006)
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Copper Delivery in Mitochondria
There are two major mitochondrial targets for
copper delivery cytochrome oxidase and
superoxide dismutase
superoxide dismutase
copper chaperones
copper chaperones
copper chaperones
copper chaperones
cytochrome oxidase
B.B.A. 1763, 759 (2006)
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Metallothioneinsequence and metal ion binding
Metallothionein controls the levels of free metal
ions in cells
This protein has a very high cysteine content
which is used to form a number of metal ion
binding clusters similar to the Fe/S clusters
This enables metallothioneins to bind and
eliminate multiple metal ions quite efficiently
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Copper Metabolism in Drosophila
Under low copper conditions metallotranscription
factors (MTF) do not activate the metallothionein
genes (Mtn) and instead are translocated to the
cytoplasm However, some MTFs do activate the
Ctr1B gene which produces Cu importers General
metal importers and copper reductases also
function to increase cellular Cu levels Cu is
shuttled to copper-containing proteins (SOD and
CcO) by copper chaperones (Atx1, CCS Cox17)
B.B.A. 1763, 737 (2006)
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Copper Metabolism in Drosophila
Under high copper conditions more MTFs are
recruited to the nucleus to activate the Mtn
genes to produce metallothioneins In spite of
MTF-1 the binding of a repressor protein (in red)
slows expression of the Ctr1B gene that produces
Cu importers The Cu efflux transporter (ATP7)
changes cellular locations to pump Cu out of the
cells The copper chaperones (Atx1, CCS Cox17)
continue to delivery Cu to their target proteins
B.B.A. 1763, 737 (2006)
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Proteins involved in Copper Metabolism

• Transcuprein
• Hemocyanin
• Albumin
• Ceruloplasmin
• Cytochrome Oxidase

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Hemocyanin
Hemocyanins are respiratory proteins containing
two copper atoms that reversibly bind a single
oxygen molecule (O2)
These proteins also contain a bound Ca ion that
functions in a structural role
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Hemocyanin structure
A view of the overall structure showing the bound
Cu (dark spheres) and Ca (light sphere)
Binuclear Cu center
Ca binding site
Protein Sci. 2 597-619 (1993)
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Human serum albumin
Serum albumin contains a large binding site that
is capable of transporting lipids and hydrophobic
drugs
This protein also has a Cu binding site at the
amino-terminus that functions to transport copper
Amino-terminal Cu transport site
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Ceruloplasmin structure
Ceruloplasmin is synthesized in the liver
containing 8 atoms of copper in its
structure Ceruloplasmin carries the majority of
the copper in our plasma Ceruloplasmin also
exhibits a copper-dependent oxidase activity
This activity is associated with oxidation of
iron, therefore assisting its transport in the
plasma by transferrin, which can only carry iron
in the ferric state
Two views of the ceruloplasmin structure showing
some of the bound copper
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Cytochrome oxidase
This is the terminal electron acceptor in the
electron transport chain and is responsible for
reducing oxygen to water
Protein subunits Cox1 blue Cox2 red Cox3
yellow peripheral subunits
relative positioning of metal cofactors Fe-heme
and Cu redox cofactors Mg and Zn structural
role
B.B.A. 1763, 759 (2006)
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Links between Cu and Fe metabolism
In addition to the simultaneous requirement for
both Cu and Fe cofactors in cytochrome oxidase
there are several other links between copper and
iron metabolism
A single membrane bound reductase (Fre1) is
responsible for the reduction of both iron and
copper There are separate transporters that bring
the reduced copper and iron into cells However,
the reoxidation of iron is catalyzed by ferrous
oxidase, a copper requiring enzyme
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Roles of Zinc
Zinc is involved in control and regulation
processes in multicellular organisms This
includes links to hormone expression, cellular
triggering events and a wide range of enzyme
catalyzed processes
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Zinc Transport

• Zinc uptake requires transmembrane transporter
  proteins to facilitate zinc movement
• In bacteria there are three families of
  transporters
• ABC transporters (ATP-binding cassette
  transporters)
• RND transporters (resistance-nodulation-cell
  division efflux pumps)
• CorA transporters (transports Zn2 and Mg2 ions)

• In eukaryotes there are two different transporter
  families
• ZIP proteins
• CDF proteins

We will focus our attention on the structural
organization and function of these eukaryotic
zinc transporters
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Zinc Import and Export
Each of these families of eukaryotic zinc
transporters contain multiple membrane spanning
helices and a histidine rich domain that binds
zinc
zinc binding site
The Zip family of transporters contain 8
transmembrane helices and transport zinc into the
cytoplasm
zinc binding site
The CDF family are antiporters that use either a
proton or a potassium ion gradient to transport
zinc out of the cytoplasm
B.B.A. 1763, 711 (2006)
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Elimination of Excess Zinc

• plant cells use P-type ATPases for zinc export
• mammalian cells use CDF proteins for zinc efflux
• yeast cells do not eliminate excess zinc,
  instead they store it in intracellular organelles
  for detoxification

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Zinc Metabolism in Yeast
ZIP transporters in blue CDF transporters in
red Zap1 is a transcriptional activator that will
upregulate the genes for these transporters under
conditions of limiting zinc vacuoles are used
for zinc detoxification and other organelles in
yeast (zincosomes) are used for zinc storage
B.B.A. 1763, 711 (2006)
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Membrane Distribution of Cu, Fe Zn Transporters
Plant cell
Cu transporters are shown in blue Fe transporters
are shown in red Zn transporters are shown in
purple The direction of metal ion transport is
given by the arrows
B.B.A. 1763, 595 (2006)
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Molybdenum Cofactor Assembly
Molybdenum cofactors are required for a number of
essential enzyme catalyzed reactions, including
reduction of nitrate, oxidation of sulfite, and
purine base metabolism
Different metal ions play important roles in
molybdenum cofactor assembly
B.B.A. 1763, 621 (2006)
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Summary

• Copper plays important roles in a wide range of
  biological processes
• Several proteins are responsible for binding and
  transporting copper, with ceruloplasmin playing
  the major role
• Copper levels are controlled by metal responsive
  transcription factors that increase Cu importer
  proteins under low copper levels and increase
  metallothioneins under high copper levels
• Copper and iron metabolism are linked by several
  redox proteins and metal ion transporters
• Zinc transport is facilitated by different
  families of transporters in both prokaryotic and
  eukaryotic organisms
• Molybdenum cofactors require the presence of Mg,
  Cu, and Fe for complete assembly