Pb2 : Sequestration with phytochelatins

1

• Pb2 Sequestration with phytochelatins
• Cd2, Me2 Sequestration with metallothioneins
• Co2 Import block, Sequestration with other
  metalloproteins
• Hg2 Organomercurials Cleavage of the Hg-C
  bond, reduction to volatile Hg(0)
• Ag deposition as insoluble Ag(0) or Ag2S

2

• Pb2
• Toxic effects lead inhibits the aminolaevulinic
  acid (ALA) synthetase, ALA dehydratase, and
  ferrochelatase. Especially, the lack of available
  Fe2 leads to excess protoporphyrin and anaemia.
• Organic lead has no effect on haem synthesis, but
  is lipid soluble, neurotoxic and passes the
  blood-brain barrier.

3

• Pb2
• Lead and cadmium (and other thiophile metals) can
  be bound by cysteine rich peptides
• PCn (g-Glu-Cys)n-Gly Phytochelatins
• hPCn (g-Glu-Cys)n-b-Ala Homophytochelatins

4

• Pb2
• Phytochelatins ocurr in plants, fungi, algae,
  marine diatoms. They are cytosolic, in plants
  mainly in roots.
• Synthesis is done by g-glutamylcysteine
  dipeptidyl transpeptidase (EC. 2.3.2.15) from
  GSH.
• (g-Glu-Cys)n-Gly GSH -gt (g-Glu-Cys)n1-Gly Gly

5

• Pb2
• Phytochelatins

6

• Pb2
• Phytochelatins, metal binding capacity
• PC3 binds 1 Pb2 or 1-2 Cd2
• PC4 binds 1-2 Pb2 or 2-3 Cd2
• The lower binding capacity for lead is likely due
  to its larger ion radius (Pb(octahedral) 133 pm
  vs. Cd(octahedral) 109 pm) and its higher
  coordination number (Pb 6-8, Cd 4-8).

7

• Pb2
• Later stages of exposure
• Heavy metals are first bound to GSH.
  Phytochelatins are synthesized within hours and
  Me-PCn complexes are formed. After days, PCn
  levels decrease, probably, further resistance
  mechanisms are induced.

8

• Cd2, Me2
• Sequestration by metallothioneins
• Binding of metals to metallothioneins is a
  general mechanism in bacteria, lower eukaryotes,
  plants, animals. They protect the cells from free
  metal ions and their deleterious effects (SH
  binding, O2 activation). They are small
  (6000-7000 Da) proteins with high Cys content
  (ca. 20) and a capacity to bind ca. 7 metal
  ions.

9
Intracellular metal binding by metallothioneins
Molecular weight 6000 8000 Da Aromatic
amino acid residues none Cysteins gt20,
some gt30 Metals bound Zn, Cu, Cd, Hg
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• Sequence motifs in metallothioneins
• Cluster A
• Hsa MT 1A MDPNCSCATGGSCTCTGSCKCKECKCNSC
• Hsa MT 4 MDPRECVCMSGGICMCGDNCKCTTCNCKTC
• Ncr MT GDCGCSGASSCNCGSGCSCSNCGSK
• Ath MT 1A-like MADSNCGCGS SCKCGDSCSCEKNYNKEC
• DNCSCGS NCSCGSNCNC
• Cluster B
• Hsa MT 1A KKSCCSCCPMSCAKCAQGCICKGASEKCSCCA
• Hsa MT 4 RKSCCPCCPPGCAKCARGCICKGGSDKCSCCP

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(No Transcript)
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• ClusterA in rat metallothioneins

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• Compare the right side of clusterA with Zn-S
  coordination in sphalerite (cubic ZnS, three
  alternating layers of S2ions, chair conformation
  of the six-ring)

14

• Compare clusterA with Zn-S coordination in
  wurtzite (hexagonal ZnS, two alternate layers of
  S2ions, chair and boat six-rings)

15

• ClusterB in rat metallothioneins

16

• Compare clusterB with Zn-S coordination in
  wurtzite (hexagonal ZnS)

17

• Co2
• In Neurospora crassa tests with 60Co show that
  the metal is incorporated and not leachable with
  EDTA (Ballentine, Stephens, 1951). No binding
  partner (or even protein) was identified at the
  time.
• Venkateswerlu and Sastry (1970s) found strains
  with blocked (passive) cobalt uptake. Normally,
  iron is imported actively (ATP dependent iron
  utilization) and cobalt is erroneously taken up
  with it.
• A cobaltoprotein is identified in a cobalt
  resistant strain (Sajani, Mohan, 1999).

18

• Co2
• The cor strain of Neurospora crassa accumulates a
  8200 Da protein (cobalt binding protein, CBP) at
  up to 12 of total protein which binds cobalt.
  Cys (29), Gly (17), Glu (14), and Asp (5) are
  the major amino acids. The protein has a high
  carbohydrate content (30). Ca. 10 Co per CBP are
  bound (70 mg Co / mg protein). Containing
  several aromatic amino acids (6 Tyr, 4 Trp),
  this is no (normal) metallothionein.

19

• Co2
• Co2 is specifically bound. Ni, Fe, Cu, and Fe
  are not detectable.
• The bound Co leads to absorption peaks at 350 nm
  and a shoulder at 440 nm.

20

• Co2 Resistance scheme

Cobaltoprotein
Passive uptake
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active cysteines

• Hg2 components of the mercuric ion resistance
  operon Tn501 (merRTPAD)
• MerP, MerT transporter
• MerA mercuric reductase
• MerR,MerD regulator of expression
• Genes in other operons
• MerB organomercury lyase
• MerF, MerC various transporters

path of mercury
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• Hg2 mercuric reductase (MerA)
• Hg2 NADPH --gt NADP H Hg(0)

The catalytic domain of MerA (ca. 450 aa) is
related to FAD-dependent disulfide
oxidoreductases (most prominent glutathion
reductase). Whereas Hg2 is an inhibitor for
glutathion reductase, it is a good substrate for
MerA.
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• Hg2 mercuric reductase (MerA)
• The active site contains 2-4 Cys and a FAD.
• The preferred coordination geometry for Hg2 is
  linear. Ligand exchange in mercury complexes
  normally proceeds via a trigonal planar complex
  (Hg(SR)3- for example).

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• Hg2 mercuric reductase (MerA)
• Hg2 is first transferred from large substrates
  to the two more exposed cysteines (outer complex)
  , then handed inwards and finally
  bound by the cysteines close to FAD and reduced
  .

25

• Hg2 mercuric reductase (MerA)
• In the case of small substrates (HgBr2 or
  Hg(CN)2), Hg2 can be accepted directly by the
  inner cysteines (bypass route).

26

• Hg2 mercuric reductase (MerA)
• The trigonal complex dissociates slowly with CN-
  but faster with Br- ligands.

Hg(CN)2 54 s-1 HgBr2 gt300 s-1
27

• Hg2 biotechnological detoxification of
  contaminated wastes with resistant Pseudomonas
  sp.
• Reducing the Hg-content from 3-10 mg/l to 50 mg/l

28

• Hg2
• For detoxification in medical applications
  dimercapto succinic acid (DMSA), dimercapto
  propane sulfonic acid (DMPS) and N-acetyl
  cysteine (NAC) is used.

29

• Ag
• Silver is highly toxic to most microbes and can
  be used as a biocide or antimicrobial agent.
• Example Silvercoating of clothes

30

• Ag
• Silver resistant strains can accumulate silver to
  as much as 25 of the dry weight biomass. This
  could be used for industrial recovery from wastes
  or ores.
• Pseudomonas stutzeri AG259, isolated from a
  silver mine, synthesizes silver-based crystals in
  the periplasmic space. These particles are seen
  in the transmission electron microscope

31

• Ag
• Silver-containing crystals in P. stutzeri in the
  transmission electron microscope (TEM).
• There are triangular, hexagonal, and irregularly
  shaped crystals.

32

• Ag Methods
• Determination of the composition of
  silver-containing crystals with Energy dispersive
  X-ray analysis (EDX) which elements? which
  amounts?
• Determination of the solid phase with electron
  diffraction Ag, Ag2S, modification?

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Energy dispersive X-ray analysis (EDX) Energy
level diagram for an atom, illustrating the
excitation of the K, L, M, and N shells and the
formation of Ka, Kb, La, and Ma X-rays. The
transitions are specific for each element and can
be measured by the emission spectrum of the
sample in an electron beam.
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Electron diffraction Electrons are diffracted in
crystals. Due to the strong interaction (much
stronger than with X-rays!) only very thin
specimens can be analyzed. The diffraction
pattern is characteristic for the crystal
structure.
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• Ag TEM, electron diffraction pattern, EDX

36

• Ag enlarged electron diffraction pattern,
  notice the threefold axis for the cubic densest
  packing of Ag.

37

• Ag TEM, electron diffraction pattern, EDX

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• Ag TEM, electron diffraction pattern, EDX

39

• Cr trace essential metal or only toxin?
• A Cr-containing glucose tolerating factor is
  commonly cited as the cofactor responsible for
  glucose metabolism.
• Cr supplements are sold to help in cases of
  glucose intolerance (diabetes).
• Nevertheless, no enzyme (or cofactor) has been
  found to contain Cr. Cr-containing molecules
  identified seem to be part of the detoxification
  mechanism.