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Principles of Bioinorganic Chemistry - 2004
Lecture
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2
3
4
5
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7
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9
10
11
12
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Date
9/9
(Th)
9/14 (Tu)
9/16
(Th)
9/21 (Tu)
9/23 (Th)
9/28 (Tu)
9/30 (Th)
10/5 (Tu)
10/7 (Th)
10/12 (Tu)
10/14 (Th)
10/19 (Tu)
10/21 (Th)
TBA
Lecture Topic
Intro; Choice, Uptake, Assembly of Mn+ Ions
Metalloregulation of Gene Expression
Metallochaperones; M n+-Folding, X-linking
Med. Inorg. Chem./ Metalloneurochemistry
Mössbauer, EPR, IR Spectral Fundamentals
Electron Transfer; Fundamentals
Long-Distance Electron Transfer
Hydrolytic Enzymes, Zinc, Ni, Co
CO and Bioorganometallic Chemistry
Dioxygen Carriers: Hb, Mb, Hc, Hr
O2 Activation, Hydroxylation: MMO, ToMO
Model Chemistry for O 2 Carriers/Activators
Complex Systems: cyt. oxidase; nitrogenase
Term Examination
Reading
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 9
Ch. 10
Ch. 10
TBA
Ch. 11
Ch. 11
Ch. 12
Ch. 12
Problems
Ch. 1
Ch. 2
Ch. 3
Ch. 4
Ch. 5
Ch. 6
Ch. 7
Recitations are held on Mondays at 5 PM, or a little later
on seminar days, in 18-475
Ch. 8
Ch. 9
Ch. 10
Ch. 11
Ch. 12
Metallochaperones; Metal Folding
PRINCIPLES:
•Metallochaperones guide and protect metals to natural sites
•Chaperone and target receptor protein structurally homologou
ILLUSTRATION:
•Copper insertion into metalloenzymes
Useful references:
•Copper Delivery by Metallochaperone Proteins. A. C. Rosenzweig, Acc.
Chem. Res., 2001, 34, 119-128.
•Perspectives in Inorganic Structural Genomics: A Trafficking Route for Copper.
F. Arnesano, L. Banci, I. Bertini, and S. Ciofi-Baffoni, Eur. J. Inorg. Chem.,
2004, 1583-1593.
Copper Uptake and Transport in Cells
The puzzles:
The total cellular [Cu] in yeast is 0.07 mM, none free.
How does copper find its way into metalloproteins?
The implications:
Mn, Fe, Zn have similar systems; understanding one
in detail has implications for all
Two Metallochaperone-mediated Cu Delivery Pathways
2O
-
+ 2H+
HO + O
Two well characterized pathways
Atx1 delivers Cu to transport ATPases in the secretory pathway,
which translocates it into vesicles for insertion into
multicopper oxidases such as ceruloplasmin
Mutations in human forms of these ATPases lead to
Menkes and Wilson diseases
CCS delivers copper to Cu,Zn SOD
Human Cu/Zn SOD is linked to ALS
Copper Uptake and Transport in Cells
The players:
SOD, superoxide dismutase, a copper enzyme, a
dimer containing two His-bridged Cu/Zn sites
CCS, a copper chaperone for superoxide dismutase
Ctr, family of membrane proteins that transport
copper across the plasma membrane, delivering
it to at least three chaperones: CCS, Cox17, Atx1
N-terminus has 8 putative Cu motifs (MXMXXM)
C-terminus has 2 CXC motifs
Atx1, the copper chaperone for Ccc2
Ccc2, a cation transporting ATPase; has CXXC sites
Fet3, a multicopper ferroxidase
Note the connection between Fe and Cu trafficking
Key Questions Address by Structural Bioinorganic
Chemistry (Rosenzweig, O’Halloran, Culotta)
What are the details of copper binding by
these proteins, including stoichiometry and
coordination geometry?
How do these chaperones interact with their
copper receptor proteins?
What features of the copper binding and
protein-protein interactions render each
chaperone specific for its target protein?
Structure of the Hg(II) form of Atx1
Hg
Cys 15
Cys 18
C
N
Hg(II) is exposed at the surface
of the protein, which is reasonable
for a protein that functions in metal
delivery-- metal sites in enzymes
are more buried.
Hg(II) coordinated by the 2 cysteines.
The apo protein has same structure but
with a disulfide bonds between the
cysteine residues.
More Details of the 1.2 Å Structure, Active Site
Cys 15
Thr 14
Ser 16
Val 12
2.34 Å
Hg 2.33 Å
Ser 19
Met 13
Cys 18
Lys 65
Ala 21
Structure of the Cu Hah1 Protein, the Human Homolog
C
N
First copper chaperone structure with Cu bound
The two molecules are primarily held together by
the bound metal ion and some hydrogen bonding
Extended H-Bonding Interactions
Stabilize the Structure
T11B
C12A
C15B
M10A
Cu
C15A C12B
T11A
M10B
T11B is conserved
in most related domains.
When it is not there it is
replaced by His, which
could serve the same
function.
Postulated Mechanism for Metallochaperone
Handoff of Copper to a Receptor Protein
(O’Halloran, Rosenzweig, Culotta, 2000)
HgAtx1
HgHah1
CuHah1
AgMenkes4
N
Domain I (Atx1-like)
yCCS1 Crystal Structure
Domain II (SOD1-like)
metal binding
not essential
target recognition
C
C20
229CXC231
C17
Domain III
metal delivery
crucial
Lamb, et al. Nature Struct. Biol. 1999, 6, 724-729
Dimer of Dimers Model
+
54 kDa
SOD1
32 kDa
86 kDa
homodimer is very stable
yCCS and hCCS are dimeric in the crystal and
in solution (yCCS under some conditions)
Heterodimer Model
+
54 kDa
Structures
32 kDa
43 kDa
indicate heterodimer formation is feasible
Heterodimer formation between different SOD1s
has been observed
Biophysical and biochemical studies
of complex formation
According
to gel filtration chromatography, dynamic light
scattering, analytical ultracentrifugation, and chemical
crosslinking experiments, yCCS and SOD1 form a specific
protein-protein complex
The
molecular weight of the complex, ~43 kDa, is most
consistent with a heterodimer
Higher order complexes,
such as a dimer of dimers, were
not detected
86 kDa
Lamb, et al. Biochem. 2000, 39, 14720-14727
43 kDa
Factors Affecting Heterodimer Formation
The heterodimeric complex formed with a mutant of SOD1
that cannot bind copper, H48F-SOD1, is more stable
Heterodimer formation is facilitated by zinc
Heterodimer formation is apparently independent of whether
copper is bound to yCCS
Heterodimer formation between
Cu-yCCS and wtSOD1 in
the presence of zinc is accompanied by SOD1 activation
These
data suggest that in vivo copper loading occurs via a
heterodimeric intermediate
Lamb, et al. Biochem. 2000, 39, 14720-14727
Crystals of the yCCS/H48F-SOD1
heterodimeric complex
Table 1 Crystallogra phic statistics
Data collection
Resolution range (Å)
Unique observations
Total observations
Completeness (%)
Rsym
% > 3 (I)
12.0 - 2.9
32,933
119,535
98.8 (99.6)
0.109 (0.351)
69.9 (29.2)
Refinement
Resolution range
P3221
a = b = 104.1 Å, c = 233.7 Å
Solved by molecular replacement
Lamb, et al., Nature Structural Biology (2001),
8(9), 751-755.
Number of reflections
R-factor
R-free
Number of protein, nonhydrogen
atoms
Number of nonprotein atoms
Rms bond length (Å)
Rms bond angles (°)
Average B value (Å2)
12.0 – 2.9
30,885
0.217
0.260
5,956
25
0.007
1.40
27.9
SOD1 homodimer
Domain I
yCCS homodimer
Domain III
Domain II
H48F-SOD1 monomer
yCCS monomer
C146
F48
C57
C229
C231
Mechanism of metal ion transfer
yCCS Domain I probably does
not directly deliver the metal ion
yCCS Domain III is well positioned
in the heterodimer to insert the
metal ion
His 63
His 46
His 120
Cys 57
Cys 229
Transient intermonomer disulfide
formation may play a role in yCCS
function
His 48
Cys 231
Metal Folding of Biopolymers
PRINCIPLES:
•Metal ions organize the structures of biopolymers
•In binding proteins, metal ions typically shed water
molecules
•In binding nucleic acids, aqua ligands remain for H-bonding
•Metal-mediated biopolymer folding facilitates interactions
•Cross-link formation underlies metallodrug action
•High
coordination numbers are used for function
ILLUSTRATIONS:
•Zinc finger proteins control transcription
•Ca2+, a second messenger and sentinel at the synapse
•Cisplatin, an anticancer drug
Zinc Fingers - Discovery, Structures
A. Klug, sequence gazing, proposed zinc fingers for TFIIIA,
which controls the transcription of 5S ribosomal RNA.
Zn2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein.
Y or F – X –CC – X2,4 –CC – X3 – F – X5 – L – X2H– H – X3,4
H – H – X2
The coordination of two S and 2
N atoms from Cys and His
residues was supported by
EXAFS; Zn–S, 2.3 Å; Zn–N, 2.0 Å.
Td geometry.
The protein folds only when zinc
is bound; > 1% of all genes have
zinc finger domains.
X-ray Structure of a Zinc Finger Domain
Structure of a Three Zinc-Finger Domain of Zif 268
Complexed to an Oligonucleotide Containing its
Recognition Sequence
The Specificity of Zinc for Zinc-finger Domains
Kd value:
Metal ion:
2 pM 5nM 2mM 3mM
Zn2+ Co2+ Ni2+ Fe3+
For [Co(H2O)6]2+
+ 3/5 o
- 2/5 o
LFSE = -5(2/5 o) + 2(3/5 o) = -4/5 o+ 2P (small)
= -7440 cm-1 (since  o = 9300 cm-1) = -21.3 kcal mol-1
For [Co(Cys)2(His)2]
+ 2/5 t
- 3/5 t
LFSE = -4(3/5 t) + 3(2/5 t) = -6/5 t + 2P (small)
= -5880 cm-1 (since  t = 4900 cm-1) = -16.8 kcal mol-1
Thus Co2+ loses 4.8 kcal mol-1 in going from aqueous solution
2+ does not.
to the zinc finger environment; Zn