Download Metal Ions in Biological Systems

F8390
Metalloproteins: Structure and Function
1. Introduction
1.1. Metalloproteins: Functions in Biological Chemistry
1.2. Some fundamental metal sites in metalloproteins
2. Mononuclear zinc enzymes: Carbonic anhydrase
3. Metalloproteins reacting with oxygen
3.1. Why do aerobic organisms need metalloproteins?
3.2. Oxygen transport proteins & Oxygenases
3.2.1. Hemoglobin, Myoglobin Cytochrome P450
3.2.2. Hemerythrin & Ribonucleotide Reductase R2 &
Methane monooxygenase diiron subunits
3.2.3. Hemocyanin & Tyrosinase
4. Electron transfer proteins
4.1. Iron-sulfur proteins
4.2. Blue copper proteins
5. Conclusion
1. Introduction
1.1 Metalloproteins : Functions in Biological Chemistry
- Catalysis of hydrolysis and dehydration by zinc
enzymes: Carbonic anhydrase
- Catalysis of electron transfer reactions:
Cytochromes, non-heme-iron-enzymes,
blue Cu-proteins, iron-sulfur proteins
- Transport of atom groups (e.g., O2):
Hemoglobin, Hemerythrin, Hemocyanin)
- Signal transduction: Calmodulin (Ca2+-binding
regulatory protein)
1.2. Some fundamental metal sites in metalloproteins
Metal site
1. Metal complexes of porphyrins and corrins
- Iron porphyrins
= Hemoglobin & Myoglobin
= Cytochromes
- Vitamin B12 = Cobalt corrinoid
2. Bridged bimetallic complexes
- Fe2 clusters
= Hemerythrin
= Methane Monooxygenase
= Ribonucleotide Reductase RR2
- Cu2 clusters
= Hemocyanin
Function
O2 transport
Redox catalysts
Radical catalyst
Methyltransferase
O2 transport
Hydroxylase
Radical generation
O2 transport
1.2. Some fundamental metal sites in metalloproteins Continued
- Mn2 clusters
= O2-evolving complex
= Mn-Catalase
Photosystem II
H2O2 disproportionation
- Zn2 clusters
= Zinc aminopeptidases
Peptide cleavage
- Ni2 clusters
= Urease
Hydrolysis of urea
3. Fe-S clusters
Electron transfer
4. Mo-pterin
Xanthine oxidase
3. Zinc fingers
DNA binding
1.2. Some fundamental metal sites in metalloproteins: Exemples. Iron porphyrines
anchoring points to protein
Hemoglobin/myoglobin
Cytochrome c (involved in respiratory chain)
1.2. Some fundamental metal sites in metalloproteins: Co-corrin complex in cobalamin
R= 5‘-Ado coenyzme B12
III
- Organometallic compound (M-C bond)
- 9 chiral centers
1.2. Some fundamental metal sites in metalloproteins: Diiron clusters
Ribonucleotide reductase R2 unit
Hemerythrin
Methane monooxygenase
hydroxylase protein
1.2. Some fundamental metal sites in metalloproteins: Exemples. Cu2 and Mn2 clusters
Hemocyanin (oxygen transport)
Cuff et al.,J.Mol.Biol.1998
Mangenese catalase
(Whitaker et al., Eur. J. Biochem. 2003, 270, 1102-1116)
1.2. Some fundamental metal sites in metalloproteins: Exemples. Zn2 and Ni2 clusters
Aminopeptidase from Aeromanas proteolytica Urease: catalytic cycle
(Stamper et al., Biochemistry 2004, 43, 9620-9628)
http://www.cup.uni-muenchen.de/ac/kluefers/
homepage/L_bac.html
1.2. Some fundamental metal sites in metalloproteins: Exemples. Fe-S clusters
4Fe-4S cluster
http://www.steve.gb.com/science/enzymes.html
1.2. Some fundamental metal sites in metalloproteins: Exemples. Zn-fingers
Coordination of zinc in a zinc finger
E
s
Zinc finger of the estrogen receptor
t
is responsible DNA-binding
r
o
g
e
n
Zinc finger:
http://www.infobiogen.fr/services/chromcancer/Deep/TranscripFactorsID20043.html
r
Zinc finger
of estrogen receptor: http://www.web-books.com/MoBio/Free/Ch4F2.htm
e
Estrogen
receptor mechanism
c
http://www.cancer.gov/cancertopics/understandingcancer/estrogenreceptors/
e
p
t
2. Mononuclear zinc enzymes: Carbonic anhydrase
Zinc is essential to all forms of life, with an average adult human containing  3 g of zinc. The
influence of Zn derives from its presence in enzymes. An understanding of the roles that Zn
plays in biological systems requires a detailed appreciation of how the chemistry of Zn is
modulated by its coordination environment. The most common structural motif in Zn enzymes is
one in which a tetrahedral Zn center is attached to the protein backbone by three amino acid
residues, with the fourth site being occupied by the catalytically important water (or hydroxide)
ligand. Importantly, His binds to metals as a neutral molecule, whereas Cys, Asp, and Glu bind
after deprotonation, as Cys-, Asp-, and Glu- anions.
http://www.columbia.edu/cu/chemistry/groups/parkin/zinc.html
pKa = 7.0
-
+
+
+
2+
pKa = 8.26
-
-
pKa = 8.9
Carboxypeptidase A
0
-
-
-
pKa
pKa==11.2
11.2
-
2-
-
-
P.Andersson et al, Eur. J. Biochem. 113, 425-433 (1981)
W.N.Lipscomb, N. Sträter, Chem. Rev. 96, 2375-2433 (1996).
-
Carbonic anbydrase is a zinc-containing enzyme that catalyzes the reversible
hydration of carbon dioxide: CO2 + H2O  HCO3- + H+.
In the absence of a catalyst, this hydration reaction proceeds with an effective firstorder rate constant of 0.01 s-1 at 37°C, pH 7. This is too slow for physiological
processes. For example, CO2 must be almost instantaneously converted into HCO3in muscles to be transported in the blood. Conversely, HCO3- in the blood must be
dehydrated to form CO2 for exhalation as the blood passes through the lungs.
Carbonic anhydrases accelerate CO2 hydration dramatically. The most active
enzymes, typified by human carbonic anhydrase II, hydrate CO2 at rates as high as
kcat = 106 s-1, or a million times a second.
Carbonic anbydrase is a monomeric 29 kD protein consisting of 260 amino
acids. Zn2+ in the active site is coordinated by three histidine residues and a
H2O/OH- ligand.
 Download the structure of human CA II from the protein database, code 1CA2
Highlight the Zn ion
Highlight the coordinating His94, His96, His119 ligands, and the H2O ligand
To a buffer molecule
Catalytic cycle of carbonic anhydrase
His64 is used as a
„proton shuttle“
between Zn-OH2 and
buffer molecules
Nucleophilic attack of
water on Zn,
elimination of HCO3-
 Use VMD to highlight His64
Comment your observation
Zn-stabilized OH- ion
carries out a nucleophilic
attack on CO2 carbon
Possible pathway for H+ transfer from Zn-OH2 to His64
C. K. Tu, D. .N. Silverman, Biochemistry 1982, 21, 6353-6360
 Use data from the preceding slide « Examples for Zinc enzymes and proteins to
produce a plot of pKa values for the coordintaed water molecule as a function of
the charge of the PtL3 fragment. Interpret the result.
Tetrahedral zinc sites from zinc proteins: Plot of pKa of Zn-OH2 as a
function of the charge of the PtL3 fragment
[H3O] [B- ]
K
[H2O][HB]
pK (H O)
a
12
2
[H3O] [B- ]
Ka 
[HB ]
ΔG   RT ln K 
qZnL3  qOH 
qZnL3  qH 2O
 1e



qZnL3
r
r
r
1e
RT ( pK a  ln[ H 2 O])  
qZnL3
r
11
10
9
8
7
6
-1
0
1
2
Complex charge [e]
3
 In the absence of other effects, pKa
is in fact expected to be a linear
function of the complex charge.