1. F8390
Metalloproteins: Structure and Function
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
Hemoglobin, Myoglobin Cytochrome P450
Hemerythrin & Ribonucleotide Reductase R2 &
Methane monooxygenase diiron subunits
Hemocyanin & Tyrosinase
4. Electron transfer proteins
4.1. Iron-sulfur proteins
4.2. Blue copper proteins
5. Conclusion

2. 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)

3. 1.2. Some fundamental metal sites in metalloproteins
Metal site Function
Metal complexes of porphyrins and corrins
- Iron porphyrins
= Hemoglobin & Myoglobin O2 transport
= Cytochromes Redox catalysts
- Vitamin B12 = Cobalt corrinoid Radical catalyst Methyltransferase
2. Bridged bimetallic complexes
- Fe2 clusters
= Hemerythrin O2 transport
= Methane Monooxygenase Hydroxylase
= Ribonucleotide Reductase RR2 Radical generation
- Cu2 clusters
= Hemocyanin O2 transport

4. 1.2. Some fundamental metal sites in metalloproteins Continued
- Mn2 clusters
= O2-evolving complex Photosystem II
= Mn-Catalase 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

5. 1. 2. Some fundamental metal sites in metalloproteins: Exemples
1.2. Some fundamental metal sites in metalloproteins: Exemples. Iron porphyrines
anchoring points to protein
Cytochrome c (involved in respiratory chain)
Hemoglobin/myoglobin

6. 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

7. 1.2. Some fundamental metal sites in metalloproteins: Diiron clusters
Ribonucleotide reductase R2 unit
Hemerythrin
Methane monooxygenase
hydroxylase protein

8. Hemocyanin (oxygen transport)
1.2. Some fundamental metal sites in metalloproteins: Exemples. Cu2 and Mn2 clusters
Mangenese catalase
(Whitaker et al., Eur. J. Biochem. 2003, 270, )
Hemocyanin (oxygen transport)
Cuff et al.,J.Mol.Biol.1998

9. Aminopeptidase from Aeromanas proteolytica Urease: catalytic cycle
1.2. Some fundamental metal sites in metalloproteins: Exemples. Zn2 and Ni2 clusters
Aminopeptidase from Aeromanas proteolytica
(Stamper et al., Biochemistry 2004, 43, )
Urease: catalytic cycle
homepage/L_bac.html

10. 1. 2. Some fundamental metal sites in metalloproteins: Exemples
1.2. Some fundamental metal sites in metalloproteins: Exemples. Fe-S clusters
4Fe-4S cluster

11. Estrogen receptor mechanism
1.2. Some fundamental metal sites in metalloproteins: Exemples. Zn-fingers
Coordination of zinc in a zinc finger
Estrogen receptor mechanism
Zinc finger of the estrogen receptor
is responsible DNA-binding
Zinc finger:
Zinc finger of estrogen receptor:
Estrogen receptor mechanism

12. 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.

13. - - - - - - - - - - - - + + + 2+ pKa = 8.9 pKa = 7.0 pKa = 7.0
Carboxypeptidase A - - - 2- - - - -
pKa = 11.2
pKa = 11.2 - - -
P.Andersson et al, Eur. J. Biochem. 113, (1981)
W.N.Lipscomb, N. Sträter, Chem. Rev. 96, (1996).

14. 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 first-order 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 HCO3- in 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.

15. 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

16. Catalytic cycle of carbonic anhydrase
To a buffer molecule
His64 is used as a „proton shuttle“ between Zn-OH2 and buffer molecules
Nucleophilic attack of water on Zn, elimination of HCO3-
Zn-stabilized OH- ion carries out a nucleophilic attack on CO2 carbon
 Use VMD to highlight His64
Comment your observation

17. Possible pathway for H+ transfer from Zn-OH2 to His64
C. K. Tu, D. .N. Silverman, Biochemistry 1982, 21,

18.  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
 In the absence of other effects, pKa is in fact expected to be a linear function of the complex charge.