Metalloproteins: Structure and Function

1. Introduction1.1. Metalloproteins: Functions in Biological Chemistry1.2. Some fundamental metal sites in metalloproteins

2. Mononuclear zinc enzymes: Carbonic anhydrase

3. Metalloproteins reacting with oxygen3.1. Why do aerobic organisms need metalloproteins?3.2. Oxygen transport proteins & Oxygenases3.2.1. Hemoglobin, Myoglobin Cytochrome P4503.2.2. Hemerythrin & Ribonucleotide Reductase R2 &Methane monooxygenase diiron subunits 3.2.3. Hemocyanin & Tyrosinase

4. Electron transfer proteins4.1. Iron-sulfur proteins4.2. Blue copper proteins

5. Conclusion

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- 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)

1.1 Metalloproteins : Functions in Biological Chemistry

1. Introduction

1.2. Some fundamental metal sites in metalloproteins

1. Metal complexes of porphyrins and corrins- Iron porphyrins

= Hemoglobin & Myoglobin O2 transport= Cytochromes Redox catalysts

- Vitamin B12 = Cobalt corrinoid Radical catalystMethyltransferase

2. Bridged bimetallic complexes- Fe2 clusters

= Hemerythrin O2 transport= Methane Monooxygenase Hydroxylase= Ribonucleotide Reductase RR2 Radical generation

- Cu2 clusters= Hemocyanin O2 transport

Metal site Function

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

1.2. Some fundamental metal sites in metalloproteins: Exemples. Iron porphyrines

Hemoglobin/myoglobinCytochrome c (involved in respiratory chain)

anchoring points to protein

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

Hemerythrin

Ribonucleotide reductase R2 unit

Methane monooxygenasehydroxylase 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(Stamper et al., Biochemistry 2004, 43, 9620-9628)

Urease: catalytic cyclehttp://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 clusterhttp://www.steve.gb.com/science/enzymes.html

1.2. Some fundamental metal sites in metalloproteins: Exemples. Zn-fingers

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Zinc finger: http://www.infobiogen.fr/services/chromcancer/Deep/TranscripFactorsID20043.htmlZinc finger of estrogen receptor: http://www.web-books.com/MoBio/Free/Ch4F2.htmEstrogen receptor mechanismhttp://www.cancer.gov/cancertopics/understandingcancer/estrogenreceptors/

Coordination of zinc in a zinc finger

Zinc finger of the estrogen receptoris responsible DNA-binding

2. Mononuclear zinc enzymes: Carbonic anhydraseZinc 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

Carboxypeptidase A

pKa = 7.0 pKa = 8.9

pKa = 11.2

- - -

-

-

--

-

--

-

-

2+ + + +

0 - 2-

pKa = 8.26pKa = 7.0

pKa = 11.2

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 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.

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

Catalytic cycle of carbonic anhydrase

Use VMD to highlight His64 Comment your observation

His64 is used as a „proton shuttle“ between Zn-OH2 and buffer molecules

Zn-stabilized OH- ion carries out a nucleophilic attack on CO2 carbon

To a buffer molecule

Nucleophilic attack of water on Zn, elimination of HCO3

-

Possible pathway for H+ transfer from Zn-OH2 to His64

C. K. Tu, D. .N. Silverman, Biochemistry 1982, 21, 6353-6360

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

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pKa (H

2O)

Complex charge [e]

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