1. From: Cécile Claude, „Enzyme Models of Chloroperoxidase and Catalase“, Inaugural Dissertation, Universität Basel, 2001 Hemoproteins: Axial Ligands and Functions

2. 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 F8390

3. Hemoproteinproximal ligand E m for Fe II /Fe III (mV) Fe III /Fe II (aq.) Fe III /Fe II - +770 Human hemoglobin Fe III /Fe II His+150 Microperoxidase11-CO Fe III /Fe II His+100 Chloroperoxidase Fe III /Fe II Cys - -150 NO synthase neuronal Fe III /Fe II Cys - -250 Horse-radish peroxidase Fe III /Fe II His -280 Cytochrome P450 2C5 Fe III /Fe II Cys - -330 Catalase Fe III /Fe II Tyr - -460 Source: C. Capeillere-Blandin, D. Matthieu & D. Mansuy, Biochem. J. 2005, 392, 583-587 Modification of the Fe II /Fe III redox potential by the protein environment Strong reductants Strong oxidantsFe II (Red.) stable Fe III (Ox.) stable Different metalloproteins need different redox potential for their function. Cytochrome P450 needs to access the unusual oxidation state Fe(V) to be able to oxidize even unreactive substrates. Therefore, it uses the negatively charged cysteine ligand which donates electrons to Fe and stabilizes the high oxidation state. One of strategies that proteins employ to modify the redox potential is using different proximal ligands.

4. antibiotic local anesthetic steroid hormone carcinogen from fungi Alkaloid from Taxus brevifolia, potent anti-cancer drug Examples of Cytochrome P450 substrates Hydroxylation at: -aliphatic carbons -aromatic carbons -double bonds -heteroatoms

5. Cytochrome P450cam (Campher-5-monooxygenase; pdb-code 1T86) access for substrate and O 2

7. e - from putidaredoxin Catalytic cycle of cytochrome P450cam Substrate RH binds into the hydrophobic pocket and pushes H 2 O out from coordination site E  ~ -0.17 V O 2 binds to the empty coordination site Low-spin Fe III E m ≤ -0.3 V Redox parnter of P450cam: putidaredoxin (Fe-S protein) E m ≈ -0.2 V http://www.cup.uni-muenchen.de/ac/kluefers/homepage/L_bac.html

8. Conclusion In many cases, metalloproteins use the same or similar active site for different purposes. The strategies to confer a particular activity to a given site include - Allowing/disallowing access of substrates to the active site (including the dynamics of diffusion of substrate/product) -Modifying the electrostatic potential by mutating the amino acids coordinated to the metal or surrounding the binding pocket

9. Practical training - Download from the pdb database the structures of bacterial cytochrome P450cam 1t86 and 1dz8 http://www.rcsb.org/pdb/home/home.do - Display the structures using VMD - Use the command „chain A“ in Graphics/Representation“ to display only the monomer A - Use the command „chain A and resname HEM“ in Graphics/Representation“ to highlight the heme group - Observe whether the two crystal structures contain the campher and/or oxygen molecule trapped near the active site - Use the command „chain A and resname CAM“ in Graphics/Representation“ to highlight the campher molecule -Use the command „chain A and resname OXY“ in Graphics/Representation“ to highlight the O 2 molecule - Examine how the two carboxylate groups of heme are anchored in the protein backbone - Examine how the campher substrate is fixed in the acess channel