From: Cécile Claude, „Enzyme Models of Chloroperoxidase and Catalase“, Inaugural Dissertation, Universität Basel, 2001

Hemoproteins: Axial Ligands and Functions

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

F8390

Hemoprotein proximal ligand Em for FeII/FeIII (mV)

FeIII/FeII (aq.) FeIII/FeII - +770

Human hemoglobin FeIII/FeII His +150

Microperoxidase11-CO FeIII/FeII His +100

Chloroperoxidase FeIII/FeII Cys- -150

NO synthase neuronal FeIII/FeII Cys- -250

Horse-radish peroxidase FeIII/FeII His -280

Cytochrome P450 2C5 FeIII/FeII Cys- -330

Catalase FeIII/FeII Tyr- -460

Source: C. Capeillere-Blandin, D. Matthieu & D. Mansuy,Biochem. J. 2005, 392, 583-587

Modification of the FeII/FeIII redox potential by the protein environment

Strong reductants

Strong oxidants FeII (Red.) stable

FeIII (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.

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

Cytochrome P450cam (Campher-5-monooxygenase; pdb-code 1T86)

access for substrate and O2

e- from putidaredoxin

e- from putidaredoxin

Catalytic cycle of cytochrome P450camSubstrate RH binds into the hydrophobic pocket and pushes H2O out from coordination siteE ~ -0.17 V

O2 binds to the empty coordination site

Low-spin FeIII Em≤ -0.3 V

Redox parnter of P450cam:putidaredoxin (Fe-S protein)Em≈ -0.2 V

http://www.cup.uni-muenchen.de/ac/kluefers/homepage/L_bac.html

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

Practical training

- Download from the pdb database the structures of bacterial cytochrome P450cam 1t86 and 1dz8http://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 O2 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