1. Redox catalysis
Flavin
NAD(P)
PQQ
Heme
Fe/S mV

2. Heme ubiquitous in nature and of vital importance in eukaryotes
complex of transition element iron with four nitrogen atoms of tetrapyrrole
ubiquitous in nature and of vital importance in eukaryotes
differences in biological specificity, redox chemistry and substrate specificity
wide variety of biological reactions/functions, versatile biocatalysts

3. Versatility of heme biochemistryN O H Fe
oxygen
transport
electron
transport
sensing
signalling
NO transport
oxidation
reactions
dehalogenation
reactions
reduction
reactions
oxygenation
reactions

4. Heme spectral properties
nm: "Soret” band
nm: "A" and "B" band(s)
sensitive to oxidation state and/or ligation

5. Heme structures A B C D
iron-protoporphyrin IX D1
P460 O
siro

6. Heme binding in cytochromes

7. Covalently bound heme

8. Heme-based biocatalysts
Catalases
H2O2 + H2O O2 + 2 H2O
Peroxidases
AH2 + H2O A + 2 H2O
Cytochrome P450s
RH + O2 + NADPH + H ROH + H2O + NADP+

9. Tuning of catalytic function
of heme cofactor
type of heme and axial ligands
reduction state Fe cation
accessibility of the active site
activation of reactants
redox potential of the heme

10. Axial ligands protein proximal distal catalase Tyr o
distal ligand
sixth ligand
Methionine
horse cytochrome c
proximal ligand
fifth ligand
Histidine
protein proximal distal
catalase Tyr o
peroxidase His His
P Cys o
cyt c His His/Met
globin His His

11. Cytochrome P450 Most versatile biological catalyst known
RH + O2 + NADPH + H+ ROH + H2O + NADP+
Most versatile biological catalyst known
Monomer of 55 kDa
Induced by xenobiotics
150 isoforms
Microsomes, membrane bound
About 30 families
20 genes per eukaryotic species
Also present in microbes

12. P450 substrates Bio-compounds Xenobiotics steroids fatty acids
eicosanoids
lipid hydroperoxides
retinoids
acetone
Xenobiotics
drugs, antibiotics
solvents
carcinogens
antioxidants
odorants
alcohols
dyes, pesticides
petroleum products

13. Cytochrome P450
Substrate deeply buried, wide range of size/shape/flexibility
Enzyme breathing
Conserved heme binding pocket, heme-thiolate protein
Variations in S-binding site, redox partners
Many genes in databank (>500 in SWISS-PROT)
More than different substrates

14. Cytochrome P450 hydroxylation epoxidation peroxygenation oxidation
Different type of reactions
hydroxylation epoxidation
peroxygenation oxidation
dealkylation dehalogenation
deamination isomerization

15. P450cam structure

17. P450 O-O bond cleavage Push mechanism
solvent
Lys +
NH2 +
NH3
Arg
NH2 O H
push: Cys acts as strong electron donor.
Protons delivered by solvent channel
Thr positions OH to stabilize OOH
Cys and Thr are conserved! H
Asp O O H - O HO
Thr O
distal
proximal
FeIII S
Cys

18. Peroxidases Analytical chemistry: coupled enzyme assays
Many applications
Analytical chemistry: coupled enzyme assays
Immunochemistry
Biosensor construction
Decolorization: textile, paper and pulp industry
Food processing and storage

19. Peroxidase reaction cycle
compound II
compound I

20. Peroxidase O-O bond cleavage mechanism
Push-pull mechanism NH N
H2N
push: provided by proximal His whose electron donor capabilities are enhanced by H-bonding with Asp.
pull: distal His accepts H+ from oxygen atom binding to Fe and transfers it to other oxygen atom.
Arg helps to stabilize developing negative charge on outer oxygen
all four residues are conserved! H H + O
H2N O
distal
proximal
FeIII N d- N H O O-

21. Peroxidases: substrate specificity
no direct access of substrates to Fe(IV)=O center
1e- oxidations preferred
complex with ferulic acid

22. Peroxidase versus P450 1 electron oxidation Fe-O not accessible
higher redox potential
His ligand
monooxygenation
Fe-O accessible
lower redox potential
Cys ligand