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mV
Flavin
NAD(P)
Fe/S
Heme
Redox catalysisRedox catalysis
PQQ
HemeHeme
• 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
electrontransport
oxidationreactions
reductionreactions
oxygenationreactions
Versatility of heme biochemistryVersatility of heme biochemistry
oxygentransport
dehalogenationreactions
sensingsignalling
N
OHO
Fe
N
N
N
OHO
NO transport
390 - 450 nm: "Soret” band
450 - 700 nm: "A" and "B" band(s)sensitive to oxidation state and/or ligation
Heme spectral propertiesHeme spectral properties
OP460 siro
Heme structuresHeme structures
A B C D
D1
iron-protoporphyrin IX
Heme binding in cytochromesHeme binding in cytochromes
Covalently bound hemeCovalently bound heme
• Catalases
H2O2 + H2O2 O2 + 2 H2O
• Peroxidases
AH2 + H2O2 A + 2 H2O
• Cytochrome P450s
RH + O2 + NADPH + H+ ROH + H2O + NADP+
Heme-based biocatalystsHeme-based biocatalysts
Tuning of catalytic function Tuning of catalytic function of heme cofactorof 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
Axial ligandsAxial ligands
Methionine
Histidine
distal ligandsixth ligand
proximal ligandfifth ligand
horse cytochrome c
protein proximal distalcatalase Tyr operoxidase His HisP450 Cys ocyt c His His/Metglobin His His
• 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
Cytochrome P450Cytochrome P450
RH + O2 + NADPH + H+ ROH + H2O + NADP+
P450 substratesP450 substrates
Bio-compounds
• steroids• fatty acids• eicosanoids• lipid hydroperoxides• retinoids• acetone
Xenobiotics
• drugs, antibiotics• solvents• carcinogens• antioxidants• odorants• alcohols• dyes, pesticides• petroleum products
• 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 300.000 different substrates
CytochromeCytochrome P450P450
Different type of reactions
• hydroxylation epoxidation
• peroxygenation oxidation
• dealkylation dehalogenation
• deamination isomerization
CytochromeCytochrome P450 P450
P450cam structureP450cam structure
P450 O-O bond cleavageP450 O-O bond cleavage
FeIII
S
O
H
HO
Push mechanism
distal
proximal
push: Cys acts as strong electron donor.
Protons delivered by solvent channel
Thr positions OH to stabilize OOH
Cys and Thr are conserved!Cys
Thr O
O
H H
solvent
O
O
Asp
NH2Arg
NH2
+
NH3
Lys
+
-
Many applications
• Analytical chemistry: coupled enzyme assays
• Immunochemistry
• Biosensor construction
• Decolorization: textile, paper and pulp industry
• Food processing and storage
PeroxidasesPeroxidases
PeroxidasePeroxidasereaction cyclereaction cycle
compound Icompound II
Peroxidase O-O bond cleavage mechanismPeroxidase O-O bond cleavage mechanism
N
NH
FeIII
N
N
H
O-
-
O
O
O H H
H2N
H2N
Push-pull mechanism
distal
proximal
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!
+
no direct access of substrates to Fe(IV)=O center
1e- oxidations preferred
Peroxidases: substrate specificityPeroxidases: substrate specificity
complex with ferulic acid
PeroxidasePeroxidase versusversus P450P450
• 1 electron oxidation• Fe-O not accessible
• higher redox potential• His ligand
• monooxygenation• Fe-O accessible
• lower redox potential• Cys ligand
Fe
O
SFe
O
S