Redox catalysis Flavin NAD(P) PQQ Heme Fe/S mV -400 0 +400

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

Versatility of heme biochemistry N O H Fe oxygen transport electron transport sensing signalling NO transport oxidation reactions dehalogenation reactions reduction reactions oxygenation reactions

Heme spectral properties 390 - 450 nm: "Soret” band 450 - 700 nm: "A" and "B" band(s) sensitive to oxidation state and/or ligation

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

Heme binding in cytochromes

Covalently bound heme

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

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

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 P450 Cys o cyt c His His/Met globin His His

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

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

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 300.000 different substrates

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

P450cam structure

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

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

Peroxidase reaction cycle compound II compound I

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-

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

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