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-4000 +400 mV Flavin NAD(P) Fe/S Heme Redox catalysis PQQ.

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Presentation on theme: "-4000 +400 mV Flavin NAD(P) Fe/S Heme Redox catalysis PQQ."— Presentation transcript:

1 -4000 +400 mV Flavin NAD(P) Fe/S Heme Redox catalysis PQQ

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

4 390 - 450 nm:"Soret band 450 - 700 nm: "A" and "B" band(s) sensitive to oxidation state and/or ligation Heme spectral properties

5 O P460 siro Heme structures ABCD D1D1 iron-protoporphyrin IX

6 Heme binding in cytochromes

7 Covalently bound heme

8 Catalases H 2 O 2 + H 2 O 2 O 2 + 2 H 2 O Peroxidases AH 2 + H 2 O 2 A + 2 H 2 O Cytochrome P450s RH + O 2 + NADPH + H + ROH + H 2 O + NADP + Heme-based biocatalysts

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 Methionine Histidine distal ligand sixth ligand proximal ligand fifth ligand horse cytochrome c proteinproximaldistal catalase Tyr o peroxidase His His P450 Cys o cyt c HisHis/Met globin His His

11 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 P450 RH + O 2 + NADPH + H + ROH + H 2 O + NADP +

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

13 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

14 Different type of reactions hydroxylationepoxidation peroxygenationoxidation dealkylationdehalogenation deaminationisomerization Cytochrome P450

15 P450cam structure


17 P450 O-O bond cleavage Fe III 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 NH 2 Arg NH 2 + NH 3 Lys + -

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

19 Peroxidase reaction cycle compound I compound II

20 Peroxidase O-O bond cleavage mechanism N NH Fe III N N H O- O- - O O O H H H2NH2N H2NH2N 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! +

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

22 PeroxidaseversusP450 1 electron oxidation Fe-O not accessible higher redox potential His ligand monooxygenation Fe-O accessible lower redox potential Cys ligand Fe O S O S

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