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

-400 0 +400

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