Activation of biomolecules

Activation of small biomolecules

Activation of small inorganic biomolecules in order to make them reactive is necessary both for aerobic and anaerobic organisms.

These reactions provide the necessary energy for their life.

Aerobic organisms: O2 (to water), N2 (to NH3), H2O and CO2 (photosynthesis) activation

Anaerobic organisms: H2, CO, CO2, CH4 activation

The reactions are catalysed by metal ions with variable oxidation states: Fe, Cu, Mo, Mn, V, Ni containing metalloenzymes.

Triptophane dioxygenase

NH

H2C

CH

NH2

COOH

NH

HC

H2C CH

NH2

COOH

O

18O

+18O2

In the resting state the enzyme hem contains a high spin FeII and the coordination position 6 is empty.

When the substrate is bound to the enzyme conformation changes and it will be able for reversible O2 binding,

[SFeO2] transition complex is formed, in which oxygen is in the form of O2

•-.

After an oxygen insertion step of FeIII- O2•- with the double bound

substrate, a rearrangement and finally a cleavage of the bonds occur.

Cytochrome P450

The electrontransfer component of the monooxygenase enzymes.

R−H + O2 + 2 e− + 2 H+ → R−OH + H2O

Solubilisation of compounds containing C-H bonds, Metabolism of lipids and other compoundsNomenclature: λmax (CO adduct) = 450 nm (instead of the usual 420 nm)

Structure: M ~ 45.000

Resting state: FeIII (low spin)N = 5 (Cys-S axial coordination)(coordination site 6: labile water molecule → oxygen

binding site)

Mechanism: FeIII−OH2 (ε=−300 mV) → FeIII, R−H (ε=−173 mV) → FeII, R−H → FeIII−O2

−, R−H → FeIII−O22−,R−H →

FeVO, R−H (vagy FeIVOP·+) → FeIII−OH2 + R−OH

Cytochrome P450

The Figure depicts the adduct formed with thio-camphor

It catalyses hydroxylation of thio-camphor.

The catalytic cycleof cytochrome P450

The key steps:

Formation of the reactive oxenoid oxoferryl(V) (= FeVO) or oxoferryl(IV)-porphyrin-radical

(= FeIVO−P·+).

A comparative table of the iron and copper containing proteins

Function Fe protein

(h: heme protein)

(nh: non-heme protein)

Cu protein

O2 transport hemoglobin (h) hemoerythrin (nh)

hemocyanin

oxygenase function cytochrome P-450 (h) methane monooxygenase (nh) pyrocatechin dioxygenase (nh)

tyrosinase quercetinase (dioxygenase)

oxidase inhibition peroxidases (h) peroxidases (nh)

amine oxidase laccase

electron transport cytochromes (h) blue Cu proteins antioxidative function peroxidases (h)

bacterial superoxide dismutase (nh) superoxide dismutase (Cu, Zn)

NO2– reductase heme containing nitrite reductase (h) Cu-containing nitrite reductase

Type I: Blue copper proteinsResting state: Cu(II), paramagnetic,unusual vis and EPR parametersε ~ 100 εnormál A║ << Anormal

Type II: Non-blue copper proteinsSpectral parameters characteristic of the tetragonally

distorted Cu(II) complexes (light blue proteins)

Type III: EPR inactive copper proteins- Cu(II) dimers (antiferromágnetic coupling)- Cu(I) state

CuA: Mixed valence copper proteinsCu(I) - Cu(II) pair

Copper states in proteins

More familiar copper proteins

Name Total I. II. III. Function

Plasstocyanin 1 1 - - electron-Azurin 1 1 - - transferStellacyanin 1 1 - -

Superoxide-dismutase 2 - 2 - enzymeMetallo- Cu storagethioneine 1-10 - 100% detoxification

Hemocyanin >10 - - 100% O2-transport

Tyrosinase 4 - - 4 oxygenase

Cerulo-plasmin 6 2 1 2+1 Cu-transportLaccase 4 4 1 2 oxidaseAscorbic acidoxidase 8 3 1 4

Occur mostly in plants (preparation from algae)Have important role in photosynthesis as electron transfer proteins.Characteristics:

- low molecular mass (M ~ 10 000, ~ 100 am acid + 1 CuII)- Intense blue color λ ~ 600 nm, ε ~ 3000 - 5000- EPR activ, low A|| coupling constant- high redox potential (ε ~ + 0.3-0.7 V)

(easy reduction)Mechanism:

Cu(II) - SR Cu(I) + .SRfast reaction

Structure:Cu(II) in unusual environment distorted tethedron(usually: 2 his +1 cys + 1 met)

Blue copper proteins

Characteristics: paramagnetic, ESR activ

pale blue ( 10 -100) Cu(II), d9

Structure/bonding: - tetragonally distorted octahedron

(like Cu(H2O)62+ or CuL4(H2O)2)- there is one labile ligand in the coord. sphere

(e.g. a water molecule in axial or equatorial position substrate binding site

Occurrance: CuZn-SODNon-blue oxidases (pl. galactose oxidase, amin oxidase)Blue copper oxidases (I + II + 2 III)

Non-blue copper proteins

ESR inactiv copper proteins

Structural characteristics:In the resting state they contain either 2 close, but independent CuI-ions, or 2 antiferromegnetically coupled CuII-ions.In the coordination sphere of Cu there are usually 3 N(His) donor atoms, while at the fourth position the substrate/O2 is bound.

Occurrance:hemocyanin: oxygen carrierenzymes: tyrosinase (mixed monooxygenase/oxidase function)

blue copper oxidases: e.g. ceruloplasmin, laccase, ascorbic acid oxidase, etc.occurs with type 1 and 2 copper(type 2 and 3 form a trimer)

Tyrosinase enzyme I.

Structure: Similar to hemocyanin but it contains only two subunits

(= 2 + 2 copper).

Action: 2 CuI + O2 CuII−O22−−CuII

reversible oxygen transfer but in enzymatic reaction.The enzyme acts in a mixed function catalytic reaction:- monophenolase (monooxygenase) activity- diphenolase (oxidase) activity

The different function from hemocyanin can be explained by the different protein character.

Similar structure, but different function occurs in the groups of iron proteins: e.g.. hemerythrin (O2 transport) and

methane monooxigenase or ribonucleotide reductase

OH

R

+ O2 + AH2

OH

R

OH

OH

R

OH

2 + O2 2

O

O

R

+ 2H2O

Tyrosinase enzyme II.

+ A + H2O

a/ monophenolase(monooxygenase)function

b/ diphenolase(oxidase) function

Blue copper oxidases I.

They catalyse the reduction of dioxygen to water by 4 electrons:

O2 + 4H+ + 4e− → 2 H2O

Structure:In the resting state they contain min. 4 CuII:

I. + II. + 2 III.The type II and III copper atoms usually forms a trimer unit.Besides these, the proteins may contain further copper centres.

Important oxidases:laccase (phenol oxidase): 4 copper atomsascorbic acid oxidase: 8 copper atomsceruloplasmin: 5 - 8 copper atoms (1 trimer + 2(5) type I)

Blue copper oxidases II.

The „trimer” activ centre of ascorbic acid oxidase:

The CuII → CuI reduction is accompanied by an increase in bond lengths. The fourth Cu (type I) is situated rather far (1300 pm) from the trimer.

Superoxide dismutase (SOD) enzymesThey catalyse dysproportionation of the superoxide anion:

2 O2− → O2 + O2

2− Main types:

CuZn-SOD: in eukaryots (cells with nucleus)Fe-SOD: in prokaryots (cells without nucleus)Mn-SOD: in prokaryots + mitochondrionNi-SOD: most recent (certain microorganisms)

Human SOD enzymes: SOD 1: cytoplasm (CuZn)SOD 2: mitochondrion (Mn)SOD 3: extracellular (CuZn)

Characteristic features of CuZn-SOD:Composition: 2 subunits (M 16.000/subunit)

(1Cu + 1Zn)/subunitStructure: Zn(II) (distorted tetrahedron), structure maker

Cu(II) (Type II: tetragonal), redoxy centre

All CuII complexes have low level of SOD activity.

Zn(II): structure maker CoII, CdII or CuII may substitute (in vitro) it without ceasing activity of the enzyme

Cu(II): participate actively in the redoxy process without the metal ion the enzyme is not active

The catalytic reaction mechanism:(temporary splitting of the Cu-His(61) bond)

Cu2+(His–)Zn2+ + O2– + H+ Cu+ + (HisH)Zn2+ + O2

Cu+ + O2– + H+ + (HisH)Zn 2+ Cu2+(His–)Zn2+ + H2O2

gross process: 2O2

– + 2H+ H2O2 + O2

Mechanism of CuZn-SOD

2 O2– + 2 H+ H2O2 + O2

SOD

His 61

His 78

His 69

H2O

Zn2+

Cu2+

His 44

His 46His 118

Asp 81

Structure of CuZn-SOD

Cytochrome c oxidase

Function: Terminal enzyme of the respiratory chain, catalysis the fourelectron reduction of dioxygen to water. Additionally, it generates a membrane proton gradient thatsubsequently drives the synthesis of ATP.

Structure: One of the most complex metalloproteins.It consists of 13 subunits (M ~ 100.000), some of them serve only to bind to the membrane.

The metal ion containing subunits:Zn and Mg – structure makers2 Fe: cytochrome a and cytochrome a3

3 Cu: CuA (2 copper) and CuB (1 copper)

Structure of CuA and CuB

CuA: mixed valence dimer

CuB: monomeric CuII centre, similar to a Type 2 copper, but

the His ligands are in trigonal pyramidal arrangement.

Peroxidases and the catalase

• In resting state haloperoxidases contain FeIII-hem,• The peroxide oxidases the hem centre and an oxoferrilcentre

(FeIV=O) is formed,• 1 electron comes from the hem or the protein and the radical

Compound I is formed,• This reacts with H2O2 through disproportionation or RH2 substrate or

the redoxi partners (cytochrome, H2O2, Cl-, MnII) and reduced,• The FeIV=O centre gives a water and returns to the FeIII-hem state.

Catalase orperoxidase

Compound I.

disproportionation

product

cit-cox, MnIII, ClO-

cit-cred, MnII, Cl- {

product

H2O2

H2O2

RH2

RH2

Vanadium

Biological role

2. Vanadium containing enzymesHaloperoxidases (Vilter, 1984)

isolated from red and brown algae species

Haloperoxidases

Chemical/Biological transformation of N-compounds

1. Nitrogen fixation (industrial): metal oxide catalysts, T ~ 400 oC, p ~ 100-200 barN2 + 3 H2 2 NH3

2. Nitrogen fixation (biological): (Mo containing nitrogenase enzyme)N2 + 10H+ + 8 e− → 2 NH4

+ + H2

3. Nitrification: NH4

+ + 2O2 → NO3− + H2O + 2H+

4. Denitrification:(Mo containing nitrate reductase enzyme + Cu and hem)2NO3

− + 12H+ + 10e− → N2 + 6H2O

1. Observation, Isolation: Certain bacteria living in symbiosis with the root system of legumin-ous plants are able to utilise the dinitrogen of the air isolation ofNitrogenase enzyme from these bacteria.

2. Model systems: N2 complexes and their catalytic activity

e.g. RuII(NH3)5N22+, CoI(N2)(H–)(PPh3)3other metals and their oxidation state: (Mo0, W0, ReI, IrI, RhI.....)activity: –

3. Structure and action of the nitrogenase:N2 + 8H+ + 8e– + 16Mg-ATP 2NH3 + H2 + 16Mg-ADP + 16”P”

- Iron-molibden cofactor: FeMo-co - Vanadium containing nitrogenase- Metal free nitrogenases

Nitrogenase and nitrogen fixation

P-cluster

M-cluster

Catalytic centres of the nitrogenase enzyme

It consists of two [Fe4S4] (P-cluster) and one [Fe4S4 – Fe3MoS3] (M-cluster).The N2 probably binds to the Mo, the energy of the reduction isprovided by the hydrolysis of ATP.

(Hales and coworkers, 1986)

Azotobacter chroococcum isolated from

the A. vinelandii bacterium

It is active in the case of the lack of

molibdenum

Xanthobacter autothrophycus accumulate

vanadium in Mo deficient environment.

Vanadium-nitrogenase

Biological role

N2 fixation/reduction process

V-nitrogenase:

N2+10e+10H++ 24MgATP = 2NH3 + 3H2 + 24MgADP

+ 24Pi

(N2+8e+8H++ 16MgATP = 2NH3 + H2 + 16MgADP + 16Pi)

Structure of Fe-V-S cluster in vanadium-nitrogenase enzyme

Fe

S V

S Fe

SFeS

SS

OOO

A vanádium-nitrogenáz enzimben található Fe-V-S klaszter szerkezete

Tungsten is not considered as an essential element.

Tungsten containing enzymes were identified in some heat-

resistant organisms: these contains W-co (tungsten-cofactor)

Which corresponds to the Mo-co.

Its spreading in nature is uncertain, but is certainly not too frequent!

Based on their heatshock resistancy it can be assumed that they

appearred in the early stage of life but later the tungsten was

substituted by molibdenum.

(It might happened because of the difference in the availability

of the two metals or the kinetics of their substitution reactions).

Tungsten containing enzymes

Ellenőrző kérdések

1. Milyen kismolekulák aktiválására van szükség a biológiai rendszerekben, és milyen fémionoknak van ezen folyamatokban kitüntetett szerepe?

2. Hasonlítsa össze a dioxigenázokat és a monooxigenázokat!

3. Hogyan alkalmazkodik a réz kémiai környezete a biológiai funkcióhoz a réztartalmú fehérjékban? A réztartalmú fehérjék fajtái és funkciói.

4. A vanádium biológiai szerepe.

5. A N2 fixálása.6. A vas és a réztartalmú fehérjék funkcionális

összehasonlítása.