Activation of biomolecules Activation of small biomolecules Activation

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: O 2 (to water), N 2 (to NH 3), H 2 O and CO 2 (photosynthesis) activation Anaerobic organisms: H 2, CO 2, CH 4 activation The reactions are catalysed by metal ions with variable oxidation states: Fe, Cu, Mo, Mn, V, Ni containing metalloenzymes.

Triptophane dioxygenase In the resting state the enzyme hem contains a high spin Fe. II and the coordination position 6 is empty. When the substrate is bound to the enzyme conformation changes and it will be able for reversible O 2 binding, [SFe. O 2] transition complex is formed, in which oxygen is in the form of O 2 • -. After an oxygen insertion step of Fe. III- O 2 • - with the double bound substrate, a rearrangement and finally a cleavage of the bonds occur.

Cytochrome P 450 The electrontransfer component of the monooxygenase enzymes. R−H + O 2 + 2 e− + 2 H+ → R−OH + H 2 O Solubilisation of compounds containing C-H bonds, Metabolism of lipids and other compounds Nomenclature: λmax (CO adduct) = 450 nm (instead of the usual 420 nm) Structure: M ~ 45. 000 Resting state: Fe. III (low spin) N = 5 (Cys-S axial coordination) (coordination site 6: labile water molecule → oxygen binding site) Mechanism: Fe. III−OH 2 (ε=− 300 m. V) → Fe. III, R−H (ε=− 173 m. V) → Fe. II, R−H → Fe. III−O 2−, R−H → Fe. III−O 22−, R−H → Fe. VO, R−H (vagy Fe. IVOP·+) → Fe. III−OH 2 + R−OH

Cytochrome P 450 The Figure depicts the adduct formed with thio-camphor It catalyses hydroxylation of thio-camphor.

The catalytic cycle of cytochrome P 450 The key steps: Formation of the reactive oxenoid oxoferryl(V) (= Fe. VO) or oxoferryl(IV)porphyrin-radical (= Fe. IVO−P·+).

A comparative table of the iron and copper containing proteins

Copper states in proteins Type I: Blue copper proteins Resting state: Cu(II), paramagnetic, unusual vis and EPR parameters ε ~ 100 εnormál A║ << Anormal Type II: Non-blue copper proteins Spectral 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 Cu. A: Mixed valence copper proteins Cu(I) - Cu(II) pair

More familiar copper proteins Name Total Plasstocyanin Azurin Stellacyanin I. III. Function 1 1 1 - - electrontransfer 2 100% enzyme Cu storage detoxification 100% O 2 -transport Superoxidedismutase Metallothioneine 2 - 1 -10 - Hemocyanin >10 - - Tyrosinase 4 - - 4 2 4 1 1 1 4 Ceruloplasmin Laccase Ascorbic acid oxidase 6 4 8 3 oxygenase 2+1 2 Cu-transport oxidase

Blue copper proteins 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 Cu. II) - 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) +. SR fast reaction Structure: Cu(II) in unusual environment distorted tethedron (usually: 2 his +1 cys + 1 met)

Non-blue copper proteins Characteristics: paramagnetic, ESR activ pale blue ( 10 -100) Cu(II), d 9 Structure/bonding: - tetragonally distorted octahedron (like Cu(H 2 O)6 2+ or Cu. L 4(H 2 O)2 ) - there is one labile ligand in the coord. sphere (e. g. a water molecule in axial or equatorial position substrate binding site Occurrance: Cu. Zn-SOD Non-blue oxidases (pl. galactose oxidase, amin oxidase) Blue copper oxidases (I + II + 2 III)

ESR inactiv copper proteins Structural characteristics: In the resting state they contain either 2 close, but independent Cu. I-ions, or 2 antiferromegnetically coupled Cu. II-ions. In the coordination sphere of Cu there are usually 3 N(His) donor atoms, while at the fourth position the substrate/O 2 is bound. Occurrance: hemocyanin: oxygen carrier enzymes: 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 Cu. I + O 2 Cu. II−O 22−−Cu. II 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 (O 2 transport) and methane monooxigenase or ribonucleotide reductase

Tyrosinase enzyme II. a/ monophenolase (monooxygenase) function + A + H 2 O b/ diphenolase (oxidase) function

Blue copper oxidases I. They catalyse the reduction of dioxygen to water by 4 electrons: O 2 + 4 H+ + 4 e− → 2 H 2 O Structure: In the resting state they contain min. 4 Cu. II: 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 atoms ascorbic acid oxidase: 8 copper atoms ceruloplasmin: 5 - 8 copper atoms (1 trimer + 2(5) type I)

Blue copper oxidases II. The „trimer” activ centre of ascorbic acid oxidase: The Cu. II → Cu. I 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) enzymes They catalyse dysproportionation of the superoxide anion: 2 O 2− → O 2 + O 22− Main types: Cu. Zn-SOD: in eukaryots (cells with nucleus) Fe-SOD: in prokaryots (cells without nucleus) Mn-SOD: in prokaryots + mitochondrion Ni-SOD: most recent (certain microorganisms) Human SOD enzymes: SOD 1: cytoplasm (Cu. Zn) SOD 2: mitochondrion (Mn) SOD 3: extracellular (Cu. Zn) Characteristic features of Cu. Zn-SOD: Composition: 2 subunits (M 16. 000/subunit) (1 Cu + 1 Zn)/subunit Structure: Zn(II) (distorted tetrahedron), structure maker Cu(II) (Type II: tetragonal), redoxy centre All Cu. II complexes have low level of SOD activity.

Mechanism of Cu. Zn-SOD Zn(II): structure maker Co. II, Cd. II or Cu. II 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) Cu 2+(His–)Zn 2+ + O 2– + H+ Cu+ + (His. H)Zn 2+ + O 2 Cu+ + O 2– + H+ + (His. H)Zn 2+ Cu 2+(His–)Zn 2+ + H 2 O 2 gross process: 2 O 2– + 2 H+ H 2 O 2 + O 2

Structure of Cu. Zn-SOD 2 O 2– + 2 H + H 2 O 2 + O 2

Cytochrome c oxidase Function: Terminal enzyme of the respiratory chain, catalysis the four electron reduction of dioxygen to water. Additionally, it generates a membrane proton gradient that subsequently 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 makers 2 Fe: cytochrome a and cytochrome a 3 3 Cu: Cu. A (2 copper) and Cu. B (1 copper)

Structure of Cu. A and Cu. B Cu. A: mixed valence dimer Cu. B: monomeric Cu. II centre, similar to a Type 2 copper, but the His ligands are in trigonal pyramidal arrangement.

Peroxidases and the catalase H 2 O 2 Catalase or peroxidase H 2 O 2 Compound I. RH 2 disproportion ation product cit-cred, Mn. II, Clcit-cox, Mn. III, Cl. ORH 2 product • In resting state haloperoxidases contain Fe. III-hem, • The peroxide oxidases the hem centre and an oxoferrilcentre (Fe. IV=O) is formed, • 1 electron comes from the hem or the protein and the radical Compound I is formed, • This reacts with H 2 O 2 through disproportionation or RH 2 substrate or the redoxi partners (cytochrome, H 2 O 2, Cl-, Mn. II) and reduced, • The Fe. IV=O centre gives a water and returns to the Fe. III-hem state.

Vanadium Biological role 2. Vanadium containing enzymes Haloperoxidases (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 o. C, p ~ 100 -200 bar N 2 + 3 H 2 2 NH 3 2. Nitrogen fixation (biological): (Mo containing nitrogenase enzyme) N 2 + 10 H+ + 8 e− → 2 NH 4+ + H 2 3. Nitrification: NH 4+ + 2 O 2 → NO 3− + H 2 O + 2 H+ 4. Denitrification: (Mo containing nitrate reductase enzyme + Cu and hem) 2 NO 3− + 12 H+ + 10 e− → N 2 + 6 H 2 O

Nitrogenase and nitrogen fixation 1. Observation, Isolation: Certain bacteria living in symbiosis with the root system of leguminous plants are able to utilise the dinitrogen of the air isolation of Nitrogenase enzyme from these bacteria. 2. Model systems: N 2 complexes and their catalytic activity e. g. Ru. II(NH 3)5 N 2 2+, Co. I(N 2)(H–)(PPh 3)3 other metals and their oxidation state: (Mo 0, W 0, Re. I, Ir. I, Rh. I. . . ) activity: – 3. Structure and action of the nitrogenase: N 2 + 8 H+ + 8 e– + 16 Mg-ATP 2 NH 3 + H 2 + 16 Mg-ADP + 16”P” - Iron-molibden cofactor: Fe. Mo-co - Vanadium containing nitrogenase - Metal free nitrogenases

Catalytic centres of the nitrogenase enzyme It consists of two [Fe 4 S 4] (P-cluster) and one [Fe 4 S 4 – Fe 3 Mo. S 3] (M-cluster). The N 2 probably binds to the Mo, the energy of the reduction is provided by the hydrolysis of ATP. P-cluster M-cluster

Vanadium-nitrogenase (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.

Biological role N 2 fixation/reduction process V-nitrogenase: N 2+10 e +10 H++ 24 Mg. ATP = 2 NH 3 + 3 H 2 + 24 Mg. ADP + 24 Pi (N 2+8 e +8 H++ 16 Mg. ATP = 2 NH 3 + H 2 + 16 Mg. ADP + 16 Pi) Structure of Fe-V-S cluster in vanadium-nitrogenase enzyme

Tungsten containing enzymes Tungsten is not considered as an essential element. Tungsten containing enzymes were identified in some heatresistant 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).

Ellenőrző kérdések 1. 2. 3. 4. 5. 6. Milyen kismolekulák aktiválására van szükség a biológiai rendszerekben, és milyen fémionoknak van ezen folyamatokban kitüntetett szerepe? Hasonlítsa össze a dioxigenázokat és a monooxigenázokat! Hogyan alkalmazkodik a réz kémiai környezete a biológiai funkcióhoz a réztartalmú fehérjékban? A réztartalmú fehérjék fajtái és funkciói. A vanádium biológiai szerepe. A N 2 fixálása. A vas és a réztartalmú fehérjék funkcionális összehasonlítása.