Activation of biomolecules

1. Activation of biomolecules

2. 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.

3. Triptophane dioxygenase 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.

4. 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 compounds Nomenclature: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

5. Cytochrome P450 The Figure depicts the adduct formed with thio-camphor It catalyses hydroxylation of thio-camphor.

6. The catalytic cycle of cytochrome P450 The key steps: Formation of the reactive oxenoid oxoferryl(V) (= FeVO) or oxoferryl(IV)-porphyrin-radical (= FeIVO−P·+).

7. A comparativetable of theiron and coppercontainingproteins

8. Copper states in proteins Type I: Bluecopper proteins Resting state:Cu(II), paramagnetic, unusualvis 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 (antiferromágneticcoupling) - Cu(I) state CuA:Mixed valence copper proteins Cu(I) - Cu(II) pair

9. More familiarcopperproteins NameTotal I.II.III. Function Plasstocyanin 1 1 - - electron- Azurin 1 1 - - transfer Stellacyanin 1 1 - - Superoxide- dismutase 2 - 2 -enzyme Metallo- Cu storage thioneine 1-10 - 100% detoxification Hemocyanin >10 - -100% O2-transport Tyrosinase 4 - - 4 oxygenase Cerulo- plasmin 6 2 1 2+1 Cu-transport Laccase4 4 1 2 oxidase Ascorbic acid oxidase 8 3 1 4

10. 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 CuII) - Intenseblue 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)

11. Non-blue copper proteins Characteristics: paramagnetic, ESR activ pale blue (  10 -100) Cu(II), d9 Structure/bonding: - tetragonally distorted octahedron (like Cu(H2O)62+ or CuL4(H2O)2) - there is one labile ligand in the coord. sphere (e.g. a water molecule in axial or equatorial position substratebinding site Occurrance: CuZn-SOD Non-blue oxidases (pl. galactose oxidase, amin oxidase) Blue copper oxidases(I + II + 2 III)

12. ESR inactivcopperproteins 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 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)

13. 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

14. Tyrosinaseenzyme II. a/ monophenolase (monooxygenase) function + A + H2O b/ diphenolase (oxidase) function

15. 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 atoms ascorbic acid oxidase: 8 copper atoms ceruloplasmin: 5 - 8 copper atoms (1 trimer + 2(5) type I)

16. Bluecopperoxidases 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.

17. Superoxide dismutase (SOD) enzymes They catalyse dysproportionation of the superoxide anion: 2 O2−→ O2 + O22− Main types: CuZn-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 (CuZn) SOD 2: mitochondrion (Mn) SOD 3: extracellular (CuZn) Characteristic features of CuZn-SOD: Composition: 2 subunits (M  16.000/subunit) (1Cu + 1Zn)/subunit Structure:Zn(II) (distorted tetrahedron), structure maker Cu(II) (Type II: tetragonal), redoxy centre All CuII complexes have low level of SOD activity.

18. Mechanism of CuZn-SOD Zn(II):structure maker CoII, CdIIor 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)Zn2+ Cu2+(His–)Zn2+ + H2O2 gross process: 2O2– + 2H+  H2O2 + O2

19. Structure of CuZn-SOD 2 O2– + 2 H+ H2O2 + O2

20. 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 a3 3 Cu: CuA (2 copper) and CuB (1 copper)

21. 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.

22. disproportionation H2O2 H2O2 Catalase or peroxidase RH2 product Compound I. cit-cred, MnII, Cl- cit-cox, MnIII, ClO- RH2 product 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.

23. Vanadium Biological role 2. Vanadium containing enzymes Haloperoxidases (Vilter, 1984) isolated from red and brown algae species

24. Haloperoxidases

25. Chemical/Biological transformation of N-compounds • Nitrogen fixation (industrial): metal oxide catalysts, T ~ 400 oC, p ~ 100-200 bar N2 + 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

26. Nitrogenaseand nitrogenfixation • 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 of • Nitrogenase enzyme from these bacteria. • 2. Model systems: • N2 complexes and their catalytic activity • e.g. RuII(NH3)5N22+,CoI(N2)(H–)(PPh3)3 • other 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

27. 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 is provided by the hydrolysis of ATP. P-cluster M-cluster

28. Vanadium-nitrogenase (Hales and coworkers, 1986) Azotobacter chroococcumisolated from theA. vinelandii bacterium It is active in the case of the lack of molibdenum Xanthobacter autothrophycus accumulate vanadium in Mo deficient environment.

29. Biological role N2fixation/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

30. Tungstencontainingenzymes 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).

32. Ellenőrző kérdések 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 N2 fixálása. A vas és a réztartalmú fehérjék funkcionális összehasonlítása.