UBB sigla

„BABEŞ-BOLYAI” UNIVERSITY

CLUJ-NAPOCA

 

Faculty of Chemistry and Chemical Engineering

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Sclerotinia sclerotiorum laccase: biochemical characterization and applications

- PhD thesis public summary-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PhD Candidate: Augustin-Cătălin Moţ

PhD Supervisor: Prof. Dr. Florin Dan Irimie

 

 

 

CLUJ-NAPOCA – 2012


 

 

UBB sigla

„BABEŞ-BOLYAI” UNIVERSITY

CLUJ-NAPOCA

 

Faculty of Chemistry and Chemical Engineering

http://profile.ak.fbcdn.net/hprofile-ak-snc4/592212_372289489493977_1554058963_n.jpg

 

 

Augustin-Cătălin Moţ

 

 

Sclerotinia sclerotiorum laccase: biochemical characterization and applications

- PhD thesis -

 

 

Doctoral committee

 

President: Prof. Dr. Mircea Dărăbanţu, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca

PhD Supervisor: Prof. Dr. Florin Dan Irimie, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca

 

Reviewers:

 

CS I Dr. ŞTEFAN EUGEN SZEDLACSEK, Institute of Biochemistry, Romanian Academy, Bucharest

Prof. Dr. CARMEN SOCACIU, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca

Conf. Dr. RADU SILAGHI-DUMITRESCU, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, Cluj-Napoca

 

 

 


 

 

 

instrumente_structurale

 

MECTS

Logo_UBB

UNIUNEA EUROPEANĂ

GUVERNUL ROMÂNIEI

MINISTERUL MUNCII, FAMILIEI ŞI

PROTECŢIEI SOCIALE

AMPOSDRU

Fondul Social European

POSDRU 2007-2013

Instrumente Structurale

2007-2013

 

OIPOSDRU

UNIVERSITATEA BABEŞ-BOLYAI

CLUJ-NAPOCA

 

 

 

 

Investing in people!

Ph.D. scholarship, Project co-financed by the SECTORAL OPERATIONAL PROGRAM FOR HUMAN RESOURCES DEVELOPMENT 2007 – 2013

 

Priority Axis 1. "Education and training in support for growth and development of a knowledge based society"

 

Key area of intervention 1.5: Doctoral and post-doctoral programs in support of research.

Contract nr.: POSDRU/88/1.5/S/60185 – “Innovative doctoral studies in a Knowledge Based Society”

 

Babeş-Bolyai University, Cluj-Napoca, Romania

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

 

 

 

Dedicated to my beloved wife, Rodica and to my joyful daughter, Olga.



 

Table of Contents

Table of Contents. ix

List of Figures. xiii

List of Tables. xxi

Abbreviations. xxiii

Acknowledgements. xxv

Aims of the thesis. xxvii

General Introduction. xxix

Chapter 1. Literature survey. 1

1.1. Introduction. 1

1.2. Overall structure of laccases 2

1.21. Architectural features of laccases 2

1.2.2. C-terminus in asco-laccases 5

1.2.3. Laccases with quaternary structure. 7

1.2.4. Laccases glycosilation. 8

1.3. Active sites structure of laccases 9

1.3.1. Type 1 copper active site. 9

1.3.1.1. Spectroscopic features of type 1 copper center 10

1.3.1.2. Structure of type 1 copper center 11

1.3.1.3. Redox potential of the type 1 copper 12

1.3.1.4. Substrate binding pocket 16

1.3.2. Trinuclear active site. 17

1.4. Catalytic mechanism of laccases 19

1.4.1. Oxygen reduction to water 19

1.4.2. Substrate oxidation. 23

1.5. Laccases purification, characterization and applications 24

1.5.1. Natural sources of laccases and their physiologic roles 24

1.5.2. Purification of laccases 28

1.5.3. Laccases characterization. 29

1.5.4. Applications of laccases 30

1.6. Sclerotinia sclerotiorum as laccase source candidate. 36

1.7. Copper complexes as models for laccase active sites 38

1.6.1 Model compounds for type 1 copper site. 39

1.6.2. Model compounds for type 2/3 copper sites 41

Chapter 2. Laccase is upregulated via stress pathways in the phytopathogenic fungus Sclerotinia sclerotiorum.. 43

2.1. Introduction. 43

2.2. Materials and methods 44

2.2.1. Chemicals and reagents 44

2.2.2. Culture media and growth conditions 44

2.2.3. Screening for laccase inducers 45

2.2.4. Carbon and nitrogen sources 45

2.2.5. Yeast extract as laccase inducer 46

2.2.6. Chelidonium majus extract as laccase inducer 47

2.2.7. pH and its role in laccase induction. 47

2.2.8. Biomass measurements 47

2.2.9. Laccase activity measurements 47

2.2.10. Data analysis 48

2.3. Results and discussions 48

2.3.1. Screening for laccase inducers 48

2.3.2. Carbon and nitrogen sources as laccase regulators 48

2.3.3. Yeast extracts enhance laccase production. 52

2.3.4. Influence of Chelidonium majus extract upon laccase production. 56

2.3.5. pH as regulator of laccase biosynthesis in Sclerotinia sclerotiorum.. 58

2.4. Conclusions 60

Chapter 3. Isolation, purification and characterization of S. sclerotiorum laccase. 61

3.1. Introduction. 61

3.2. Materials and Methods 62

3.2.1. Media and grow conditions 62

3.2.2. Enzyme isolation. 62

3.2.3. Enzyme assay and protein determination. 63

3.2.4. Enzyme characterization. 63

3.2.4.1. Electrophoresis 63

3.2.4.2. Molecular weight determination. 63

3.2.4.3. Spectral studies 64

3.2.5. Statistics 64

3.2.6. Mass spectrometric characterization of the laccase. 64

3.3. Results and discussion. 66

3.3.1. Culturing and laccase purification. 66

3.3.2. Characterization. 68

3.4. Conclusions 77

Chapter 4. Insights into S. sclerotiorum laccase mechanisms. 79

4. 1. Introduction. 79

4.2. Materials and methods 81

4.2.1. Chemicals 81

4.2.2. Laccase purification. 81

4.2.3. Adducts preparation. 82

4.2.4. UV-vis and fluorescence measurements 83

4.2.5. EPR measurements 84

4.3. Results and discussions 84

4.3.1. Blue laccase isolation. 84

4.3.2. Substrate-specific adduct colors 86

4.3.3. ABTS binds to a Tyr residue. 88

4.3.4. Enzymatic activity. 92

4.3.5. Stability. 94

4.4. Conclusions 96

Chapter 5. Application of S. sclerotiorum laccase in prooxidant evaluation of phenolics and propolis extracts. 97

5.1. Introduction. 97

5.2. Material and methods 100

5.2.1. Chemicals 100

5.2.2. Hemoglobin and laccase purification. 101

5.2.3. Enzyme kinetics measurements 101

5.2.4. Pro-oxidant and antioxidant activity measurements 102

5.2.5. Quercetin radical investigation by UV-vis and EPR spectroscopies 103

5.2.6. Propolis extracts preparation and investigation. 103

5.2.7. Cyclic voltametry measurements 104

5.2.8. HPLC-MS and MS investigations 104

5.2.9. Folin-Ciocalteu and hemoglobin/ascorbate peroxidase activity inhibition. 105

5.2.10. Lypofilicity data. 106

5.2.11. Statistics 106

5.3. Results and discussions 107

5.3.1. Characterization of flavonoid substrates 107

5.3.2. EPR and UV-vis detection of a species assigned as a flavonoid radical 109

5.3.3. Laccase-induced prooxidant reactivity of flavonoids on hemoglobin. 113

5.3.4. Applications on antioxidant and pro-oxidant activities of Romanian propolis extracts 118

5.3.4.1. Antioxidant evaluation of propolis extracts 118

5.3.4.1. Pro-oxidant evaluation of propolis extracts 130

5.4. Conclusions 133

Chapter 6. Models and theoretical approaches on laccase active sites. 135

6.1. Type 1 copper active site investigation. 135

6.2. Type 2/3 copper active site investigation. 138

General conclusions. 149

References. 151

List of publications. 175

 


 


 

List of Figures

Figure 1. The three domains A (red), B (blue) and C (green) of the Trametes Trogii (a) and the two domains A (red) and B (blue) of the SLAC laccase from Streptomyces coelicolor. The copper ions appear as orange spheres (b).The three domains of the Bacillus subtilis laccase (A (red), B (blue) and C (green)) showing the specific external loop connection and the lid-like structure of the third domain (c). Schematic presentation of several multicopper oxidases (LAC, AO, SLAC, CER, NR), adapted from (Skalova et al. 2009), based on their domain numbers and copper sites as well. Monomeric chains are distinguished by different shades of grey while the copper centers are depicted as black (T2/T3 types) and grey (T1 type) spheres (d). 3

Figure 2. Multiple sequence alignments of several laccases (TvLAC - Trametes versicolor (1GYC), ThLAC - Trametes hirsuta (3FPX), CcLAC - Coprinopsis cinerea (1HFU), CgLAC - Coriolopsis gallica (2VDZ), TtLAC - Trametes trogii (2HRH), CzLAC - Coriolus zonatus (2HZH), RlLAC - Rigidoporus lignosus (1V10), LtLAC - Lentinus tigrinus (2QT6), CmLAC - Cerrena maxima (3DIV), MaLAC - Melanocarpus albomyces (2Q9O), TALAC - Thielavia arenaria (3PPS); pdb codes of the proteins are given in parentheses) showing the laccase signature sequences that are forming four conserved regions (up) which are illustrated in the Coriolopsis gallica laccase as R1, R2, R3 and R4 (down). The four copper ions are shown as spheres. 4

Figure 3. C-terminus position of M. albomyces (i), T. arenaria (ii), B. subtilis (iii), T. hirsuta (iv) laccases. The first two have the N-terminal aminoacid towards the trinuclear cluster (one of the coppers from the cluster may be partly observed as orange ball) and is essential for enzyme activity while the last two have no role determined being positioned towards the surface of the protein (a). Sequence alignement of the C-termini of laccases from: M. albomyces (MaLAC, Q70KY3), N. crassa (NcLAC, P06811), C.globosum (CgLAC, Q2HBW4), C. parasitica CpLAC, (Q03966), P. anserina (PaLAc, P78722), G. graminis var. tritici (GgLAC, Q8TFE2), B. fuckeliana (BfLAC, Q96WM9), S. minor (SmLAC, A1YJE8), S. sclerotiorum (SsLAC1, A7EIG9), S. sclerotiorum (SsLAC2, A1YJE9), T.versicolor (TvLAC, Q12718), T. hirsuta (ThLAC, Q02497), T. arenaria (TaLAC, 3PPS – PDB code). In parentheses are the protein abbreviation and the Uniprot accession code. The highly conserved residues are red-colored while the medium conserved residues are blue-colored. The C-terminus motif of the asco-laccases DSGX is marked by two red arrows (b). 6

Figure 4. The sugar unit bond to Asn89 is stabilizing the three domains of Thielavia arenaria laccase (A, B and C) and the C-terminus tail by forming hydrogen bonds with Asn555 and Ser180 (a) while the sugar unit from Asn202 is stabilizing domains A and B by forming hydrogen bonds with Asn6, Arg11, Arg71, Tyr216 (b). 9

Figure 5. T1Cu and T2/T3Cu trinuclear center active sites of the Trametes versicolor laccase. The inter-site electron pathway is marked by black arrows; the distance between the oxygen atom of the cysteine and Nδ1 of His452 is also marked (2.78 Ĺ). The copper atoms are depicted as spheres. 10

Figure 6. Several laccase T1Cu geometries (distances given in Ĺ). First row: distorted tetrahedron geometries for SLAC, CueO and CotA laccase. Second row: distorted trigonal planar geometries for Letinus tigrinus and Thielavia arenaria laccases. One or two hydrophobic aminoacid such as Ile, Leu or Phe can be observed in the vicinity of the T1Cu center. 12

Figure 7. The movement of a helical segment (residues 455–461) in the Trametes versicolor laccase. According to Piontek (2002), the strong hydrogen bond between Ser113-Glu460 leads to an elongated His458-T1Cu distance. Some of the residues from the background have been removed for clarity. Distances are given in Ĺ. 15

Figure 8. Substrate binding pocket of Melanocarpus albomyces (3FU7), Trametes versicolor (1KIA), Bacillus subtilis (1UVW), Trametes trogii (2HRG) laccases binding 2, 6-dimethoxy-p-benzoquinone, 2, 5-xylidine, ABTS and p-toluate respectively (distances are in Ĺ). The ligand of the T1Cu center (depicted as orange sphere. The presumed hydrogen bonds are drawn in dotted lines. 17

Figure 9. Trinuclear center in the Thielavia arenaria laccase. The T2 and the two T3 copper atoms are depicted as grey spheres. Histidines 505 and 503 (yellow) are within the highly conserved HCH tripeptide motif and conduct the electrons from the T1 copper to the trinuclear centre. 18

Figure 10. Mechanism of oxygen reduction to water by laccases. The peroxy intermediate is a two-electron reduced species, and the native intermediate is a four-electron reduced species. 21

Figure 11. The possible mechanism of action of laccase mediator systems on non-phenolic compounds: via electron transfer route (ET) and via hydrogen atom transfer (HAT). 23

Figure 12. Laccase catalytic cycle in absence (up) and presence (down) of a mediator. 32

Figure 13. Life cycle of phytopathogenic fungus S. sclerotiorum (adapted from http://www.sclerotia.org/lifecycle/) 38

Figure 14. Coordination geometries of a set of 105 copper complexes (Cambridge Structural Database System) having 2 nitrogen and 1 sulfur ligands determined by X-ray diffractometry. The number of structures for each structure was obtained from (Gray et al. 2000) while the structures representations were obtained from (Averill and Eldredge 2006). 40

Figure 15. Right: Absorption spectrum of plastocyanin (left ε scale) and “normal” D4h [CuCl4]2- (right ε scale). Left: X-band EPR spectrum of plastocyanin (top) and D4h [CuCl4]2- (bottom) (Solomon et al. 2004). 40

Figure 16. Two most important type 1 copper models and their LUMO orbitals calculated with SCF XR-scattered wave (Xα-sw) (orbitals obtained and adapted from Solomon et al. 2004) 41

Figure 17. Dinucleating ligands with a phenoxo bridge create a type 3 copper model which can react with dioxygen quasi-reversibly in the Cu(I) state. R – H or Me, X and Y – 2-pyridyl, 1-pyrazolyl, 1-(3,5-dimethypyrazolyl) in different combinations. 42

Figure 18. Images of S. sclerotiorum mycelia grown on different carbon and nitrogen sources as labeled. Their maturation is 8 days from inoculation (at maximum laccase activity). The carbon and nitrogen sources influence the maturation time, sclerotia number and mycelia morphology. 50

Figure 19. Left: C/N influence upon laccase production (exponential decay fitting), triangle point: no carbon sources added; Right: Dependence of laccase activity on nitrogen and carbon sources concentration. The graph is based on twelve different experimental points and was constructed using 3D Surface Plot option (Statistica 7) and Distance Weighted Least Squares fitting procedure, stiffness 0.35 out of 0-1 interval. 52

Figure 20. Top: The variation of laccase activity in time at three different yeast extract concentrations which was added in day 4 after inoculation (ANOVA test, p<0.001, t-test, 0.006<p<0.016). The inoculated media were based on tryptose/saccharose. Control refers to the media with no supplementation. Inset – biomass measured as dried mycelium collected in day 8 after inoculation. Bottom: Pictures of mycelia of representative samples, before being collected and dried. 53

Figure 21. Laccase activity variations in time (from day 3 to day 8, from inoculation) at different yeast extract content, in presence of no extra nitrogen sources and 10 g/L saccharose as carbon source. Inset – biomass of the fungus measured as dried mycelium weight. The maximum laccase activity is reached at days 6-7 and at about 7 g/L yeast extract, over this value the activity is diminished. To a liquid medium containing the previously mentioned salts, trace elements and saccharose (5 g/L) and glucose (5 g/L), yeast extract was added at several concentrations (2, 4, 7, 11, 15 g/L). Each concentration was done in triplicates. After sterilization, cooling and inoculation (day 1), the laccase activity was monitored on days 3-8. The experiment was repeated twice. 54

Figure 22. Top: Biomass of the samples collected and dried in day 9 (ANOVA test, p=0.005). Bottom: The variations of laccase activity from day 5 to day 9 after supplementation with yeast extract, yeast extract fractions F1, F2, F3 lipid fraction according to Materials and Methods, and their mixture in day 3 after inoculation (ANOVA test, p<0.0000). Controls refer to culture samples with no supplementation. t-test confirms the highly significant differences between extract, mixture, F3 and Ref., p<0.001 and no differences between F1, F2, Lipids and Control p>0.46). 55

Figure 23. Images of mycelia of S. sclerotiorum at 9 days from inoculation grown tryptose/saccharose medium up to day 3, then supplemented with yeast extract, yeast extract fractions F1, F2, F3 according to Materials and Methods, lipid fraction and their mixture. Reference refers to culture samples with no supplementation. 55

Figure 24. Influence of medium pH at sterilization (glucose/saccharose/yeast extract medium) upon laccase activity (ANOVA test, p<0.001), biomass (ANOVA test, p<0.001), sclerotia number and mycelium morphology of S. sclerotiorum fungus grown for 9 days after inoculation. The pH at inoculation was set to 5.53±0.06. 56

Figure 25. Influence of C. majus extract on laccase activity (expressed as laccase activity/mg dried mycelium, ANOVA test: differences are significant, p=0.03) and number of sclerotia developed at day 6 of maturation (ANOVA test, p<0.0000, t-test confirms highly significant differences between extract treated samples and control, 0.0000<p<0.0017). The C. majus extract was added to a tryptose/saccharose medium on day 3 after inoculation. Ctr - controls refer to the samples with no supplementation. 57

Figure 26. Top: Biomass measured as dried mycelium weight of S. sclerotiorum at 9 days from inoculation grown tryptose/saccharose medium up to day 3, then supplemented with C. majus extract at different concentrations. Bottom: Images of two representatives of these mycelia. Ref. refer to culture samples with no supplementation. 58

Figure 27. SDS-PAGE and the corresponding electrophoretic protein profiles of the extracelluar medium at day 6 (tryptose/saccharose medium (control – ctr.)) supplemented with C. majus extract on day 3, at different volume concentrations. Mainly regulated proteins are indicated by arrows up (positive regulation) and down (negative regulation). 58

Figure 28. Kinetic profiles of the pH variation of the extracellular medium of S. sclerotiorum fungus. The experimental data (averages of three distinct samples for each pH) are fitted with a Bolztmann sigmoid function whose t80% values are plotted in inset. 59

Figure 29. Influence of medium pH at inoculation upon laccase activity (measured at 120 h after inoculation) is statistically significant (ANOVA test, p=0.025) and not significant (ANOVA test, p=0.8) upon biomass (dried mycelium weight) of the S. sclerotiorum fungus. The pH at sterilization was 9.5 and was the same for all samples. No significant differences were observed in the number of sclerotia for these tested samples. 60

Figure 30. Determination of optimum ammonium sulphate saturation for laccase precipitation from the culture medium.. 66

Figure 31. Purification of laccase by anion exchange chromatography (HiTrap, GeHelathcare). The laccase was eluted from the column at ~ 27% buffer B. Buffer A: tris 20 mM pH 7, buffer B: tris 20 mM pH 7, NaCl 1M.. 67

Figure 32. Purification of laccase by hydrophobic ionic interaction (Phenyl-5PW, Tosoh). The laccase was eluted from the column at ~ 87% buffer A. Buffer A: (NH4)2SO4, tris 20 mM pH 7, buffer B: tris 20 mM pH 7. 67

Figure 33. Molecular weight determination of final pure laccase by analytical size exclusion chromatography. The molecular weight markers are mentioned in Materials and methods section. 68

Figure 34. Left: Monitoring the laccase purity by 14% SDS-PAGE. 1 – Crude extract; 2 – Anion exchage step; 3 – Hydrophobic ionic interaction chromatography step; Lac – Size exclusion chromatography, final step. M – Markers. Laccase apparent molecular weight ~65 kDa. LacABTS – SDS-PAGE separated laccase (without heat treatment to avoid copper loss) stained with ABTS in 50 mM acetate buffer pH 4, appears as a wide green band due to ABTS+● formation. Right: Concentrated and diluted final pure laccase samples on 14% SDS-PAGE. 69

Figure 35. First panel: UV-vis spectra of final pure laccase showing the 330 nm feature which is specific to T3-type binuclear center. The ~610 nm band is not detectable even in high concentration solution (inset). Second panel: S. sclerotiourum laccase superimposed on a blue laccase as reference (from Streptomyces coelicolor). 70

Figure 36. Fluorescence spectra of the purified laccase. The emission spectrum (centered at a maximum of 440 nm) was obtained by excitation at 330 nm and excitation spectrum (maxima at 280 and 330 nm) by 420 nm emission. The laccase concentration was ~ 10 µM in MES buffer 20 mM. 71

Figure 37. EPR spectra of purified laccase, showing specific features for a T1 copper center - g ~ 2.2 and A ~ 83 x 10-4 cm-1 – as well as T2 copper center features (g ~ 2.4, A ~ 178 x 10-4 cm-1). The experimental spectrum was simulated using the POWFIT software. 72

Figure 38. pH dependence of the laccase activity with several substrates (first panel) and temperature dependence using ABTS as substrate (second panel) (inset – Arrhenius plot on the 25 -55C interval). Third panel – activation followed by exponential decay inactivation of laccase activity exposed at 40C and 50C (inset). 73

Figure 39. Multiple sequence alignment of several laccases designated by the PDB codes (organism source: 3PPS - Thielavia arenaria, 1GW0 – Melanocarpus albomyces, 1KYA – Trametes versicolor, 3FPX – Trametes hirsuta, 2QT6 – Lentinus tigrinus, 1V10 – Rigidoporus lignosus) and the S. sclerotiorum laccase designated by the uniprot code A7EM18. The alignment was obtained by MultAlin version 5.4.1 software (Corpet 1988). 75

Figure 40. CD spectrum of S. sclerotiorum laccase (9.4 µM) as purified in 5 mM TAPS pH 7.8. 76

Figure 41. UV-vis spectra of blue and yellow forms of the laccase of S. sclerotiorum in 25 mM MES buffer pH 6.3. The protein concentration is 7.5 µM. 85

Figure 42. EPR spectra of blue laccase form revealing both the type 1 copper center and the type 2 copper center. Conditions: 25 mM MES pH 6.3, 60 µM laccase. 86

Figure 43. UV-vis spectra of the blue (a) and yellow (b) laccases, and of the red guaiacol (c), orange TMB (d), and purple ABTS (e) laccase adducts. The spectra are normalized at 280 nm. The approximate concentration of the protein (possible ε280nm variation) is 8 µM. 87

Figure 44. ABTS-tyrosine (black) and ABTS-laccase (grey) UV-vis spectra at pH 6.3 (25 mM MES). 88

Figure 45. Tyrosine and ABTS form an adduct in a 1:1 ratio (left panel), as deduced from a Job’s plot. The solution spectra (right panel) are measured after overnight reaction of ABTS with tyrosine in different molar reaction ratios ([Tyr]/[ABTS]) as labeled, in presence of 15 nM laccase, 50 mM citrate buffer, pH 4. 89

Figure 46. pH titration of ABTS-tyrosine (circles) and ABTS-laccase (rhombuses) adducts. The pKa values are calculated by fitting the experimental data with a sigmoidal model. B: UV-vis changes of ABTS-laccase adduct while pH decreases. 89

Figure 47. Active sites of S. sclerotiorum laccase as modeled having M. albomyces laccase as template. The Tyr426 found close to the substrate pocket (between two loops at ~ 4Ĺ away from substrate pocket (2,6-dimethoxyphenol found in M. albomyces substrate pocket)) may be susceptible to form the adduct. 91

Figure 48. Reduction of pure yellow laccase (a) with dithionite in excess (b) and re-oxidation by exposition to air (c) monitored by UV-vis spectroscopy. Inset: Blue form (solid black), guaiacol-laccase form (solid gray) and difference spectra for yellow laccase (dotted): a – b (isolated oxidized – reduced), c – b (re-oxidized – reduced), and a – c (isolated oxidized – re-oxidized). 91

Figure 49. Michaelis-Menten curves for blue (A), yellow (B), ABTS (C), guaiacol (D), TMB (E) laccases and their Eadie-Hofstee linearization plots (as insets) using Q0H2 as substrate. Km and kcat values are listed in Table 9. 92

Figure 50. Fluorescence spectra (emission while exciting at 330 nm and excitation while followed at 420 nm) of blue, yellow and ABTS-laccase. Protein solutions are in 20 mM TrisHCl pH 6.8. 94

Figure 51. Exponential decays of activity of yellow, blue and ABTS-laccase while exposed at 50C. The half-lives are 3.5, 0.8 and 1.1 h respectively while at 40C are 8.2, 5.6 and 6.8 h respectively. 95

Figure 52. Titration of yellow, blue and ABTS-laccase with guanidine hydrochloride monitored by the shift of the maximum band of the emission spectrum while the sample was excited at 280 nm, using fluorescence spectroscopy. The experimental data was fitted with sigmoidal curves, yielding inflection points: yellow laccase (rhombuses) 2360 ± 67; ABTS-laccase (circles) 2616 ± 36; blue laccase (triangles) 2835 ± 57. 95

Figure 53. A: Quercetin (40 µM) oxidation by laccase (113 nM) in 100 mM pH 6 acetate buffer; spectra from only a few time points are shown, for clarity. Inset – appearance (black) and disappearance (grey) of the absorbance proposed to be assigned to the quercetyl radical (centered on 540 nm) during the reaction. B: Kinetic profile of quercetin oxidation at 365 nm (circles) and of quercetin radical at 540 nm (rhombuses). Inset – first derivative of 365-nm kinetic profile. 109

Figure 54. A: Formation and disappearance of quercetin radical during the oxidation of quercetin by laccase (226 nM) in 100 mM acetate buffer pH 6 monitored at 540 nm. The quercetin concentration is varied from 5 µM to 100 µM. B: Variation of spectral maximum of the radical (rhombuses) and the time required to reach it (circles). 110

Figure 55. A. Formation and disappearance of quercetyl radical during the oxidation of quercetin (40 µM) by laccase in acetate buffer at pH 6, monitored at 540 nm. The laccase concentration is varied from 56 nM to 906 nM. B: Variation of spectral maximum of the radical (rhombuses) and the time required to reach it (circles). 110

Figure 56. Variation of the area under the curve for the ~540 nm signal, vs. the quercetin concentration (rhombuses) while keeping the laccase concentration constant at 226 nM, and vs. the laccase concentration (circles) while keeping quercetin concentration constant at 40 µM. 111

Figure 57. A: EPR spectra of DMPO-phenoxyl radical spin adduct of luteolin, kaempherol, quercetin and propolis extract at 95 K after 4 minutes turnover with laccase 90 nM in 100 mM acetate buffer, pH 6, 100 U/mL CAT, 100 U/mL SOD. The reaction was stopped by freezing in liquid nitrogen. B: EPR spectra of DMPO-phenoxyl radical spin adduct of quercetin after different time of turnover (stopped with sodium fluoride), at room temperature. 112

Figure 58. Hemoglobin (100 µM) oxidation during successive addition of quercetin (3x2.4 µM) in presence of laccase (283 nM) and 83 U/mL SOD and 150 U/mL CAT in 100 mM pH 6 acetate buffer (some intermediate spectra were removed for clarity). Inset – kinetic profile of 577 nm band, specific to oxyHb. 114

Figure 59. Variation of the oxy-Hb oxidation rate with the time elapsed from the start of the reaction. The reaction was initiated by addition of laccase (113 nM) into a quercetin (40 µM) solution. At this time point the oxy-hemoglobin (34 µM) was added and the initial rate of the hemoglobin oxidation was measured monitoring the 540 nm band (also specific to oxy-hemoglobin). The measurements were done in triplicate, error bars are in SE terms. Inset – correlation of the measured rate to absorbance at 540 nm (a measure of quercetyl radical concentration). 114

Figure 60. Kinetic profile of oxy-hemoglobin oxidation (34 µM Hb, 83 U/mL SOD, 150 U/mL CAT, 113 nM laccase in 100 mM acetate buffer pH 6) monitored at 577 nm. The compound concentration is 2 µM. 115

Figure 61. A. Kinetic profile of oxy-hemoglobin (10 µM) oxidation (monitored at 577 nm) change with quercetin concentration and B. calibration curve obtained by area under the curve (AUC) vs. ln[quercetin] used for calculation of quercetin equivalents which leads to the prooxidant quercetin factor (pQF) value. 116

Figure 62. Correlation of the proposed QFs and the %DPPH250s for the studied propolis samples. 121

Figure 63. UV-vis electronic spectra of some propolis extracts (a). The 353 nm absorbance correlates with the antioxidant capacity and with 1630 cm-1 absorbance (R = 0.889, p < 0.0001) (b). 123

Figure 64. Grouping of the propolis samples according to their origin using the scores of the principal components obtained after the PCA was applied on the 240 - 410 nm UV-vis spectroscopic range (a) and on the 1454 - 1780 cm-1 IR spectroscopic range (b), DPPH kinetic profile (c). 124

Figure 65. HPTLC silica gel chromatographic plates exposed at 366 nm after analysis of some studied propolis samples. The mobile phase was toluene:ethyl acetate:formic acid in 30:12:5 volumetric ratios (first panel); the second sample (from left to right, first T24) chromatogram in all three channels RGB. The four compounds identified were caffeic acid (1), quercetin (2), apigenin (3) and galangin (4) (second panel). 126

Figure 66. Time profile of ascorbic acid consumption by peroxide catalyzed by hemoglobin, monitored at 290 nm; times of reagent addition are indicated by arrows. 127

Figure 67. EPR spectra of base-treated propolis extracts at varying concentrations. Inset shows that the double-integrated EPR signal areas are directly dependent upon the concentration of natural extract added to the reaction mixture. 127

Figure 68. Free radical signals detected by EPR in various propolis samples of different origin. Inset - arithmetic difference between two signals reveals the existence of two main components present within the sample. For sample number see Table 1. 128

Figure 69. EPR signals of radicals generated in ethanolic solutions of related or found in compounds from propolis by sodium hydroxide treatment. Inset – luteolin and sample 11 similarities of EPR signals. The silent EPR spectrum of 6-chloro-7-methylflavone is identical with flavone and 2’-methoxyflavone. 129

Figure 70. Box and Whisker plot of pQFs for some propolis samples from various geographical locations and floral. The measurements were done at 25 µg/mL propolis, 10 µM oxy-hemoglobin, 113 nM laccase, and 83 U/mL SOD and 150 U/mL CAT and were performed in duplicates. 132

Figure 71. First derivative of the kinetic profile of the oxidation of oxy-hemoglobin monitored at 577 (34 µM Hb, 83 U/mL SOD, 150 U/mL CAT, 113 nM laccase in 100 mM acetate buffer pH 6, 1 µM flavonoid) in presence of individual or binary mixture with quercetin of luteolin (A), kaempherol (B), 5,7-dihydroxyflavone (C), galangin (D). 133

Figure 72. General structure of laccases (based on pdb ID 2Q9O), highlighting the four copper ions and the coordination environment around the type 1 copper center. 135

Figure 73. Model of the type 1 copper of laccases, employed in the present work. Arrows indicate bonds around/along which distortions were performed in spectroscopic calculations. 136

Figure 74. The main orbitals responsible for the blue color of the laccase type 1 center, according to ZINDO/S-CI calculations. 137

Figure 75. Catalytic cycle that may in principle be operating for copper-containing oxygenases. Dashed arrows indicate alternative pathways which may all lead to product. The protein-derived ligands are considered to be three histidinines, as seen in some but not in all dioxygen-activating copper enzymes. 140

Figure 76. UV-vis spectra of CuTPPs (2 μM) in 50 mM acetate pH 5, with 200 mM nitrite, 475 μM ABTS, 40 mM guaiacol, 10 mM dithionite. 142

Figure 77. EPR spectra of CuTPPs (250 μM) in in 50 mM acetate pH 5, with 40 mM nitrite, 2 mM ABTS, 80 mM guaiacol, 10 mM dithionite, 40 mM imidazole. 143

Figure 78. Spin densities computed for copper(II)-porphyrinate models with the metal four-coordinated (left) or five-coordinated (axial ligation by a water molecule – center) and compared to an Fe(III)-nitrite porphyrinate model. 144

Figure 79. Models employed to evaluate to what extent copper (III) may be accessed in a biologically-relevant coordination environment 145

 



 

List of Tables

Table 1. Redox potentials of various laccases and corresponding sequence alignments. The conserved HCH tripeptide, axial ligand (last) are marked in bold. N.d refers to laccases whose sequence was not determined. The pair of aminoacids marked in bold and italics is involved in the Piontek hypothesis (see text). Unless stated (UP – uniprot database, PDB – Protein Data Bank), the sequence codes are from GenBank. E0 is measured vs. NHE......................... 14

Table 2. Recent cultivation conditions and purification procedures with their yield and fold factors for several laccase purifications from different organisms.............................................. 29

Table 3. Statistical data of Km and kcat values for most used laccase substrates...................... 30

Table 4. Some characterization data regarding some recently purified laccases. The references for each organism can be found in Table 2......................................................................... 31

Table 5. Effects of various potential laccase inducers upon the laccase activity in S. sclerotiorum................................................................................................................................... 49

Table 6. Biomass and laccase activity variations when different carbon and nitrogen sources were used............................................................................................................................ 50

Table 7. Concentration/purification of laccase using salt precipitation and chromatographic methods...................................................................................................................... 68

Table 8. Substrate catalytic parameters of the purified laccase obtained by non-linear fitting model using Origin 8............................................................................................................. 74

Table 9. Michaels-Menten parameters for three substrates for five forms of the purified laccase obtained by Eadie–Hofstee linearization in case of biphasic cases (Q0H2) and non-linear fitting using GraphPad for normal curves................................................................................ 93

Table 10. Prooxidant, antioxidant, enzymatic kinetic parameters and redox potentials of the studied compounds................................................................................................................ 108

Table 11. Floral, geographical origins and description of the studied propolis samples......... 119

Table 12. Correlation coefficients between several calculated parameters of the kinetic profile of DPPH bleaching assays and some relevant IR and UV-vis absorbances......................... 121

Table 13. Distinct bands in FT-IR spectra found in propolis extracts................................... 122

Table 14. Geographical origins and their numbering the propolis samples taken into this section study (interaction with hemoglobin)........................................................................... 125

Table 15. Correlation coefficients between various antioxidant measurement methods. “DPPH” denotes the percent decrease in DPPH absorbance in 680 seconds. GAE denotes the gallic acid equivalents, in mg/mL. HAPX denotes the ratio between rates of ascorbate consumption by hemoglobin and peroxide in the absence and presence of propolis, respectively. EPR denotes the area under the EPR signal; as determined by double integration. Details are found in Materials and Methods............................................................................................... 130

Table 16. Several parameters regarding binary mixture experiments................................... 133

 



 

Abbreviations

Ĺ – ĺngström (1Ĺ=10-10 m);

ABTS – a well known laccase substrate: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid);

ANOVA – analysis of variance;

APCI – atmospheric-pressure chemical ionization;

AUC – area under the curve;

C/N – carbon to nitrogen ratio;

cAMP – cyclic adenosine monophosphate;

CAT – catalase;

CD – circular dichroism;

CER – ceruloplasmin;

CotA – the protein (with laccase activity) encoded by the gene with the same name involved in spor coat dvelopement of Bacillus subtilis;

CueO – copper efflux oxidase (the laccase from Escherichia coli);

CuTPP – [5, 10, 15, 20-tetrakis(N-methylpyridyl-4)porhinato]copper(II) tetratosylate);

2,6-DMP – 2,6-dimethoxyphenol;

D4h – denotes a symmetry group;

Da – daltons;

DAD – diode array detector;

DMPO – 5,5-dimethyl-pyrroline N-oxide;

DPPH – 2,2-diphenyl-1-picrylhydrazyl;

DTT – dithyotreitol;

E – normal electrode potential;

EPR – electron paramagnetic resonance;

ESI – electrospray ionization;

FPLC – fast protein liquid chromatography;

(FT)IR – Fourier transformed infrared;

GAE – gallic acid equivalent;

GuHCl – guanidine hydrochloride;

HAPX – hemoglobin-ascorbate peroxidase;

Hb – hemoglobin;

HOMO – highest occupied molecular orbital;

kcat – catalytic constant (turnover number);

Km – Michaelis-Menten constant;

LAC – three domain laccase (common laccase);

LC(-MS) – liquid chromatography (-mass spectrometry);

LMCT – ligand to metal charge transfer;

LUMO – lowest unoccupied molecular orbital;

MES – 2-(N-morpholino)ethanesulfonic acid used as buffer;

MOPS – 3-(N-morpholino)propanesulfonic acid used as buffer;

NHE – normal hydrogen electrode;

nm – nanometer;

NMR – nuclear magnetic resonance;

NR – nitrite reductase;

p – the probability of obtaining a statistic test;

PAGE – polyacrylamide gel electrophoresis;

PBS – phosphate buffer saline;

PCA –Principal Component Analysis;

PDB – protein data bank;

Ph-OH – generic formula for phenolic compound;

pQF – prooxidant quercetin factor;

Q0 – dimethoxy-5-methyl-p-benzoquinone;

QF – quercetin factor;

QM/MM – quantum mechanical and molecular mechanics;

RGB – red green blue color channel;

RNS – reactive nitrogen species;

ROS – reactive oxygen species;

RP – reversed phase;

rpm – rotations per minute;

RSD – relative standard deviation;

SD – standard deviation;

SDS – sodium dodecyl sulfate;

SLAC – small laccase (one domain laccase);

SOD –superoxide dismutase;

T1Cu – type 1 copper;

T2Cu – type 2 copper;

T3Cu – type 3 copper;

TAPS – 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid used as buffer;

TEAC – trolox equivalent antioxidant capacity;

TMB – tetramethylbenzidine;

TRIS – 2-amino-2-hydroxymethyl-propane-1,3-diol used as buffer;

U – enzymatic unit;

UV-vis – ultra violet and visible;

V – volt;

WE –working electrode;


 

Acknowledgements

 

 

 

 

I want to give thanks to prof. dr. Florin Dan Irimie for allowing me to be his PhD student and for all his time afforded whenever it was required. I am very gratefully to conf. dr. Radu Silaghi-Dumitrescu who was a wonderful scientific supervisor, for all his impartial and kind help, advice and scientific guidance offered from his nice and respectful conviction. Many thanks are also given to Dr. Dirk Heering for all his scientific advice, nice time spent together and for allowing me to work in his laboratory. Prof. dr. Grigore Damian, conf. dr. Marcel Parvu, conf. dr. Costel Sarbu, dr. Iulia Lupan, dr. Alex Lupan, dr. Zsuzsa Darula, Cristina Coman are all thanked for their help and helpful discussions during the work of this thesis.

In the end I want to mention that I will remain with wonderful memories from laboratory 6 wherever I will be in future, thus all people met there are nicely hugged.

 

 

 

 


 


Aims of the thesis

 

The present thesis has five main objectives which were stated in a preliminary form before the work was started and suffered several adjustments during the work proceeded. Each of these objectives contains several main steps which were foreseen in the research thesis project or was established during the ongoing procedures.

  1. Determination of optimum conditions considering maximum laccase activity in liquid culture of Sclerotinia sclerotiorum
    • Evaluation of several types of liquid mediums upon laccase secretion;
    • Assessing the influence of numerous important parameters/conditions upon laccase production by the fungus such as: pH, N and C sources, time, several inducers;
    • Evaluation of some important physiologic relevant factors upon laccase regulation in order to bring some insights of its physiologic role;
  2. Isolation, purification and characterization of Sclerotinia sclerotiorum laccase

·      Establishment of suitable protocols for laccase isolation and purification using chromatographic and electrophoretic facilities;

·      Determination of specific activity and biochemical properties (KM, kcat, optimum pH and temperature, thermostability, substrate selectivity) of the purified enzyme;

·      Spectral characterization of the purified enzyme (UV-vis, CD, EPR, MS);

  1. Elucidation of the mechanism of the reactions catalyzed by the pure enzyme

·      Characterization of possible reaction intermediates and their kinetics;

·      Study of enzyme – substrate interaction;

  1. Applications of the Sclerotinia sclerotiorum laccase

·      Establishment of protocols suitable for evaluations of prooxidant and antioxidant activities of polyphenols and some natural extracts;

  1. Theoretical and experimental studies of model compounds for laccases

·      Evaluation of reactivity towards some ligands and laccase substrates of some copper complexes;

·      Theoretical studies of laccase copper centers concerning their reactivity and spectral behaviour.



 

General Introduction

 

 

 

P

roteins having one or more copper ions as cofactors play very important roles in cellular metabolism of all living organisms. They are involved in photosynthesis, oxidative phosphorylation, homeostasis of metal ions and catabolism of many nutrients. The main reactions involving copper proteins are electron transfer, this due to copper ability to exist in two oxidation states Cu+ and Cu2+. Copper centers in proteins have such a coordination sphere provided by the polypeptidic structure so that the transition from one oxidation state to another to be thermodynamically favorable.

           The simplest copper dependent proteins are azurins and plastocyanines, they are usually involved in electron transfer reactions. Other more complex proteins, with copper ions in the active sites, such as galactose oxidase, nitrite reductase, ceruloplasmin, ascorbate reductase, bilirubin oxidase and last but not least laccase, are involved electron transfer reactions from reduced substrates to electron deficient molecules.

Laccase (p-diphenols: dioxigen oxidoreductase) is an oxidoreductase (EC 1.10.3.2) with four copper ions in two active sites, which catalyzes the oxidation of reduced substrates usually phenols or aromatic amines, coupled with the reduction of molecular oxygen to water. Laccase is one of the oldest enzymes ever studied, it was described for the first time by Yoshida (1883) and categorized by Bertrand (1895) as a copper containing oxidase. However, only in recent decades, when it was discovered that laccases are part of the enzymatic arsenal involved in wood degradation by white rot fungi, study of these enzymes has greatly increased. A more recent interest in this enzyme is its involvement in the virulence of some phytopathogenic fungi, as is the case of the present thesis.

Currently, this enzyme is the central subject of many worldwide research groups, due to scientific curiosity and its high potential in numerous applications in biotechnology and bioanalytical chemistry.


 

Summary of the content of the thesis

 

The first chapter describes the most recent research on the overall structural features of laccases as well as on the structures and properties of the active sites, along with the currently proposed mechanisms of reaction. Laccase (p-diphenol:dioxygen oxidoreductase), one of the oldest discovered enzymes, contains four copper ions in two active sites and catalyzes a monoelectronic oxidation of substrates such as phenols and their derivatives, or aromatic amines, coupled to a four-electron reduction of dioxygen to water. The catalytic mechanism was studied for decades but is still not completely elucidated, especially in terms of the reduction of dioxygen to water. The key structural features of this enzyme are under research in several groups using techniques such as X-ray diffraction, electron paramagnetic resonance (EPR) spectroscopy, site-directed mutagenesis. The high interest in laccases is explained by the large number of biotechnological applications. Their distribution in nature, the physiologic role, most used methods for purification and biochemical properties and parameters used for their characterization are also described. Numerous applications of laccases such as textile industry, wood processing paper production, pharmaceutical and chemical industries and others are described. Some biological aspects regarding Sclerotinia sclerotiorum phytopathogenic fungus and reasons for using this organism as laccase source are presented at the end of the chapter. In the last part of the chapter some copper complexes used as models for laccase active sites are discussed.

The second chapter describes the factors affecting the production of laccase from the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary. The carbon/nitrogen ratio appears to be of great importance. Rather than a simple nutrient-rich nitrogen source, yeast extract behaves as a true laccase upregulator, apparently acting via a stress pathway. Chelidonium majus extract, a known antifungal agent, acts in a similar manner. The compound(s) in the yeast extract responsible for enhancing laccase synthesis are suggested to be hydrolysable small organic molecules. Both extracts reduce biomass and sclerotia development and enhance laccase production, leading to an increase in laccase activity by one order of magnitude compared to controls. The pH of the medium, a well-known virulence regulator for this fungus, also acts as a true laccase regulator, though via a different mechanism. The effect of pH appeared to be linked to the acidification kinetics of the extracellular medium during fungal development. A number of other known laccase inducers were found to enhance laccase production at most two-fold.

Chapter three contains information regarding the production, purification and characterization of a laccase from the phytophathogenic fungus Sclerotinia sclerotiorum. This laccase is identified by mass spectrometry with a sequence coverage of 74.9% (458/577 AA) revealing that the protein is identical or highly homologous to a predicted oxidoreductase from this species (A7EM18 in the Uniprot database); the closest homologous protein previously isolated from a fungus is the Melanocarpus albomyces, with only 35% identity. The UV-vis spectral features of this laccase classify it as a “yellow” one. The EPR spectrum nevertheless demonstrates resemblance to blue laccases – including the type 1 center not detectable in UV-vis spectra. The presence of type 3 coppers was proven by fluorescence spectrum and by 330 nm band in UV-vis. The purified laccase has an apparent molecular mass of 70 kDa and appears as a monomer. The values of KM and kcat were determined for ABTS, 2,6-dimethoxyphenol, p-phenylenediamine and guaicol and are typical of a laccase. The optimal pH value is around 4 except for ABTS, for which activity is linearly increasing with acidity. The high laccase activity in liquid culture makes Sclerotinia sclerotiorum a useful source of laccase for practical applications.

In chapter four it is provided the first evidence that the yellow laccase isolated from Sclerotinia sclerotiorum is obtained from a blue form by covalent, but nevertheless reversible modification with a polyphenolic product. Yellow laccases lack the typical blue type 1 Cu absorption band around 600 nm, but are nevertheless multicopper oxidases with laccase properties. After separating the polyphenols, a typical blue laccase is obtained. With ABTS as model substrate for this blue enzyme, a purple adduct is formed with a spectrum nearly identical to that of the 1:1 adduct of an ABTS radical and Tyr. This modification significantly increases the stability and substrate affinity of the enzyme, not by acting primarily as bound mediator, but by allosteric activation that also alters the type 1 Cu site. Thus, S. sclerotiorum yellow laccase is an intrinsically blue multi-copper oxidase that autocatalytically activates itself upon first encounter with a radical-forming aromatic substrate.

The fifth chapter contains numerous results regarding the application of the purified enzyme on antioxidant and prooxidant properties of some phenolics and propolis extracts. A transient species may be detected with UV-vis and EPR spectroscopy during turnover of a laccase with quercetin; this species is assigned as a quercetin-derived radical, based on EPR spectra as well as based on UV-vis similarities with previously reported data on a quercetyl radical obtained via a non-enzymatic route. The formation and decay of this species correlate well with the prooxidant reactivity manifested by flavonoids in the presence of laccase. An assay for the prooxidant reactivity of natural compounds is proposed based on the results reported here; this assay has the advantages of using a biologically-relevant process (hemoglobin oxidation), and of not needing added oxidizing agents such as peroxide or superoxide. Correlations, or the lack thereof, between the prooxidant parameters and the redox potentials, antioxidant capacities and lipophilicities, are analyzed. New assays for antioxidant activity of natural extracts are also described. It can be noted that the laccase employed in this study does display structural and reactivity-related similarities to a range of other proteins, which includes ceruloplasmin.

The last chapter of the thesis contains the results regarding molecular modelling of laccase active sites and the experiments describing the reactivity of some copper complexes used as models for type 2 copper sites. Laccases contain a blue mononuclear copper center known as ‘type-1’, and thought to be the primary electron acceptor from organic substrates during the catalytic cycle. A small group of laccases are also known that lack the 600 nm band and hence the blue color (“yellow laccases”). In first section it is reported the use of semiempirical (ZINDO/S-CI) calculations in order to simulate UV-vis spectral parameters for the laccase type 1 copper, attempting to assign geometrical and electronic structure elements that may control the color of this site. The ~600-nm band of the type 1 copper is confirmed to arise mainly from sulfur-to-copper charge transfer, and strong distortions allowing for its displacement by more than 200 nm and/or its dissolution are identified. In the second section some copper porphyrinates are analysed with respect to its reactivity towards some laccase substrate and some other redox active compounds. Copper porphyrinates are generally known to display a less diverse reactivity compared to their iron counterparts. It is examined a water-soluble copper porphyrinate for its ability to engage in reactions involving axial ligation to the copper or possible redox cycling. Although UV–vis spectra indicate an expected lack of reactivity, electron paramagnetic resonance spectra (EPR) reveal an unexpected wealth of changes in electronic structures at the copper, induced by potential ligands such as imidazole or nitrite, but also by seemingly unexpected candidates for ligands, such as 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and guaiacol, as well as by dithionite. An important function of many copper-containing proteins is activation of O2 and subsequent substrate oxidation. The Cu (III) oxidation state is generally considered to be less accessible because of the highly positive Cu (III)/Cu (II) redox potentials with typical amino acid ligands. In the last part it is employed density functional (DFT) calculations to explore to what extent copper (III) may be accessed in a biologically-relevant coordination environment around a mononuclear copper center, by breaking the oxygen-oxygen bond in a copper-(hydro) peroxide complex. In agreement with previous findings on copper models with related coordination patterns, the formally high-valent copper complex produced by O-O bond cleavage appears to in fact harbor both oxidizing equivalents on the ligands. The potential energy surface for such a reaction reveals that with the three-histidine binding motif at the copper, O-O bond cleavage is not impossible, but rather disfavored thermodynamically.


General conclusions

 

·  Optimal conditions under which the S. sclerotiorum laccase can be produced were determined. The carbon and nitrogen sources and C/N ratio appear to be of great importance for laccase production in this fungus. Rather than a simple nutrient-rich nitrogen source, yeast extract behaves as a true laccase inducer/upregulator, apparently acting via a stress pathway. Chelidonium majus extract, a known antifungal agent, acts in a similar manner. The pH of the medium, a well-known virulence regulator for this fungus, also acts as a true laccase regulator, though via a different mechanism. The effect of pH appears to be linked to the acidification kinetics of the extracellular medium during fungal development. Thus, evidence is shown that this enzyme is involved in stress response pathways, most likely connected to virulence.

·  Sclerotinia sclerotiorum laccase has been isolated, and its catalytic properties characterized. Notably, although this laccase can be classified as a “yellow laccase” based on the UV-vis spectrum, the “blue” T1 center is nevertheless observable in the EPR spectrum. The extent to which the S. sclerotiorum laccase may indeed allow definition of a new type of laccase (neither truly “blue”, nor truly “yellow”) remains to be explored, especially as for most yellow laccases the EPR spectra have not been reported; should such a class be confirmed, a term such as “mixed blue-yellow” might be appropriate.

·  Direct evidence for an example where a blue laccase can be converted to a yellow form in vitro by covalent modification at the T1 site, with metabolites produced by the laccase itself was provided. Moreover, this autocatalytic modification significantly improves the structural and catalytic properties of the enzyme. In essence, S. sclerotiorum yellow laccase is an intrinsically blue multi-copper oxidase that has activated itself upon first encounter with a polyphenolic substrate. A tyrosine residue was identified near the T1 site, which may be the target of such modifications.

·  A transient species may be detected with UV-vis and EPR spectroscopy during turnover of a laccase with quercetin; this species is assigned as a quercetin-derived radical, based on EPR spectra as well as based on similarities with previously reported data. Furthermore, this species correlates well with the prooxidant reactivity manifested by flavonoids in the presence of laccase. An assay for prooxidant reactivity of natural compounds is proposed based on these results, which has the advantages of using a biologically-relevant process (hemoglobin oxidation), and of not needing added oxidizing agents such as peroxide or superoxide. Correlations, or the lack thereof, between the parameters obtained from this assay and redox potentials, antioxidant capacities and lipophilicities, are discussed. It was also noted that the laccase employed in this study does display structural and reactivity-related similarities to a range of other proteins, which includes the serum ceruloplasmin, and also displays reactivity similarities with heme-containing peroxidases. In addition, a new more informative and effective scale of antioxidant capacity is obtained by applying PCA on DPPH (2, 2-diphenyl-1-picrylhydrazyl) bleaching kinetic profiles. In order to obtain comparable antioxidant activities, a non-dimensional parameter was generated which is termed the quercetin factor (QF), which defines the ratio between quercetin equivalent in mg/L of the assayed propolis sample and the corresponding propolis concentration in mg/L. Further application of this methodology to other botanical extracts will confirm this new method for assessing antioxidant activity.

·  The coordinative chemistry of copper porphyrinates may be distinctly more complex than previously described, and that EPR but not UV-vis spectroscopy is the method of choice for investigating this new chemistry.

·  Using computational methods, torsion and elongation-type deformations have been identified, which allow a “blue” tri-coordinated type 1 copper center to apparently lose its characteristic 600-nm band responsible for its blue color both by shifting it by more than 200 nm, and, in some cases, by decreasing the extinction coefficients. However, DFT calculations suggest that such distortions might also be detectable with EPR spectroscopy.

·  Unlike in related iron or manganese complexes, high-valent states appear not to be achievable via peroxo chemistry in copper complexes – even though O-O bond cleavage per se appears to entail reasonably low energy barriers; this may be interpreted to be due to a difference in redox potentials, which makes the peroxide-derived hydroxo and oxo ligands easier to oxidize than Cu (II).


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List of personal publications on thesis topic at 03rd October 2012

 

1.         Moţ A.C., Damian G., Sarbu C., Silaghi-Dumitrescu R., Redox reactivity in propolis: direct detection of free radicals in basic medium and interaction with hemoglobin, Redox Report 14 (2009) 267-274; (IF: 1.732)

2.         Moţ A.C., Silaghi-Dumitrescu R., Sarbu C., Rapid and effective evaluation of the antioxidant capacity of propolis extracts using DPPH bleaching kinetic profiles, FT-IR and UV–vis spectroscopic data, Journal of Food Composition and Analysis 24 (2011) 516–522; (IF: 2.079)

3.         Lupan A., Matyas C., Moţ A.C., Silaghi-Dumitrescu R., Can geometrical distortions make a laccase change color from blue to yellow?, Studia Universitatis Babes-Bolyai Chemia, 56 (2011) 231-238. (IF: 0.129)

4.         Moţ A.C., Pârvu M., Damian G., Irimie F.D., Darula Z., Medzihradszky K.F., Brem B., Silaghi-Dumitrescu R., A “yellow” laccase with “blue” spectroscopic features, from Sclerotinia sclerotiorum, Process Biochemistry 47 (2012) 968–975; (IF: 2.627)

5.         Moţ A.C., Syrbu S.A., Makarov S.V., Damian D., Silaghi-Dumitrescu R., Axial ligation in water-soluble copper porphyrinates: contrasts between EPR and UV–vis, Inorganic Chemistry Communications 18 (2012) 1-3; (IF: 1.972)

6.         Imre A., Moţ A.C., Silaghi-Dumitrescu R., Exploring the possibility of high-valent copper in models of copper proteins with a three-histidine copper-binding motif, Central European Journal of Chemistry 10 (2012) 1527-1533; (IF: 1.073)

7.         Moţ A.C., Silaghi-Dumitrescu R., Laccases: complex architectures for one-electron oxidations, Biochemistry (Moscow), accepted; (IF: 1.058)