Cover Page
The handle http://hdl.handle.net/1887/50245 holds various files of this Leiden University dissertation.
Author: Nami, F.
Title: Enzymatic reduction of oxygen by small laccase. A rapid freeze-quench EPR study
Issue Date: 2017-03-07
Faezeh Nami
Proefschrift
ter verkrijging van de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof.mr. C.J.J.M. Stolker, volgens besluit van het College voor Promoties
te verdedigen op woensdag 7 maart 2017 klokke 16.15 uur
door
Faezeh Nami
geboren te Bastak, Iran in 1983
Promotoren: Prof. dr. E.J.J. Groenen (Universiteit Leiden) Prof. dr. G.W. Canters (Universiteit Leiden) Promotiecommissie: Prof. dr. M. Benatti (Max-Planck-Institut für
Biophysikalische Chemie, Georg-August- Universität Göttingen, Germany)
Prof. dr. W.R. Hagen (Technische Universiteit, Delft)
Dr. A.W.J.W. Tepper (Crossbeta Biosciences bv, Utrecht)
Prof. dr. E.R. Eliel (Universiteit Leiden) Prof. dr. M.A.G.J. Orrit (Universiteit Leiden)
An electronic version of this thesis can be found at https://openaccess.leidenuniv.nl
The work described in this thesis is supported by the Netherlands Organisation for Scientific Research (NWO) that is funded by the Dutch Ministry of Education, Culture and Science (OCW).
The cover shows the 3D structure of the enzyme small laccase. The margins are
“tazhib”, an old Persian art.
Casimir PhD series, Delft-Leiden 2017-04 ISBN 978-90-8593-289-5
Contents
1. Introduction…..………1
1. 1 Motivation………...2
1.2 Multicopper oxidases……….……..2
1.3 Small laccase………4
1.4 Rapid freeze-quench……….5
1.5 EPR spectroscopy……….8
1.6 Thesis outline………..17
2. Rapid-freeze-quench EPR spectroscopy………..23
2.1 Introduction………24
2.2 Material and methods……….27
2.2.1 Preparation of rapid-freeze-quench samples………...27
2.2.2 Sample packing………...28
2.2.3 EPR measurements……….29
2.3 Results………29
2.3.1 Evaluation of the sucking method……….…..30
2.3.2 The myoglobin-azide reaction rate constant and freezing time….….32 2.4 Discussion……….…….35
2.5 Conclusion……… ….37
3. Rapid-freeze-quench EPR up to 275 GHz……….………..43
3.1 Introduction………44
3.2 Material and methods……….47
3.2.1 Rapid-freeze-quench device………...……….47
3.2.2 EPR measurements……….48
3.3 Results………48
3.3.1 Preparation and loading of rapid-freeze-quench samples…………...48
3.3.2 The myoglobin-azide reaction………52
3.4 Discussion………..57
3.5 Conclusion……….61
4. A Rapid Freeze-Quench Multi-Frequency EPR Study of an Intermediate in the Enzymatic Reduction of Oxygen by Small Laccase. The T1D and T1D Y108F mutants…….………..67
4.1 Introduction………68
4.2 Materials and methods………...73
4.2.1 Protein isolation………..73
4.2.2 Chemicals………73
4.2.3 EPR measurements……….73
4.2.4 Rapid-freeze-quench sample preparation………73
4.3 Results………75
4.3.1 Reoxidation of T1D SLAC……….75
4.3.2 Reoxidation of T1D Y108F SLAC……….………86
4.4 Discussion……….……….92
4.5 Conclusion……….…….………..101
5. A Rapid Freeze-Quench EPR Study of the Enzymatic Reduction of Oxygen by Wild-type SLAC………109
5.1 Introduction………..110
5.2 Materials and methods……….113
5.2.1 Protein isolation………113
5.2.2 Chemicals………..113
5.2.3 EPR measurements………113
5.2.4 Rapid freeze-quench sample preparation………..114
5.3 Results………..114
5.4 Discussion………118
5.5 Conclusion………...120
6. Mechanism of O2 reduction by (T1D) SLAC………125
Summary………137
Samenvatting………...………...141
List of publications………...………..145
Curriculum vitae……….………147
Acknowledgment………149
