Infrared and UV-visible time-resolved techniques for the study of tetrapyrrole-based proteins A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy (PhD) in the Faculty of Life Sciences 2013 Henry John Russell Contents List of Tables .......................................................................................................................................................................... 5 List of Figures ........................................................................................................................................................................ 6 Abstract ...................................................................................................................................................................................10 Declaration ............................................................................................................................................................................11 Copyright Statement ........................................................................................................................................................11 Acknowledgements ..........................................................................................................................................................12 Abbreviations and Symbols ..........................................................................................................................................13 Preface and Thesis Structure .......................................................................................................................................16 1. Introduction ................................................................................................................................ 19 1.1 Spectroscopy Overview ..............................................................................................................................19 1.2 UV-visible and Infrared spectroscopy ................................................................................................25 1.2.1 Static Measurement ...........................................................................................................................25 1.2.1.1 UV-vis spectrophotometer ........................................................................................................25 1.2.1.2 Fourier Transform Infrared Spectrometer .......................................................................27 1.2.2 Rapid Mixing Techniques ................................................................................................................29 1.2.2.1 Stopped-flow UV-vis spectroscopy .......................................................................................30 1.2.2.2 Stopped-flow FT-IR spectroscopy .........................................................................................31 1.2.3 Photoexcitation techniques ...........................................................................................................32 1.2.3.1 Laser-flash Photolysis ..................................................................................................................33 1.2.3.2 Ultrafast UV-vis Transient Absorption ...............................................................................36 1.2.3.3 Time-resolved Infrared Spectroscopy ................................................................................38 1.2.4 Combination of techniques for protein study ......................................................................40 1.3 Analysis of tetrapyrrole-based proteins by UV-vis and IR spectroscopy.........................41 1.3.1 Cobalamins .............................................................................................................................................44 1.3.1.1 B12-catalysis ......................................................................................................................................45 1.3.1.2 B12 photolysis ...................................................................................................................................49 1.3.1.3 Ethanolamine Ammonia Lyase ...............................................................................................51 1.3.2 Haems ........................................................................................................................................................54 1.3.2.1 Cytochrome c’...................................................................................................................................58 1.4 Thesis Aims and Objectives......................................................................................................................63 2. Stopped-flow FT-IR Application Note: Temporal Resolution and Calibration of a Stopped-flow FT-IR Spectrometer using the Ester Hydrolysis of Methyl Chloroacetate ....... 64 2.1 Introduction .....................................................................................................................................................64 2.2 Methodology ....................................................................................................................................................64 2.3 Experimental ...................................................................................................................................................64 2.4 Results and Conclusion ..............................................................................................................................67 3. Ultrafast Infrared Spectral Fingerprints of Vitamin B12 and Related Cobalamins .. 68 3.1 Abstract ..............................................................................................................................................................68 3.2 Introduction .....................................................................................................................................................69 3.3 Materials and Methods ...............................................................................................................................71 3.4 Results and Discussion ...............................................................................................................................72 3.4.1 Adenosylcobalamin ............................................................................................................................73 2 3.4.2 Methylcobalamin .................................................................................................................................77 3.4.3 Cyanocobalamin and Hydroxocobalamin. ..............................................................................80 3.4.4 Model Frequency Calculations .....................................................................................................82 3.5 Summary and Conclusions .......................................................................................................................85 3.6 Supplementary Information ....................................................................................................................86 4. Protein Motions Are Coupled to the Reaction Chemistry in Coenzyme B12- Dependent Ethanolamine Ammonia Lyase................................................................................. 94 4.1 Introduction .....................................................................................................................................................94 4.2 Materials and Methods ...............................................................................................................................96 4.3 Results and Discussion ...............................................................................................................................98 4.4 Summary and Conclusions .................................................................................................................... 104 4.5 Supplementary Information ................................................................................................................. 105 5. Modulation of ligand-haem reactivity by binding pocket residues demonstrated in cytochrome c’ over the femtosecond-second temporal range ........................................... 113 5.1 Abstract ........................................................................................................................................................... 113 5.2 Introduction .................................................................................................................................................. 113 5.3 Materials and Methods ............................................................................................................................ 117 5.4 Results and Discussion ............................................................................................................................ 120 5.4.1 Wild-type AxCytcp ........................................................................................................................... 120 5.4.2 L16A AxCytcp ..................................................................................................................................... 124 5.4.3 R124A AxCytcp .................................................................................................................................. 126 5.5 Concluding Remarks ................................................................................................................................