STRUCTURAL STUDIES OF METALLOPROTEINS USING X-RAY ABSORPTION SPECTROSCOPY AND X-RAY DIFFRACTION by Paul Joseph Ellis A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy School of Chemistry University of Sydney August, 1995 ii Contents Page Acknowledgments ix Preface x List of Abbreviations xii List of Figures xiii List of Tables xviii Summary xxi Chapter 1: X-ray Absorption Fine Structure (EXAFS) 1-12 1.1 What is EXAFS? 1 1.2 The origin of the EXAFS oscillations 2 1.3 Multiple-scattering 4 1.4 Data collection 5 1.4.1 Generating monochromatic X-rays 5 1.4.2 Transmission XAS measurements 6 1.4.3 Fluorescence XAS measurements 7 1.4.4 Polarised XAS measurements 8 1.4.5 Low-temperature XAS measurements 8 1.5 Data reduction (extracting the EXAFS) 9 1.5.1 Removing the underlying background absorbance 9 1.5.2 Normalisation 10 1.5.3 Removing the smooth edge background 10 1.5.4 Compensating for decreasing m0 11 Chapter 2: EXAFS structure analysis using the program XFIT 13-29 2.1 Introduction 13 2.2 The model 14 2.3 Calculation of the theoretical EXAFS 15 2.4 Empirical EXAFS calculation 16 2.5 Ab initio EXAFS calculation 16 2.6 Polarised EXAFS 18 iii 2.7 Fourier filtering 18 2.8 Refinement algorithm 20 2.9 Constraints and restraints 21 2.10 Refinement using more than one EXAFS data set 22 2.11 Multiple absorbing atom sites 23 2.12 Goodness-of-fit (residual) 23 2.13 Monte-Carlo error analysis 24 2.14 User-friendly interface 26 2.15 Examples 27 2.16 Data 28 2.17 Some recent XFIT analyses 28 Chapter 3: Plastocyanin 30-35 3.1 Introduction 30 3.2 X-ray diffraction studies of plastocyanin 31 3.3 Implications for oxidation and reduction 33 3.4 Variation with pH of the ratio of the low- and high-pH 34 forms of reduced Pc 3.5 Other "blue" copper proteins: why do EXAFS? 35 Chapter 4: Collection of EXAFS data from oxidised and reduced 36-40 plastocyanin 4.1 Preferred crystal orientations for collecting polarised 36 EXAFS from poplar Pc. 4.2 Polarised EXAFS from oriented single crystals of oxidised Pc 36 4.3 Unpolarised EXAFS from frozen solutions of reduced Pc at 37 pH 4.8 and 7.2 4.4 Polarised EXAFS from oriented single crystals of reduced Pc 38 at pH 4.5 and 7.2 4.5 Data reduction 39 iv Chapter 5: General description of the EXAFS analyses of plastocyanin 41-51 5.1 Criteria for the analyses 41 5.2 The X-ray crystal structures 41 5.2.1 Constructing the EXAFS models 41 5.2.2 XRD restraints 43 5.3 Typical geometry of the ligand sidechains 43 5.3.1 Changes in the starting model due to the ligand geometry 45 restraints 2 5.4 Typical values of E0, S0 , and the Debye-Waller factors 46 5.4.1 A note on E0 46 2 5.4.2 E0 and S0 restraints 46 5.4.3 Debye-Waller factors restraints 47 5.5 The observed EXAFS 47 5.5.1 Weighting of the EXAFS curves relative to each other 47 5.5.2 Weighting of the EXAFS relative to the restraints 48 5.5.3 Choosing the Fourier-filtering windows 48 5.5.3.1 EXAFS windows 49 5.5.3.2 Fourier-transform window 50 Chapter 6: EXAFS analysis of oxidised plastocyanin 52-64 6.1 Original analysis 52 6.2 Reanalysis 53 6.3 Analysing the refinements 58 6.3.1 Refinement with the atoms fixed at the XRD coordinates 58 6.3.2 The refinements included in the final average 59 6.3.3 The average Cu-ligand distances 59 6.4 Estimating the uncertainty in the Cu-ligand distances 60 6.4.1 Monte-Carlo error analysis 60 6.4.2 Cu-ligand bond distances from the crystal 2 EXAFS 61 6.4.3 Uncertainty in the crystal orientations 61 6.4.4 EXAFS analysis of model compounds 61 6.4.5 Uncertainty in E0 63 6.4.6 Estimates of uncertainty 63 6.5 Empirical analysis 63 6.6 Conclusion 64 v Chapter 7: EXAFS analysis of reduced plastocyanin 65-83 7.1 General note on the analysis of EXAFS from reduced Pc 65 7.1.1 Estimating the [L]:[H] ratio in crystals of reduced Pc at 295 K 65 7.1.2 Reduced Pc solutions at 10 K 68 7.1.3 Potential errors resulting from the uncertain [L]:[H] ratios 68 7.2 The final analysis 68 7.3 Analysing the refinements 78 7.3.1 Refinement with the atoms fixed at the XRD coordinates 78 7.3.2 The refinements included in the final average 79 7.3.3 The average Cu-ligand distances 79 7.4 Estimating the uncertainty in the Cu-ligand distances 80 7.4.1 Uncertainty in the Cu-ligand distances due to uncertainty 80 in the [L]:[H] ratios 7.4.2 Uncertainty in E0 80 7.4.3 Estimates of uncertainty 81 7.5 Independent analyses of polarised and unpolarised EXAFS 82 data 7.6 Conclusion 83 Chapter 8: EXAFS analyses of plastocyanin: discussion 84-89 8.1 Cu-ligand distances determined by EXAFS 84 8.2 Changes with oxidation state and pH 84 8.3 Implications for oxidation/reduction 85 8.4 Comparison of the EXAFS and XRD Cu-ligand distances 85 8.5 Comparison with other EXAFS analyses of Pc 87 8.6 Conclusion 88 8.7 Future work 88 Chapter 9: The crystal structure of leghemoglobin 90-108 9.1 Introduction: nitrogen fixation 90 9.2 Leghemoglobin 91 9.3 Crystallographic studies of leghemoglobin 93 9.4 Crystallographic studies of ferric soybean leghemoglobin a 93 nicotinate vi 9.5 Rerefinement of the ferric soybean leghemoglobin a 94 nicotinate model 9.5.1 Protocol for the new refinement 95 9.5.2 Generating the initial model 96 9.5.3 Refinement 97 9.6 Estimates of precision 106 9.7 Ramachandran plots 107 Chapter 10: The structure of ferric soybean leghemoglobin a nicotinate 109-120 10.1 Gross structure of the molecule 109 10.2 Secondary and tertiary structure 114 10.3 Temperature factors 115 10.4 The heme pocket 115 10.5 Hydrogen-bonding to the heme ligands 119 Chapter 11: The structure of lupin Lb II in relation to its properties 121-148 11.1 Identifying the key features of lupin Lb II 121 11.2 Basic ligand association and dissociation processes in 123 monomeric hemoglobins 11.3 Structural factors influencing association and dissociation 123 in sperm whale Mb 11.3.1 Key residues in sperm whale Mb 124 11.3.2 Heme accessibility 125 11.3.3 Stabilisation of water and ligand molecules by hydrogen 127 bonding 11.3.3.1 Obstruction of the binding site by water 127 11.3.3.2 Stabilisation of a bound ligand 127 11.3.4 Sidechains potentially hindering the binding site 128 11.3.4.1 Phenylalanine CD1 128 11.3.4.2 The distal valine E11 128 11.3.4.3 The distal histidine E7 129 11.3.5 Heme pocket size 129 11.3.6 Heme reactivity and the orientation of the proximal 130 histidine imidazole vii 11.4 Lupin Lb II 131 11.4.1 Comparison of the expected and observed differences 131 between Lb and Mb 11.4.2 Distal cavity size, mobility of the distal histidine and 133 flexibility of the pocket 11.4.3 Large distal cavity 134 11.4.4 The effect of a deletion on the mobility of the distal histidine 135 11.4.5 The flexibility of the globin backbone 137 11.4.6 Effects of the large distal cavity, mobile distal histidine and 141 flexible pocket 11.4.6.1 The ability to bind bulky ligands with high affinity 141 11.4.6.2 High rates of diffusion 141 11.4.6.3 Rapid Fe-ligand bond disruption 142 11.4.6.4 Rapid bond formation 142 11.4.7 Proximal histidine orientation 143 11.4.8 Heme conformation 144 11.4.8.1 Heme ruffling 144 11.4.8.2 Conformation of the heme vinyl substituents 147 Chapter 12: The structure of soybean Lb a in relation to its properties 149-156 12.1 Distal cavity size in soybean Lb a 149 12.2 Distal histidine mobility and heme pocket flexibility 149 12.3 Heme conformation 151 12.4 Proximal histidine orientation 152 12.5 An alternate pathway for ligand molecules? 152 12.5.1 The E7/E10 pathway 152 12.5.2 The alternate pathway 153 12.6 Structural and functional homology between soybean Lb a 155 and lupin Lb II 12.7 Conclusion 156 viii Appendices: Contents 4- Appendix A: EXAFS Evidence that the CuCl6 Ion in (3-chloroanilinium)8(CuCl6)Cl4 Has an Elongated Rather than Compressed Tetragonal Geometry. Appendix B: Extended X-Ray Absorption Fine Structure, Crystal Structures at 295 and 173 K, and Electron Paramagnetic Resonance and Electronic Spectra of Bis[tris(2-pyridyl)-methane]copper(II) Dinitrate. References ix Acknowledgements It is impossible to properly thank in this limited space the many people who have contributed to the work described herein and so I can only offer here my gratitude to a few whose contribution has been particularly conspicuous. I must first thank my supervisor, Prof. Hans Freeman, both for his invaluable guidance over the past several years, and for his practical help in conducting the experiments.