STRUCTURAL STUDIES OF USING X-RAY ABSORPTION AND X-RAY DIFFRACTION

by

Paul Joseph Ellis

A thesis submitted in partial fulfilment of the requirements for the degree of

School of 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. His characteristic attention to detail has been especially valuable to me during the preparation of this thesis and no other colleague has made a greater contribution to its final form.

My next thanks go to Prof. Keith Hodgson, Dr. Britt Hedman and the members of the Hodgson group, both past and present, who not only played a major role in the collection of the XAS data, but have also been a valuable source of instruction and advice.

My gratitude is extended to the members of the Crystal Structure Group, particularly to Drs. Trevor Hambley and J. Mitchell Guss for their kind and generous help.

Thanks also to my postgraduate colleagues, especially Dr. Thomas Maschmeyer, Dr. Barry Fields, and Emma Proudfoot for their assistance and encouragement.

I gratefully acknowledge helpful discussions with Drs. Graham George, and Ingrid Pickering, and Cyril Appleby, and Prof. James Penner-Hahn.

For their support I gratefully acknowledge: (i) The Department of Employment, Education and Training for the award of an APRA scholarship, (ii) The Stanford Synchrotron Radiation Laboratory, which is supported by the U.S. Department of Energy, for providing synchrotron beam time, (iii) The Australian Nuclear Science and Technology Organisation for providing travel funding, (iv) The Australian Research Council and the Dr. Joan R. Clark Research Fund for funding supporting the research, and (v) The staff of the Visualisation Laboratory, VisLab, in the School of Physics at the University of Sydney for their unstinting help and generous access to their computers.

Finally, it is my pleasure to thank my parents, brothers and sister for their help, encouragement and inspiration. Without them, none of this would have been possible. x

Preface

This thesis is a report of original research undertaken by the author and is submitted for the degree of Doctor of Philosophy at the University of Sydney.

The research comprised three projects. The first was concerned with the technique of structural determination by the analysis of X-ray absorption fine structure (EXAFS). The second was concerned with the application of this technique to the "blue" copper protein plastocyanin. The third concerned the refinement of the X-ray diffraction crystal structure of the plant O2-binding protein leghemoglobin.

All of the work detailed in this thesis was conducted by the candidate except where noted in this preface.

Structure of the thesis:

Chapter 1 Is a brief review of the and measurement of EXAFS.

Chapter 2 Describes the interactive EXAFS analysis program XFIT. This program, developed solely by the candidate, has been described in a published paper upon which Chapter 2 draws heavily:

Ellis, P. J., and Freeman, H. C. (1995) XFIT - an Interactive EXAFS Analysis Program. Journal of Synchrotron Radiation, 2, in press.

The original version, drafted by the candidate, was similar in form and content to the published paper, completed in collaboration with Prof. H. C. Freeman and incorporating comments by Dr. J. Penner-Hahn.

Chapter 3 Is a brief review of the structure and function of the "blue" copper protein plastocyanin and the motivation behind the use of EXAFS.

Chapter 4 Describes the retrieval of published polarised EXAFS data from oxidised poplar (Populus nigra var. italica) plastocyanin and collection of unpolarised and polarised EXAFS data from reduced poplar plastocyanin at low and high xi

pH. The candidate was responsible for the preparation of the samples. The crystals were oriented with the assistance of Prof. H. C. Freeman, and the XAS data was collected with the assistance of Prof. H. C. Freeman, Prof. K. O. Hodgson, Dr. Britt Hedman, and members of the Hodgson group at Stanford University.

Chapter 5 Is a general description of the analysis of the plastocyanin EXAFS data.

Chapter 6 Completes the description of the analysis of the polarised EXAFS data from oxidised plastocyanin.

Chapter 7 Completes the description of the analysis of the polarised and unpolarised EXAFS data from reduced plastocyanin at low and high pH.

Chapter 8 Is a discussion of the Cu-site dimensions obtained from the EXAFS analyses. These dimensions are also compared to those obtained previously by X-ray diffraction and EXAFS analysis.

Chapter 9 After a brief review of the function and properties of the monomeric plant hemoglobin "leghemoglobin", Chapter 9 describes the refinement of the X-ray diffraction crystal structure of soybean (Glycine max) leghemoglobin a nicotinate. As detailed in this chapter, the refinement used data collected by previous investigators and drew on an earlier, unsuccessful, refinement.

Chapter 10 Describes the refined structure of soybean leghemoglobin a nicotinate.

Chapter 11 Is an analysis of the major structural differences between sperm whale (Physeter catodon) myoglobin, a model for monomeric hemoglobins, and lupin (Lupinus luteus) leghemoglobin II, the only other leghemoglobin to have been crystallographically characterised.

Chapter 12 Is an examination of the soybean leghemoglobin a structure in the light of the key features of lupin leghemoglobin II identified in the previous chapter. xii

List of Abbreviations

ADP adenosine diphosphate ATP adenosine triphosphate B temperature factor CuIPc high-pH form of reduced plastocyanin CuIIPc oxidised plastocyanin DAFS diffraction anomalous fine structure EPR electron paramagnetic resonance e.s.d. estimated standard deviation EXAFS X-ray absorption fine structure HCuIPc low-pH form of reduced plastocyanin Lb leghemoglobin Mb myoglobin MIR multiple isomorphous replacement NMR nuclear magnetic resonance Pc plastocyanin 3- Pi inorganic phosphate (PO4 ) r.m.s. root mean square SSRL Stanford Synchrotron Radiation Laboratory XAFS X-ray absorption fine structure XAS X-ray absorption spectroscopy XRD X-ray diffraction xiii

List of Figures Page

Figure 1.1 The K X-ray absorption edge of CuO. 1

Figure 1.2 K-edge EXAFS from CuO. 2

Figure 1.3 A photoelectron wave emitted from the absorbing atom is 3 back-scattered by a scattering atom.

Figure 1.4 Generation of the monochromatic X-ray beam. 5

Figure 1.5 Typical apparatus for a transmission XAS experiment. 6

Figure 1.6 Typical apparatus for a fluorescence XAS experiment. 7

Figure 1.7 Typical apparatus used in collecting polarised XAS data from oriented 8 single crystals.

Figure 1.8 The K X-ray absorption edge of CuO and the background absorbance 9 estimated by fitting a quadratic curve to the absorbance between 8479 eV and 8928 eV.

Figure 1.9 Normalised K X-ray absorption edge of CuO and the featureless 10 background edge absorbance estimated using a polynomial spline curve.

Figure 1.10 Difference between the normalised K-edge absorption from CuO and 11 the featureless background edge absorbance estimated using a polynomial spline curve.

Figure 1.11 Normalised absorbance calculated using expression (1.13) compared to 12 the normalised K-edge absorbance of CuO.

Figure 1.12 K-edge EXAFS from CuO. 12

Figure 2.1 Fourier filtering in XFIT. 19 xiv

Figure 2.2 Schematic representation of the Monte-Carlo error analysis of 25 three parameters.

Figure 3.1 Schematic representation of photosynthetic electron transport at the 30 thylakoid membrane.

Figure 3.2 Stereo a-carbon diagram of Pc. 31

Figure 3.3 The Cu site of CuIIPc as determined by XRD at 1.33 Å. 32

Figure 3.4 The Cu site of HCuIPc at pH 3.8 as determined by XRD at 1.9 Å. 32

Figure 3.5 Cu-ligand distances in Pc as determined by X-ray diffraction plotted 34 as functions of pH and oxidation state.

Figure 5.1 Models used in the multiple-scattering EXAFS analyses of Pc. 43

Figure 5.2 Ideal geometry of the ligand sidechains. 44

Figure 5.3 EXAFS window used with the ê//c-polarised EXAFS from 49 reduced Pc at pH 7.2.

Figure 5.4 The Fourier-transform window used with every data set. 50

Figure 6.1 Cu-ligand distances from the multiple-scattering analyses of EXAFS 55 from an oriented single crystal of CuIIPc as functions of the EXAFS weighting factor.

Figure 6.2 R-factors for the fit between the calculated EXAFS and the observed 55 EXAFS from an oriented single crystal of CuIIPc as functions of the EXAFS weighting factor.

Figure 6.3 ê//b-polarised EXAFS from CuIIPc and the difference between the 56 observed and calculated EXAFS for each refinement. xv

Figure 6.4 ê//c-polarised EXAFS from CuIIPc and the difference between the 57 observed and calculated EXAFS for each refinement.

Figure 7.1 Cu-ligand distances from the multiple-scattering analyses of EXAFS 74 from oriented single crystals and frozen solutions of reduced Pc as functions of the EXAFS weighting factor.

Figure 7.2 R-factors for the fit between the calculated EXAFS and the observed 75 EXAFS from oriented single crystals and frozen solutions of reduced Pc as functions of the EXAFS weighting factor.

Figure 7.3 EXAFS from reduced Pc at pH 4.8 and, ê//a-, ê//b- and ê//c-polarised 76 EXAFS from reduced Pc at pH 4.5 and the difference between the observed and calculated EXAFS for each refinement.

Figure 7.4 Unpolarised, ê//a-, ê//b- and ê//c-polarised EXAFS from reduced Pc at 77 pH 7.2 and the difference between the observed and calculated EXAFS for each refinement.

Figure 9.1 Schematic representation of oxygen transport and nitrogen fixation in 92 the Rhizobium/legume symbiosis.

Figure 9.2 Progress of the refinement, illustrated by graphing the crystallographic 105 R-factor as a function of PROLSQ cycle.

Figure 9.3 Luzatti plot for soybean Lb a nicotinate refined at 2.3 Å resolution. 105

Figure 9.4 Ramachandran plots for the two molecules in the asymmetric unit of 108 soybean Lb a nicotinate.

Figure 10.1 Alpha carbon diagram of soybean Lb a. 109

Figure 10.2 The sequence of soybean Lb a. 110

Figure 10.3 Temperature factors (B) averaged over the backbone atoms of 115 each residue. xvi

Figure 10.4 Protoporphyrin IX, viewed from the distal side of the heme pocket. 116

Figure 10.5 Nicotinate ligand. 116

Figure 10.6 The heme pocket of molecule A. 118

Figure 10.7 The heme pocket of molecule A showing all residues with 118 contacts £ 4.0 Å with the heme or nicotinate.

Figure 10.8 Superposed heme pockets of molecules A and B. 119

Figure 10.9 Hydrogen bonding to the heme ligands in molecule A. 120

Figure 11.1 The three residues within 4.0 Å of the bound O2 in sperm whale MbO2. 125

Figure 11.2 The E7/E10 pathway in Mb imidazole. 126

Figure 11.3 Hindrance of the heme NA and NC atoms by proximal histidine 130 imidazole He1 and Hd2 atoms resulting from eclipsing of NA-NC by the imidazole plane.

Figure 11.4 Superposition of lupin Lb II and sperm whale Mb. 135

Figure 11.5 Effect of the deletion of Arg CD3 on the mobility of the distal histidine. 136

Figure 11.6 The sidechain of the distal histidine relative to the heme in lupin Lb II 137 nicotinate and lupin deoxyLb II.

Figure 11.7 Superposed backbones of lupin deoxyLb II and lupin Lb II nicotinate. 138

Figure 11.8 Distance from Ca atoms of lupin FeIII Lb II nicotinate to the 138 corresponding atoms in the deoxy structure.

a Figure 11.9 Distances from the C atoms of MbO2 and LbO2 to the corresponding 139 atoms in the deoxy structures. xvii

Figure 11.10 Superposition of the residues potentially contacting an O2 ligand in 142 lupin Lb II and sperm whale Mb.

Figure 11.11 Heme ruffling in lupin Lb II nicotinate. 145

Figure 11.12 Vinyl groups of the heme in lupin Lb II and nearby sidechains. 147

Figure 12.1 Superposition of soybean Lb a nicotinate and sperm whale Mb 150 imidazole.

Figure 12.2 Imidazole positions required for hydrogen bonding between the distal 150 histidine and a nicotinate or fluoride ligand in soybean Lb a.

Figure 12.3 Superposed heme groups of soybean Lb a nicotinate and 151 lupin Lb II nicotinate.

Figure 12.4 Vinyl groups of the heme in soybean Lb a and nearby sidechains. 152

Figure 12.5 The E7/E10 and alternate pathways in soybean Lb a nicotinate. 154

Figure 12.6 Superposition of soybean Lb a nicotinate and lupin Lb II nicotinate. 155 xviii

List of Tables Page

Table 3.1 Summary of the Cu-site dimensions determined by X-ray crystal 33 structure analysis of reduced (CuI) and oxidised (CuII) poplar Pc.

Table 4.1 Summary of XAS data collection from Pc. 40

Table 5.1 Atoms comprising the models used in the EXAFS analyses of Pc. 42

Table 5.2 Atomic coordinates relative to the Cu atom in the X-ray crystal 45 structures and in the starting models for the EXAFS analyses.

Table 5.3 Windows used with the EXAFS from Pc. 51

Table 6.1 Cu-ligand parameters obtained from the empirical analysis of CuIIPc 52 single-crystal EXAFS data by Scott et al. (1982).

Table 6.2 Summary of the multiple-scattering analyses of the EXAFS data from 54 an oriented single crystal of CuIIPc.

Table 6.3 Cu-ligand distances yielded by the multiple-scattering analyses of 60 EXAFS from an oriented single crystal of CuIIPc.

2 Table 6.4 Cu-ligand distances, E0 and S0 values obtained from the EXAFS 62 I I analyses of [Cu (diethylthiourea)3]2SO4 and Cu (N-methylimidazole)2BF4.

Table 6.5 Estimated uncertainty in the Cu-ligand distances and the contributing 64 factors.

Table 7.1 EXAFS R-factors for polarised EXAFS from reduced Pc from a 66 refinement using a [L]:[H] ratio of 100:0 at pH 4.5 and 0:100 at pH 7.2.

Table 7.2 Estimates of the [L]:[H] ratio in crystals of reduced Pc obtained using 67 the EXAFS and the X-ray crystal structures and the values used in the final analysis. xix

Table 7.3 Summary of the multiple-scattering analyses of the EXAFS data from 70 oriented single crystals and frozen solutions of reduced Pc.

Table 7.4 Cu-ligand distances yielded by the multiple-scattering analyses of 79 polarised and unpolarised EXAFS from reduced Pc.

Table 7.5 Cu-ligand distances in reduced Pc as functions of [L]:[H] ratio. 81

Table 7.6 Estimated uncertainty in the Cu-ligand distances and the contributing 82 factors.

Table 7.7 Cu-ligand distances obtained from independent analyses of the 82 unpolarised and polarised EXAFS data from reduced Pc.

Table 8.1 Cu-ligand distances determined by the EXAFS analyses of poplar Pc. 84

Table 8.2 Cu-ligand distances from XRD and EXAFS analyses of Pc and the 85 differences between them.

Table 8.3 Cu-ligand distances obtained from published EXAFS analyses of Pc. 87

Table 9.1 Summary of the X-ray data collection, unit cell and symmetry of 94 soybean Lb a nicotinate.

Table 9.2 Summary of the refinement procedure. 100

Table 9.3 Weighting parameters and r.m.s. deviations from target values in the 104 restrained least-squares refinement.

Table 9.4 Fe-ligand distances in molecules A and B of soybean Lb a nicotinate. 107

Table 10.1 Intramolecular hydrogen bonds and salt bridges in soybean 111 FeIII-Lb a nicotinate.

Table 10.2 Residues potentially in contact with the heme or nicotinate. 117 xx

Table 11.1 Crystallographically-characterised derivatives of lupin Lb II and 122 sperm whale Mb deposited in the Protein Data Bank.

Table 11.2 The effects of specific structural changes on the rate constants for 124

O2 binding and diffusion in sperm whale Mb.

Table 11.3 Expected effects of the structural differences between sperm whale 132

Mb and lupin Lb II on the rate constants for O2 diffusion and binding.

Table 11.4 Volume of the distal cavities of lupin Lb II and sperm whale Mb. 134

Table 11.5 Difference between the distance from the Ca atom of the proximal 140 histidine to the Ca atom of the distal histidine and the corresponding distance in the deoxy state for derivatives of lupin Lb II and sperm whale Mb.

Table 11.6 Distance from the terminal methyl atom Cg2 of Val E11 to the binding 143 II site in lupin Lb II and sperm whale Mb for the Fe deoxy, O2 and CO derivatives.

Table 11.7 Displacement of FeII and proximal histidine Ne2 from the heme plane 145

in the deoxy, O2 and CO forms of Mb and Lb.

Table 11.8 Monomeric hemoglobins with ruffled hemes. 146

Table 12.1 Overall association (k'O2) and dissociation (kO2) rate constants, and 155

dissociation constant (KO2) for O2 binding to soybean Lb a, lupin Lb II and sperm whale Mb at pH 7.0. xxi

Summary

EXAFS analysis using the program XFIT

XFIT is an interactive and user-friendly program for the analysis of X-ray absorption fine structure (EXAFS) curves. XFIT incorporates in a single package a number of features available in other existing programs: ab initio EXAFS calculation (using FEFF 4.06/6.01), empirical EXAFS calculation (as in XFPAKG), allowance for polarisation, use of Fourier filtering and the application of constraints and restraints. Additional features not previously available are: simultaneous refinement with respect to several data sets, simultaneous refinement of several absorber sites and Monte-Carlo error analysis. Applications including the analysis of EXAFS data from mixtures and the analysis of DAFS (diffraction anomalous fine structure) data are indicated.

Analysis of polarised and unpolarised EXAFS from poplar plastocyanin

Poplar plastocyanin (Pc) is a small (99 amino acid, 10 kDa) Cu protein involved in the photosynthetic electron-transfer chain. The Cu-ligand distances in oxidised Pc (CuIIPc) and reduced Pc at high and low pH (CuIPc and HCuIPc) have been determined by multiple-scattering analysis of EXAFS from oriented single crystals of oxidised Pc, from oriented single crystals of reduced Pc at pH 4.5 and 7.2, and from frozen solutions of reduced Pc at pH 4.8 and 7.2:

Cu-ligand bond length (Å) Cu-ligand bond CuIIPc CuIPc HCuIPc

Cu-Nd(His37) not determined 1.96 ± 0.02 1.95 ± 0.02 Cu-Nd(His87) 1.93 ± 0.02 2.01 ± 0.04 - Cu-Sg(Cys84) 2.13 ± 0.02 2.19 ± 0.01 2.16 ± 0.01 Cu-Sd(Met92) 2.70 ± 0.10 2.86 ± 0.10 2.33 ± 0.04

The distances obtained are internally consistent (i.e. the differences between CuIIPc, CuIPc and HCuIPc are consistent with expectation). Although the CuIIPc distances differ significantly from the crystallographic values, a detailed investigation of possible errors in the EXAFS analysis failed to find any explanation for the discrepancy. xxii

Crystal structure of soybean leghemoglobin a nicotinate

Soybean leghemoglobin (Lb) a is a small (143 amino acid, 16 kDa) protein facilitating the transport of O2 to respiring N2-fixing bacteria at low free-O2 tension. The crystal structure of soybean Lb a nicotinate has been refined at 2.3 Å resolution. The final crystallographic R factor is 15.8%. This structure provides strong support for the conclusion drawn from a comparison of lupin Lb II with sperm whale myoglobin that the unique properties of Lb arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.