Preface ........................................................ ix
List of Color Figures and Website Materials ................... xix
Chapter 1. Introduction
John R. Pilbrow
1 Iron-Sulfur Cluster-Containing Proteins ...................... 2
2 Molybdenum Enzymes ........................................... 3
3 Manganese-Containing Enzymes ................................. 5
4 Novel Melalloenzymes and Metalloproteins ..................... 5
5 Conclusions .................................................. 6
IRON-SULFUR-CONTAINING PROTEINS
Chapter 2. Electron Magnetic Resonance of Iron-Sulfur Proteins
in Electron-Transfer Chains: Resolving Complexity
Richard Cammack and Fraser MacMillan
1 Introduction ................................................ 12
1.1 Problems of Complex Electron-Transfer Systems .......... 13
2 Iron-Sulfur Proteins ........................................ 14
2.1 Types of Clusters ...................................... 16
3 Information from Advanced EMR ............................... 17
3.1 Relaxation Rates ....................................... 18
3.2 Identification of Cluster Ligands ...................... 18
3.3 Interactions with Protons and Paramagnets .............. 18
3.4 Further Structural Information ......................... 19
3.5 Orientation-Selective ENDOR and ESEEM .................. 19
3.6 Studies of Intact Membrane-Bound Complexes ............. 20
3.7 Methods of Isolating Spectra of Individual Components .. 21
3.8 Results from l4N ESEEM ................................. 23
4 Selected Examples from Electron-Transport Chains ............ 27
4.1 Xanthine Dehydrogenase/Oxidase as a Model .............. 27
4.2 Mitochondria and Aerobic Bacteria ...................... 29
4.3 Complex I (NADH:Ubiquinone Reductase) .................. 29
4.4 Complex II (Succinate:Quinone Reductase) and
Quinol:Fumarate Reductase .............................. 30
4.5 Complex III ............................................ 32
4.6 Microbial Anaerobic Respiration ........................ 33
5 Conclusions ................................................. 35
Chapter 3. Catalysis and Gene Regulation
Helmut Beinert .............................................. 45
Chapter 4. Iron-Sulfur Clusters in "Radical SAM" Enzymes:
Spectroscopy and Coordination
Serge Gambarelli1, Etienne Mulliez2, and Marc Fontecave
1 Introduction ................................................ 53
2 "Radical SAM" Iron-Sulfur Enzymes: An Example of
a Low-Molecular-Weight Ligand to a [4Fe-4S] Cluster ......... 54
2.1 The Pyruvate Formate Lyase System ...................... 57
2.2 Lysine 2,3-Aminomutase ................................. 58
2.3 Anaerobic Ribonucleotide Reductase ..................... 58
3 Detection of Hyperfine Coupling Interactions
in Metalloproteins .......................................... 60
3.1 ENDOR: Principles and General Considerations ........... 62
3.2 ESEEM and HYSCORE ...................................... 64
4 Analysis of Ligand Hyperfine Coupling Interactions .......... 66
5 Applications to Metalloproteins ............................. 69
5.1 Pyruvate Formate Lyase-Activating Enzyme (PFL-AE) ...... 69
5.2 Lysine 2,3-Aminomutase (LAM) ........................... 72
5.3 Anaerobic Ribonucleotide Reductase Activating Enzyme
(aRNR-AE) .............................................. 73
6 Conclusion .................................................. 75
MONONUCLEAR MOLYBDENUM ENZYMES
Chapter 5. EPR Studies of Xanthine Oxidoreductase and Other
Molybdenum-Containing Hydroxylases
Russ Hille
1 Introduction ................................................ 91
2 Historical Context .......................................... 91
3 The Active Site Structure of Xanthine Oxidoreductase ........ 93
4 Isotopic Substitution Studies .............................. 106
5 Magnetic Interactions Between Centers in Xanthine
Oxidoreductase ............................................. 111
6 Concluding Comments ........................................ 114
Chapter 6. High-Resolution EPR Spectroscopy of Mo Enzymes.
Sulfite Oxidases: Structural and Functional Implications
John H. Enemark, A.V. Astashkin, and A.M. Raitsimring
1 Introduction and Structures from X-Ray Crystallography ..... 122
2 Earlier cw EPR Investigations .............................. 125
3 Frequencies Observed in Pulsed EPR for a System of
Electron Spin S = 1/2 and Arbitrary Nuclear Spin
in Weak Interaction Limit .................................. 127
4 Pulsed EPR Techniques Used in This Work .................... 133
4.1 ENDOR ................................................. 133
4.2 ESEEM Techniques ...................................... 134
5 General Problems in Extraction of Structural Parameters
from Magnetic Resonance Parameters ......................... 138
6 Sample Preparation and Instrumentation ..................... 139
7 High-Resolution Pulsed EPR Spectra, Magnetic Resonance
Parameters, and Structural Implications for Various
Forms of SO ................................................ 140
7.1 Exchangeable Protons: Similarities and Differences
in SOs from Different Organisms ............................ 140
7.2 Groups Blocking Water Access to Mo(V) ................. 148
7.3 Nonexchangeable Protons ............................... 151
7.4 Exchangeable Oxygen Ligands ........................... 154
8 Biological Implications .................................... 159
9 Conclusion ................................................. 162
Note Added in Proof ........................................ 162
Chapter 7. Dimethylsulfoxide (DMSO) Reductase, a Member of
the DMSO Reductase Family of Molybdenum Enzymes
Graeme R. Hanson and Ian Lane
1 Introduction ............................................... 169
2 EPR Studies of Mo(V) Species ............................... 171
3 EPR Studies of DMSO Reductase .............................. 172
3.1 Respiratory DMSO Reductase ............................ 173
3.2 Periplasmic DMSO Reductase ............................ 173
3.3 Catalytic Mechanism ................................... 188
4 Conclusions ................................................ 193
MANGANESE-CONTAINING ENZYMES
Chapter 8. The Manganese-Calcium Cluster of the Oxygen-
Evolving System: Synthetic Models, EPR Studies, and
Electronic Structure Calculations
Marcin Brynda and R. David Britt
1 Introduction ............................................... 203
2 Theoretical Background for the Polynuclear Manganese
Clusters ................................................... 205
2.1 Introduction to the Spin Physics of Exchange-
Coupled Manganese Complexes ........................... 205
2.2 EPR Theory for Exchange Coupled Systems ............... 209
2.3 Computational Methods for Magnetically Coupled
Homonuclear Metal Clusters ............................ 218
3 Synthetic Models for Manganese Cluster of the OEC .......... 222
3.1 Current Structural Proposals for the Pentanuclear
Mn4Ca Cluster of the OEC ............................. 222
3.2 EPR Characteristics of the Manganese Cluster of
the OEC ............................................... 225
3.3 Synthetic Models ...................................... 228
4 Computational Studies of the OEC ........................... 248
4.1 Calculations on the Mechanistic Aspects of the
Water Oxidation with DFT .............................. 248
4.2 Mixed Molecular Mechanics/Quantum Mechanics Studies
of Water Oxidation .................................... 252
5 Conclusions and Prospectives ............................... 253
Appendix ................................................... 255
Chapter 9. Manganese Metalloproteins
Sarah J. Smith, Kieran S. Hadler, Gerhard Schenk,
Graeme R. Hanson, and Nataša Mitić
1 Introduction ............................................... 273
2 Manganese Catalases ........................................ 276
2.1 Biochemical and Structural Characterization ........... 276
2.2 Spectroscopic Characterization ........................ 278
2.3 Mechanistic Implications .............................. 284
3 Ribonucleotide Reductase ................................... 285
3.1 Biochemical and Structural Characterization ........... 285
3.2 Spectroscopic Characterization ........................ 287
3.3 Mechanistic implications .............................. 294
4 Class lb Ribonucleotide Reductases ......................... 295
4.1 Biochemical and Structural Characterization ........... 295
4.2 Spectroscopic Characterization ........................ 296
4.3 Mechanistic Implications .............................. 299
5 Manganese-Iron Oxygenases .................................. 299
5.1 Biochemical and Structural Characterization ........... 299
5.2 Spectroscopic Characterization ........................ 301
5.3 Mechanistic Implications .............................. 303
6 SoxB ....................................................... 304
6.1 Biochemical and Structural Characterization ........... 304
6.2 Spectroscopic Characterization ........................ 305
6.3 Mechanistic Implications .............................. 306
7 Bacteriophage λ Protein Phosphatase ........................ 306
7.1 Biochemical and Structural Characterization ........... 306
7.2 Spectroscopic Characterization ........................ 307
7.3 Mechanistic Implications .............................. 309
8 Purple Acid Phosphatase .................................... 310
8.1 Biochemical and Structural Characterization ........... 310
8.2 Spectroscopic Characterization ........................ 310
8.3 Mechanistic Implications .............................. 312
9 Phosphotricsterase ......................................... 312
9.1 Biochemical and Structural Characterization ........... 312
9.2 Spectroscopic Characterization ........................ 313
9.3 Mechanistic Implications .............................. 314
10 Arginase ................................................... 317
10.1 Biochemical and Structural Characterization ........... 317
10.2 Spectroscopic Characterization ........................ 317
10.3 Mechanistic Implications .............................. 322
11 Methionyl Aminopeptidase ................................... 324
11.1 Biochemical and Structural Characterization ........... 324
11.2 Spectroscopic Characterization ........................ 325
11.3 Mechanistic Implications .............................. 327
NOVEL METALLOENZYMES AND METALLOPROTEINS
Chapter 10. EPR of Cobalt-Substituted Zinc Enzymes
Brian Bennett
1 Introduction ............................................... 345
2 Review of Cobalt-Substituted Enzymes ....................... 346
3 Methods of Co(II) Insertion ................................ 348
4 EPR Experimental Techniques and Considerations ............. 350
5 Spectral Interpretation .................................... 356
6 Spectral Interpretation: A Case Study ...................... 364
7 Complementary Techniques ................................... 365
8 Conclusions ................................................ 366
Chapter 11. Hyperfine and Quadrupolar Interactions in
Vanadyl Proteins and Model Complexes: Theory and Experiment
Sarah С. Larsen and N. Dennis Chasteen
1 Introduction ............................................... 372
1.1 Coordination Chemistry of VO2+ ........................ 372
1.2 EPR Properties ........................................ 373
1.3 The Additivity Relationship for Predicting Ligand
Environments .......................................... 374
1.4 The Ground State and Ligand Hyperfine Couplings ....... 374
2 ENDOR and ESEEM of Vanadyl Model Complexes ................. 376
2.1 14N Hyperfine and Quadrupole Coupling Constants ....... 376
2.2 1H and l7O Coupling Constants ......................... 379
2.3 31P Hyperfine Coupling Constants ...................... 380
2.4 51V Nuclear Quadrupole Coupling Constants ............. 380
3 Density Functional Theory Calculations of EPR
Parameters in Vanadyl Model Complexes ...................... 381
3.1 Overview of DFT Methods for Calculations of EPR
Parameters ............................................ 381
3.2 DFT Calculations of Vanadium EPR Parameters ........... 383
3.3 DFT Calculations of Ligand Hyperfine and Quadrupole
Coupling Constants .................................... 387
3.4 Outlook ............................................... 390
4 Select Protein Studies ..................................... 391
4.1 Pyruvate Kinase ....................................... 391
4.2 S-Adcnosylmcthionine Synthetase ....................... 393
4.3 Imidazole Glycerol Phosphate Dehydratase .............. 394
4.4 ATP Synthase .......................................... 394
4.5 D-Xylose Isomerase .................................... 395
4.6 Transferrins .......................................... 397
4.7 Ferritin .............................................. 398
5 Tissues .................................................... 399
5.1 Kidney and Liver ...................................... 399
5.2 Bone .................................................. 401
6 Conclusions ................................................ 402
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