VOLUME FOUR HUNDRED AND THIRTY-SEVEN

METHODS IN

ENZYMOLOGYGlobins and Other NitricOxide-Reactive Proteins,Part B

ROBERT K. POOLE

AMSTERDAM • BOSTON • HEIDELBERG • LONDON

ELSEVIER

2008, Elsevier Inc

978-0-12-374278-0

Contributors xviiPreface xxviiVolumes in Series xxix

Section I. Nitric Oxide-Metabolising andDetoxifying Enzymes 1

1. Structural Studies on Flavodiiron Proteins 3Joäo B. Vicente, Maria Armenia Carrondo, Miguel Teixeira,and Carlos Frazäo

1. Introduction 42. Crystallization of Flavodiiron Proteins 43. Diffraction Data Collection, Structure Determination, and Refinement 74. Overall Description of Structures 85. Conclusion 16References 17

2. Biochemical, Spectroscopic, and Thermodynamic Propertiesof Flavodiiron Proteins 21Joäo B. Vicente, Marta C. Justino, Vera L. Gonsalves, Ligia M. Saraiva,and Miguel Teixeira

1. Introduction 222. Cloning of Genes Encoding Flavodiiron Proteins and

Their Truncated Domains 243. Production and Purification of Recombinant Flavodiiron Proteins 254. Biochemical Characterization of Flavodiiron Proteins 265. Spectroscopic Properties 296. Redox Properties 327. Conclusions 37Acknowledgments 42References 42

3. Kinetic Characterization of the Escherichia col/ Nitric OxideReductase Flavorubredoxin 47Jo -do B. Vicente, Francesca M. Scandurra, Elena Forte, Maurizio Brunori,Paolo Sarti, Miguel Teixeira, and Alessandro Giuffre

1. Introduction 482. Amperometric Measurements 493. Spectroscopic Measurements 514. Conclusions 61Acknowledgments 61References 61

4. Escherichia coil Cytochrome c Nitrite Reductase NrfA 63Thomas A. Clarke, Paul C. Mills, Susie R. Poock, Julea N. Butt,Myles R. Cheesman, Jeffrey A. Cole, Jay C. D. Hinton, Andrew M. Hemmings,Gemma Kemp, Christopher A. G. Söderberg, Stephen Spiro, Jessica VanWonderen, and David J. Richardson

1. Introduction 642. Measurement of Cytochrome c Nitrite Reductase-Dependent

Consumption of Nitric Oxide in Whole Cells 663. Growth of E. coil Optimized for Cytochrome c Nitrite Reductase

Production for Use in Enzyme Purification 66

4. Purification of Cytochrome c Nitrite Reductase 68

5. Assaying the Cytochrome c Nitrite Reductase 696. Crystallization of E. coil Cytochrome c Nitrite Reductase 73

7. Concluding Remarks 74Acknowledgments 76References 76

5. The Respiratory Nitric Oxide Reductase (NorBC) fromParacoccus denitrificans 79Sarah J. Field, Faye H. Thorndycroft, Andrey D. Matorin,David J. Richardson, and Nicholas J. Watmough

1. Introduction 80

2. Purification of Native NorBC from Paracoccus denitrificans 82

3. Purification of Recombinant NorBC 85

4. Amperometric Assays of NO Consumption 86

5. Pseudoazurin as an Electron Donor in Assays of NorBC 88

6. Preparation of NOR for Spectroscopic Investigation 91

7. Electron Paramagnetic Resonance Spectroscopy 96

8. Concluding Remarks 98

Acknowledgments 99

References 99

6. Redox-Controlled Dinitrosyl Formation at the Diiron-OxoCenter of NorA 103Rainer Cramm and Katja Strube

1. Introduction 1042. Genetic Context and Expression of the NorA Gene in R. eutropha 1053. Purification of NorA 1064. Disulfide Bridges in NorA 1075. Iron Analysis and Preparation of Apo-NorA 1086. Interconversion of Redox Forms of NorA 1097. Generation of NorA-DNIC In Vitro 1118. Preparation of NorA-DNIC Formed In Vivo 1139. Quantification of NO from NorA-DNIC 113

10. Outlook 114References 114

7. Purification and Functional Analysis of Fungal Nitric OxideReductase Cytochrome P450nor 117Li Zhang and Hirofumi Shoun

1. Introduction 1182. Screening of P450nor Activity 1193. Gas Analysis 1194. Purification of P450nor 1205. Nitric Oxide Reductase Activity Assay 1216. Protein Sequencing 1227. Isolation of cDNA 1238. Subcellular Fractionation of T. cutaneum 1249. Site-Directed Mutagenesis 125

10. Expression of Recombinant Proteins 12611. Purification of Recombinant Proteins 12712. Titration of NAD Analogs 12813. Stopped-Flow Rapid Scan Analysis 13014. Other Analysis 13115. Conclusion 131Acknowledgments 131References 131

8. A Quantitative Approach to Nitric Oxide Inhibition of TerminalOxidases of the Respiratory Chain 135Maria G. Mason, Rebecca S. Holladay, Peter Nicholls, Mark Shepherd,and Chris E. Cooper

1. Introduction 1362. Evaluation of Current Techniques for Measuring pNO, p0 2, and KM (0 2) 137

3. Nitric Oxide Donor Compounds 1384. Nitric Oxide Kinetics 1395. Oxygen Kinetics 1496. Optical Detection of Enzyme Intermediates in the Presence

of Oxygen and NO 151Appendices 153Acknowledgments 156References 156

Section II. Sensor Proteins 161

9. Cloning, Expression, and Purification of the N-terminalHeme-Binding Domain of Globin-Coupled Sensors 163Jennifer A. Saito, Tracey Allen K. Freitas, and Maqsudul Alam

1. Introduction 1642. Bioinformatic Search of Globin-Coupled Sensors 1643. Functional Analysis of Globin-Coupled Sensors 166Acknowledgments 171References 171

10. Oxygen-Sensing Histidine-Protein Kinases: Assays of LigandBinding and Turnover of Response-Regulator Substrates 173Marie-Alda Gilles-Gonzalez, Gonzalo Gonzalez,Eduardo Henrique Silva Sousa, and Jason Tuckerman

1. Introduction 1742. Assays 175Acknowledgments 187References 187

11. Reactions of Nitric Oxide and Oxygen with the Regulatorof Fumarate and Nitrate Reduction, a Global TranscriptionalRegulator, during Anaerobic Growth of Escherichia coil 191Jason C. Crack, Nick E. Le Brun, Andrew J. Thomson, Jeffrey Green,and Adrian J. Jervis

1. Introduction 1922. Production of 4Fe-FNR Protein 1943. Determination of Iron and Acid-Labile Sulfide Content of FNR 1974. UV-Visible Absorbance Spectra of FNR 1985. Cluster Reaction with Nitric Oxide and Oxygen 1986. Purification of 2Fe-FNR 2047. Detection of Other Reaction Products 2048. Conclusions 206References 207

12. Genome-Wide Identification of Binding Sites for the NitricOxide-Sensitive Transcriptional Regulator NsrR 211Sam Efromovich, David Grainger, Diane Bodenmiller, and Stephen Spiro

1. Introduction 2122. Strain Construction 2143. Reference and Control Samples 2164. Culture Conditions 2175. Immunoprecipitation of DNA Targets Associated with NsrR 2186. DNA Labeling, Microarray Hybridization, and Processing 2197. Visualization and Analysis of DNA Microarray Data 2208. A New Statistical Methodology for Treatment of Chip-on-Chip Data 2229. Conclusions 231Acknowledgments 231References 231

13. Characterization of the Nitric Oxide-Reactive TranscriptionalActivator NorR 235Benoit D'Autreaux, Nick Tucker, Stephen Spiro, and Ray Dixon

1. Introduction 2362. Measurement of NorR Activity In Vivo 2373. Measurement of Transcriptional Activation by NorR In Vitro 2384. Detection of the Ferrous-Nitrosyl Form of NorR by In Vivo Electron

Paramagnetic Resonance (EPR) 2405. In Vitro Reconstitution of the Iron Center in NorR 2426. Measurement of NO Affinity 2437. Standardization of the NO Electrode 2468. Determination of NorRFe(NO) K d 2479. Conclusions 248Acknowledgment 248References 249

Section III. Advanced Spectroscopic Methods 253

14. Hemoglobins from Mycobacterium tuberculosis andCampylobacter jejuni: A Comparative Study with ResonanceRaman Spectroscopy 255Changyuan Lu, Tsuyoshi Egawa, Masahiro Mukai, Robert K. Poole,and Syun-Ru Yeh

1. Hemoglobin Superfamily: An Overview 2562. Microbial Hemoglobins 2573. Resonance Raman Spectroscopy: Applications in Hemeproteins 258

4. Structures and Functions of Microbial Hemoglobins 2665. Closing Remarks 281Acknowledgments 282References 282

15. The Power of Using Continuous-Wave and Pulsed ElectronParamagnetic Resonance Methods for the Structure Analysisof Ferric Forms and Nitric Oxide-Ligated Ferrous Forms of Globins 287Sabine Van Doorslaer and Filip Desmet

1. Introduction 2882. Electron Paramagnetic Resonance in a Nutshell 2893. EPR Studies of NO-Ligated Globins 2954. EPR Studies of Ferric globins 3015. Spin-Labeling Herne Proteins 3046. Future Challenges and Possibilities 305Acknowledgments 305References 306

16. Oxygen Binding to Herne Proteins in Solution, Encapsulatedin Silica Gels, and in the Crystalline State 311

Luca Ronda, Stefano Bruno, Serena Faggiano, Stefano Bettati,and Andrea Mozzarelli

1. Oxygen-Binding Curves to Herne Proteins 3132. Determination of OBCs for Hemoglobin in Solution 3163. Determination of K l for Hemoglobin in Solution in the Absence

of Allosteric Effectors 3184. Determination of OBCs for T State Hemoglobin Gels in the Absence

and Presence of Allosteric Effectors 3185. Determination of OBCs for T State Hemoglobin Crystals 3206. Determination of OBCs for Hemocyanin in Solution and in Silica Gels 323Acknowledgments 325References 325

17. Characterization of Ligand Migration Mechanisms insideHemoglobins from the Analysis of Geminate Rebinding Kinetics 329Stefania Abbruzzetti, Stefano Bruno, Serena Faggiano, Luca Ronda,Elena Grandi, Andrea Mozzarelli, and Cristiano Viappiani

1. Introduction 3302. Principles of Nanosecond Laser Flash Photolysis 3303. Basic Experimental Layouts 3314. Encapsulation of Hbs in Silica Gels 335

5. Enhancement of Geminate Rebinding and Advantagesof Gel Encapsulation 336

6. Extraction of Kinetic Information 337Acknowledgments 342References 342

18. Ligand Dynamics in Heme Proteins Observed by FourierTransform Infrared Spectroscopy at Cryogenic Temperatures 347Karin Nienhaus and G. Ulrich Nienhaus

1. Introduction 3482. Materials 3493. Fourier Transform Infrared Cryospectroscopy 3534. Low-Temperature FTIR Spectroscopy on NO-Ligated Herne Proteins 3655. Concluding Remarks 373Acknowledgments 374References 374

19. Time-Resolved X-Ray Crystallography of Herne Proteins 379Vukica Srajer and William E. Royer, Jr.

1. Introduction 3792. Experiment 3813. Data Processing and Analysis 3854. A Case Study: Scapharca Dimeric Hemoglobin 3885. Conclusions 391Acknowledgments 393References 393

20. Structural Dynamics of Myoglobin 397M. Brunori, D. Bourgeois, and B. Vallone

1. Background 3982. Crystallographic Studies of Myoglobin States 3993. Experimental Approaches 400Acknowledgments 413References 413

21. Use of the Conjugate Peak Refinement Algorithm forIdentification of Ligand-Binding Pathways in Globins 417Stephen D. Golden and Kenneth W. Olsen

1. Introduction 4182. Exploration of Oxygen-Binding Pathways in Myoglobin 418

3. Theoretical Models 4194. Potential Energy Function 4205. Transition Pathways 4216. Methods 4257. Results 4298. Conclusions 432References 433

22. Finding Gas Migration Pathways in Proteins Using ImplicitLigand Sampling 439Jordi Cohen, Kenneth W. Olsen, and Klaus Schulten

1. Introduction 4402. Methods 4423. Example Calculation: Truncated Hemoglobin (trHb) from

Paramecium caudatum 4464. Discussion 449Acknowledgments 455References 456

23. Identification of Ligand-Binding Pathways inTruncated Hemoglobins Using Locally Enhanced SamplingMolecular Dynamics 459Stephen D. Golden and Kenneth W. Olsen

1. Introduction 4602. Molecular Dynamics 4623. Locally Enhanced Sampling Molecular Dynamics 4654. Methods 4665. Results 4686. Conclusions 471References 472

24. Nitric Oxide Reactivity with Globins as Investigated ThroughComputer Simulation 477

Marcelo A. Marti, Luciana Capece, Axel Bidon-Chanal, Alejandro Crespo,Victor Guallar, F. Javier Luque, and Dario A. Estrin

1. Introduction 4782. Molecular Dynamics (MD) Methods 479

3. Quantum Mechanical-Molecular Mechanical Methods 4854. Illustrative Examples 488

5. Ligand Migration Profiles from MSMD and PELE Simulations:Exploring Ligand Entry Pathways in M. tuberculosis trHbN 490

6. Conclusions 494Acknowledgments 495References 495

25. Microbial Responses to Nitric Oxide and Nitrosative Stress:Growth, "Omit," and Physiological Methods 499Steven T. Pullan, Claire E. Monk, Lucy Lee, and Robert K. Poole

1. Introduction 5002. Methods 5043. Nitric Oxide, NO-Releasing Agents, and Nitrosating Agents 5124. Illustrative Results from Applications of These Methods 514References 516

26. Analysis of Nitric Oxide-Dependent Antimicrobial Actionsin Macrophages and Mice 521Andres Vazquez-Torres, Tania Stevanin, Jessica Jones-Carson,Margaret Castor, Robert C. Read, and Ferric C. Fang

1. NO--Dependent Antimicrobial Actions of Murine Macrophages 5222. NO--Dependent Antimicrobial Actions of Human Macrophages 5283. NO•-dependent Antimicrobial Actions in Laboratory Mice 532References 536

27. Measuring Nitric Oxide Metabolism in the PathogenNeisseria meningitidis 539Melanie J. Thomson, Tania M. Stevanin, and James W. B. Moir

1. Introduction 5402. Safety Aspects of Handling N. meningitidis in the Laboratory 5413. Metabolism of Neisseria sp. 5414. Experimental Approaches to Analyzing Nitrogen Metabolism

Relevant to NO 5445. Simultaneous Measurement of Oxygen and NO during Pure

Culture of N. meningitidis 5476. Measurement of NO Production/Disappearance in Tissue Culture

Using Human Monocyte-Derived Macrophages 555References 558

28. Localization of S-Nitrosothiols and Assay of Nitric OxideSynthase and S-Nitrosoglutathione Reductase Activity in Plants 561Francisco J. Corpas, Alfonso Carreras, Francisco J. Esteban, Mounira Chaki,Raquel Valderrama, Luis A. Del Rio, and Juan B. Barroso

1. Introduction 5622. Determination of L-Arginine-Dependent NOS Activity by Ozone

Chemiluminescence in Plant Tissues 5633. Assay of GSNOR Activity 5664. Localization of S-Nitrosothiols and S-Nitrosoglutathione in Plant

Tissues by Confocal Laser-Scanning Microscopy (CLSM) 5675. Conclusion 571Acknowledgments 572References 572

29. Methods for Nitric Oxide Detection duringPlant-Pathogen Interactions 575E. Vandelle and M. Delledonne

1. Introduction 5762. Nitric Oxide Detection by Mass Spectrometry 5773. Nitric Oxide Detection by Laser Photoacoustic Spectroscopy 5794. Nitric Oxide Detection by Chemiluminescence 582

5. Nitric Oxide Detection by Hemoglobin Conversion 5836. Nitric Oxide Detection by Electron Paramagnetic Resonance (EPR)

Spin Trap 5857. Nitric Oxide Detection Using Diaminofluoresceins 5878. Conclusion 590References 591

30. Bioimaging Techniques for Subcellular Localization of PlantHemoglobins and Measurement of Hemoglobin-DependentNitric Oxide Scavenging In Planta 595

Kim H. Hebelstrup, Erik 0stergaard-Jensen, and Robert D. Hill

1. Introduction 596

2. Measuring Hemoglobin-Dependent NO Scavenging 596

3. Techniques for Determination of Subcellular Localizationof Plant Hemoglobins 597

4. Imaging of Hemoglobin-Dependent NO Scavenging inArabidopsis Plants 598

5. Engineering of GLB1-GFP/GLB2-GFP Constructs and MicroscopicAnalysis of A. thaliana Plants Expressing GFP-Tagged Hemoglobin 600

References 603

31. Use of Recombinant Iron-Superoxide Dismutase as A Markerof Nitrative Stress 605Estibaliz Larrainzar, Estibaliz Urarte, Inigo Auzmendi, Idoia Ariz,Cesar Arrese-Igor, Esther M. Gonzalez, and Jose F. Moran

1. Introduction 6062. I mmunodetection of Nitrated Proteins: Metal-Mediated

Tyrosine Nitration of BSA 6083. Tyrosine Nitration of Purified Recombinant Vu_FeSOD

Affects its Enzymatic Activity 6104. Tyrosine Nitration in Vu_FeSOD can be Estimated Using

Antibodies Against 3-Nitrotyrosine 6125. SIN-1-Dependent Vu_FeSOD Nitration can be Detected

by the Loss of Enzymatic Activity 612Acknowledgments 616References 616

Author Index 619Subject Index 647