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Handbook ofPorphyrin Sciencewith Applications to Chemistry, Physics,Materials Science, Engineering, Biology

and Medicine

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N E W J E R S E Y L O N D O N S I N G A P O R E B E I J I N G S H A N G H A I H O N G K O N G TA I P E I C H E N N A I

World Scientific

Handbook ofPorphyrin Science

Volume 6

NMR and EPR Techniques

Editors

Karl M. KadishUniversity of Houston, USA

Kevin M. SmithLouisiana State University, USA

Roger GuilardUniversit de Bourgogne, France

with Applications to Chemistry, Physics,Materials Science, Engineering, Biology

and Medicine

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British Library Cataloguing-in-Publication DataA catalogue record for this book is available from the British Library.

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ISBN-13 978-981-4307-18-5 (Set)ISBN-13 978-981-4307-19-2 (Vol. 6)

Typeset by Stallion PressEmail: [email protected]

All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic ormechanical, including photocopying, recording or any information storage and retrieval system now known or tobe invented, without written permission from the Publisher.

Copyright 2010 by World Scientific Publishing Co. Pte. Ltd.

Published by

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Printed in Singapore.

HANDBOOK OF PORPHYRIN SCIENCEwith Applications to Chemistry, Physics, Materials Science, Engineering, Biology and Medicine(Volumes 610)

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Contents

Preface xiiiContributing Authors xvContents of Volumes 110 xxiii

29 / NMR and EPR Spectroscopy of ParamagneticMetalloporphyrins and Heme Proteins 1

F. Ann WalkerI. Introduction and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

A. Structures and Electron Configurations of Metalloporphyrins . . . . 10II. Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

A. Proton Chemical Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191. Contact Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202. Pseudocontact (Dipolar) Shifts . . . . . . . . . . . . . . . . . . . . . . . 23

a. Pseudocontact Shifts of Metalloporphyrin Substituents . . . 23b. Measurement of Magnetic Susceptibility Anisotropies

of Ferriheme Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 26c. Residual Dipolar Couplings of Proteins for Structure

Determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293. Temperature Dependence of Contact

and Pseudocontact Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . 31B. Nuclear Relaxation and Linewidths . . . . . . . . . . . . . . . . . . . . . . 33

1. Chemical Exchange Line Broadening and EXSYCross Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2. Proton T1 and T2 Relaxation Times, as Controlledby Electron Spin Relaxation Times, T1e . . . . . . . . . . . . . . . . . 34a. Electron Spin Relaxation Times, T1e or s . . . . . . . . . . . . . 34b. Nuclear Spin-Lattice Relaxation Times, T1 . . . . . . . . . . . . 34c. Nuclear SpinSpin Relaxation Times, T2 . . . . . . . . . . . . . 38

C. Spin Density and Bonding: Mechanisms of SpinDelocalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391. The Metal Ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392. The Porphyrin Ring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

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3. The Effect of Axial Ligand Plane Orientation on theCombined Contact and Pseudocontact Shifts of Low-SpinFerriheme Proteins and Synthetic Hemins withHindered Axial Ligand Rotation. . . . . . . . . . . . . . . . . . . . . . 50

4. Mechanisms of Spin Delocalization throughChemical Bonds, and Strategies for Separationof Contact and Pseudocontact Shifts . . . . . . . . . . . . . . . . . . 55

D. Methods of Assignment of the 1H NMR Spectraof Paramagnetic MetalIoporphyrins . . . . . . . . . . . . . . . . . . . . . . 571. Substitution of H by CH3 or Other Substituent . . . . . . . . . . . 572. Deuteration of Specific Groups . . . . . . . . . . . . . . . . . . . . . . 583. 2D 13C Natural Abundance HMQC Spectra and

Specific or Complete 13C Labeling of Protoheminfor the Assignment of Heme Resonances in Proteins . . . . . . . 59

4. Saturation Transfer NMR Experiments . . . . . . . . . . . . . . . . . 625. NOE Difference Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 636. Two-Dimensional NMR Techniques . . . . . . . . . . . . . . . . . . . 64

III. Spectral Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69A. Resolution and Assignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69B. Analysis of Shifts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

1. Curvature in the Curie Plot over the Temperature Rangeof the Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77a. Zero-Field Splitting Contributions to the

Pseudocontact Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79b. Nonzero Intercepts of the Curie Plot . . . . . . . . . . . . . . . . 79

2. Empirical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803. g-Tensor Anisotropy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

IV. Effect of Metal Ion and Spin State on Bonding . . . . . . . . . . . . . . . . . 83A. Iron Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

1. Iron(I) Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832. Diamagnetic Iron(II) Porphyrins . . . . . . . . . . . . . . . . . . . . . . 84

a. Six-Coordinate Diamagnetic Complexes . . . . . . . . . . . . . 84b. Five-Coordinate Diamagnetic Complexes . . . . . . . . . . . . 85

3. Intermediate-Spin Iron(II) Porphyrins: Observed Shiftsand the Mechanism of Spin Delocalization . . . . . . . . . . . . . 85

4. Five-Coordinate High-Spin Iron(II) Porphyrins: ObservedShifts and the Mechanism of Spin Delocalization . . . . . . . . . 93a. Models of Deoxyhemoglobin and Deoxymyoglobin . . . . 93b. Models for the Reduced States of Cytochrome P450

and Chloroperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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c. Models for the Heme a3-CuB and Heme-NonhemeFe Centers of Cytochrome Oxidase and NOReductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

d. Hydroxide or Fluoride Complexes . . . . . . . . . . . . . . . . 103e. N-Alkyl (aryl) Porphyrin Complexes . . . . . . . . . . . . . . . 103f. Nitrene Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . 104g. Verdoheme Analogs: Iron(II) Complexes

of Octaethyloxaporphyrin, OEOP . . . . . . . . . . . . . . . . . 104h. N-confused or N-inverted Iron(II) Porphyrins

and Related N-modified Macrocycle Complexes . . . . . . 1055. Possible Iron(II) Porphyrin -Cation Radicals . . . . . . . . . . . 1066. High-Spin Iron(III) Porphyrins: Observed Shifts

and the Mechanism of Spin Delocalization . . . . . . . . . . . . 107a. Five-Coordinate, Monomeric Iron(III) Porphyrin

Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107b. Six-Coordinate Monomeric High-Spin Iron(III)

Porphyrin Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . 112c. Monomeric Complexes of Reduced or Oxidized

Ferrihemes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117i. Iron(III) Sulfhemins . . . . . . . . . . . . . . . . . . . . . . . . 117ii. Iron(III) Octaethyl- and Tetraphenylchlorin . . . . . . . 117iii. Two Iron(III) Octaethylisobacteriochlorin Isomers . . 121iv. An Iron(III) Monooxochlorin Complex . . . . . . . . . . 122v. Two Iron(III) Dioxooctaethylisobacteriochlorin

Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123vi. The Iron(III) Complex of Tetraphenyl-21-

Oxaporphyrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123vii. The High-Spin Iron(III) Complexes of

Mono-meso-octaethyloxaporphyrin andMono-meso-octaethylazaporphyrin. . . . . . . . . . . . . 124

d. Bridged Dimeric Complexes of High-Spin Iron(III)Porphyrins and Chlorins . . . . . . . . . . . . . . . . . . . . . . . . 125

7. Intermediate-Spin Iron(III) Porphyrins: Observed Shiftsand the Mechanism of Spin Delocalization . . . . . . . . . . . . 128

8. Low-Spin Iron(III) Porphyrins . . . . . . . . . . . . . . . . . . . . . . . 132a. Griffiths Three-Orbital Theory and Experimental

EPR Data for Low-Spin Iron(III) Porphyrinsand Related Macrocycles . . . . . . . . . . . . . . . . . . . . . . . 134

b. NMR Studies of Low-Spin Iron(III) Porphyrins Havingthe (dxy)2(dxz,dyz)3 Ground State . . . . . . . . . . . . . . . . . . . 147

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i. Effect of Porphyrin Substituents on the Patternof Spin Delocalization . . . . . . . . . . . . . . . . . . . . . . 147

ii. The Shifts of Coordinated Imidazole Ligandsand the Effect of Imidazole Deprotonationon the Pattern and Extent of Spin Delocalization . . . 151(a) Neutral Imidazole Ligands . . . . . . . . . . . . . . . . . 151(b) Imidazolate Ligands . . . . . . . . . . . . . . . . . . . . . 152

iii. Effect of Imidazole Plane Orientation on theParamagnetic Shifts of Low-Spin Iron(III)Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

iv. Bis-Ammine, Amino Ester and PhosphineComplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

v. Mixed-Ligand Complexes . . . . . . . . . . . . . . . . . . . . 160c. Observed Shifts and the Mechanism of Spin

Delocalization for the (dxz,dyz)4(dxy)1 Ground State . . . . . 161d. The Mixed Ground State Behavior of Bis-Cyanide

Complexes of Low-Spin Ferrihemes: Observed Shiftsand the Mechanism of Spin Delocalization . . . . . . . . . . 164

e. The Mixed Ground State Behavior of Bis-(pyridine)Complexes of Low-Spin Ferrihemes: Observed ShiftTrends and the Mechanism of Spin Delocalization . . . . 167

f. The Mixed Ground State Behavior of Bis-(pyridine)Complexes of Low-Spin Iron(III) Complexesof Oxophlorins and Meso-Amino Porphyrins . . . . . . . . . 174

g. Low-Spin FeIII Complexes of MesoMeso-Linked5,5-Bis(10,15,20-Triphenylporphyrin) . . . . . . . . . . . . . . 178

h. Five-Coordinate Low-Spin Iron(III) Porphyrinsand a Porphycene . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

i. Low-Spin Iron(III) Complexes of Reduced Hemes . . . . . 182j. Low-Spin Iron(III) Complexes of N-Alkylporphyrins . . . . 186k. Thermodynamics of Axial Ligation of Iron(III)

Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186l. Kinetics of Axial Ligand Exchange . . . . . . . . . . . . . . . . 187

9. Electron Exchange Between Low-Spin Iron(III)and Low-Spin Iron(II) Porphyrins . . . . . . . . . . . . . . . . . . . . 188

10. 1H and 13C NMR Spectroscopy of High- and Low-SpinFerriheme Proteins: The Nitrophorins and HemeOxygenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190a. NMR Spectroscopy of the Nitrophorins . . . . . . . . . . . . . 190

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Contents ix

i. NMR Investigations of the High-Spin Formsof the Nitrophorins from Rhodnius prolixus . . . . . . 196

ii. pH Titration of the High-Spin Nitrophorins fromRhodnius prolixus . . . . . . . . . . . . . . . . . . . . . . . . . 204

iii. NMR Investigations of the Low-Spin Formsof the Nitrophorins from Rhodnius prolixus,and Comparison to Other Heme Proteins . . . . . . . . 207

iv. Heme Ruffling of the Nitrophorins andComparison to Other Heme Proteins . . . . . . . . . . . 217

v. Nitrite Reductase Activity of Nitrophorin 7 . . . . . . . 221vi. Dimerization of NP4 . . . . . . . . . . . . . . . . . . . . . . . 222vii. NMR Spectroscopy of Apo-Nitrophorin 2 . . . . . . . . 223

b. NMR Spectroscopy of the Hemin-Containing HemeOxygenases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

i. NMR Study of High- and Low-Spin MammalianHeme Oxygenases. . . . . . . . . . . . . . . . . . . . . . . . . 227

ii. NMR Studies of Bacterial Heme Oxygenases . . . . . 230(a) Heme Propionate-Polypeptide Interactions

Dictate Regioselectivity in HOs . . . . . . . . . . . . 231(b) NMR Studies of Heme Electronic Structure

and its Potential Implications to the Mechanismof Heme Oxidation . . . . . . . . . . . . . . . . . . . . . 236

(c) NMR Spectroscopic Studies of DynamicReactivity Relationships . . . . . . . . . . . . . . . . . . 243

c. NMR Spectroscopy of Miscellaneous OtherHeme Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

11. Iron(III) Macrocycle -Cation Radicals . . . . . . . . . . . . . . . . 251a. High-Spin Iron(III) Porphyrin -Cation Radicals . . . . . . . 251b. Spin-Admixed and Intermediate-Spin Iron(III)

Porphyrin -Cation Radicals . . . . . . . . . . . . . . . . . . . . . 253c. Low-Spin Iron(III) Porphyrin -Cation Radicals . . . . . . . 254d. Iron(III) -Cation Radicals of Oxophlorins . . . . . . . . . . . 254e. Iron(III) Corrole -Radicals . . . . . . . . . . . . . . . . . . . . . . 256

12. Iron(IV) Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258a. Six-Coordinate, Bis-Methoxide Iron(IV) Porphyrins. . . . . 259b. Five- and Six-Coordinate Ferryl, (Fe=O)2+, Porphyrin

Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259c. Five-Coordinate Iron(IV) Phenyl Porphyrins . . . . . . . . . . 260d. Comparison of Iron(IV) Porphyrins and Iron(III)

Porphyrin -Radicals . . . . . . . . . . . . . . . . . . . . . . . . . . 261

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13. Iron(IV) Porphyrin -Radicals. . . . . . . . . . . . . . . . . . . . . . . 26114. Iron(V) Porphyrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

B. Ruthenium and Osmium Porphyrins. . . . . . . . . . . . . . . . . . . . . 266C. Cobalt Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

1. Low-Spin Cobalt(II) Porphyrins. . . . . . . . . . . . . . . . . . . . . . 268a. Observed Shifts and the Pseudocontact Interaction . . . . 268b. Oxidation of Cobalt(II) Porphyrins to Produce

-Radical Dimers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270c. Low-Spin Cobalt(II) Oxaporphyrins and

Porphodimethenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271d. High-Spin Cobalt(II) N-Alkylporphyrins and Alkoxy

Adducts of Oxaporphyrins . . . . . . . . . . . . . . . . . . . . . . 2722. High-Spin Cobalt(II) Complexes of a Weak-Field

Porphyrin Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2733. Alkylcobalt(III) Porphyrins: Agostic Interactions

or Paramagnetic Excited States? . . . . . . . . . . . . . . . . . . . . . 2734. A Cobalt(III) Porphyrin -Cation Radical. . . . . . . . . . . . . . . 275

D. Rhodium Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275E. Manganese Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

1. High-Spin Manganese(II) Porphyrins . . . . . . . . . . . . . . . . . 2792. High-Spin Manganese(III) Porphyrins . . . . . . . . . . . . . . . . . 2793. Low-Spin Manganese(III) Porphyrins. . . . . . . . . . . . . . . . . . 2804. Manganese(III) Corrole at Low Temperatures =

Manganese(II) Corrole -Cation Radical at AmbientTemperatures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280

5. Manganese(III) Porphyrin -Radicals and TheirTransformation to Dichloromanganese(IV) Porphyrins . . . . . 283

6. Oxomanganese(IV) Porphyrins. . . . . . . . . . . . . . . . . . . . . . 284F. Nickel Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284G. Lanthanide Porphyrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292H. Miscellaneous Metalloporphyrins which Have Extremely

Broad Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2941. Copper(II) and Silver(II) Porphyrins. . . . . . . . . . . . . . . . . . . 2942. Vanadium(IV) Porphyrins. . . . . . . . . . . . . . . . . . . . . . . . . . 2973. Chromium Porphyrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

I. Summary of Paramagnetic Shifts and Mechanisms of SpinDelocalization for the Metalloporphyrins . . . . . . . . . . . . . . . . . 299

V. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303VI. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

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30 / Heme Acquisition by Hemophores: A Lesson from NMR 339Paola TuranoList of Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

I. Biological Background. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340II. Hemophore Protein HasA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

A. Heme-Loaded HasA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3421. NMR of the Gallium(III) Derivative . . . . . . . . . . . . . . . . . . . 3442. NMR of the Iron(III) Derivative . . . . . . . . . . . . . . . . . . . . . . 344

a. 1H NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344b. Heteronuclear Detection . . . . . . . . . . . . . . . . . . . . . . . 346

i. 13C NMR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346ii. 15N NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

B. The H83A Variant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350C. Apo HasA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

III. HasAHasR Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353A. 1H15N NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

1. Chemical Shift Perturbation Mapping . . . . . . . . . . . . . . . . . 3562. Spectral Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

B. The Fate of the Heme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358IV. Interaction with Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359V. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

VI. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361VII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

31 / StructureFunction Relationships Among HemePeroxidases: New Insights from Electronic Absorption,Resonance Raman and Multifrequency ElectronParamagnetic Resonance Spectroscopies 367

Giulietta Smulevich, Alessandro Feis, Barry D. Howesand Anabella Ivancich

I. General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368A. Resonance Raman Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 370B. Multifrequency Electron Paramagnetic Resonance

Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370II. Superfamily of Plant, Fungal, and Bacterial Peroxidases . . . . . . . . . 372

A. Heme Pocket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3731. Fe(III) Resting State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3742. Extended Network of H-Bonds . . . . . . . . . . . . . . . . . . . . . . 380

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3. VinylProtein Interaction . . . . . . . . . . . . . . . . . . . . . . . . . . 3814. Imidazolate Character of the Proximal Iron Ligand. . . . . . . . 384

B. Heme Pocket in CatalasePeroxidases . . . . . . . . . . . . . . . . . . . 3861. KatG from Synechocystis . . . . . . . . . . . . . . . . . . . . . . . . . . 3902. KatG from Mycobacterium tuberculosis . . . . . . . . . . . . . . . 394

C. Calcium Binding Sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396D. Binding Sites for Substrates: Benzohydroxamic and

Salicylhydroxamic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400E. Ligand Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403F. Catalytic Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410

1. X-Ray Structures of Intermediates . . . . . . . . . . . . . . . . . . . . 4122. Resonance Raman Characterization of Intermediates . . . . . . 4163. Multifrequency EPR Spectroscopy: Identification and

Reactivity of Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . 422III. Superfamily of Animal Peroxidases . . . . . . . . . . . . . . . . . . . . . . . . 429

A. Covalent Links and Heme Structure . . . . . . . . . . . . . . . . . . . . . 430B. X-Ray Structures: An Overall View . . . . . . . . . . . . . . . . . . . . . . 431C. Resonance Raman and Electronic Absorption Spectroscopies . . 432D. Catalytic Intermediates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 436

1. Resonance Raman and Electronic Absorption Studies. . . . . . 4362. Multifrequency EPR Spectroscopy Combined with

Stopped-Flow Electronic Absorption Spectrophotometry. . . . 438IV. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442V. References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

xii Contents

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Preface

Although the porphyrin and tetrapyrrole research area was regarded as fullymatured during the 20th century, as evidenced for example by the awarding ofnumerous Nobel Prizes to its principal researchers, new advances and accom-plishments in the field still amaze us as editors. The area continues to blossom andto expand into new areas of science and applications that would probably neverhave occurred to our 20th century heroes. An earlier Porphyrin Handbook assem-bled the large amount of factual data that had been accumulated during the 20thcentury. Our new venture, the Handbook of Porphyrin Science takes a completelynew look at our research area and comprehensively details the contemporary sci-ence now appearing in the scientific literature that would indeed have been hardto predict even 10 years ago. In particular, fundamentally new methodologies andpotential commercial applications of the beautiful compounds that we all love areexemplified, fully recognizing the subtitle of the series with applications tochemistry, physics, materials science, engineering, biology and medicine.

The three of us have complementary expertise in physical chemistry, syntheticand bioorganic chemistry, and in synthetic and mechanistic organometallic chem-istry; this has enabled us to cover the whole field of porphyrin science and appli-cations, and to devise comprehensive volume and author content. As of the date ofwriting, between the three of us, we have published more than 1600 tetrapyrroleresearch articles, and hold 31 patents related to commercial applications of por-phyrin science. So we do know our field, and this has enabled us to assemble a first-rate group of experts who have written comprehensive up-to-date chapters withaccuracy and authority; we thank our authors for their cooperation and willingnessto go along with our highly ambitious schedule for production of these volumes.

We look forward to comments from our readers, and to suggestions that mightenable us to expand our basic interests and scientific coverage even further.Meanwhile, we hope that porphyrin researchers, old, new and of the future, willenjoy reading these volumes just as much as we enjoyed planning and, with thehelp of World Scientific Publishing Company, producing them from manuscript topublished article, in a timely manner.

Karl M. Kadish (Houston, Texas, USA)Kevin M. Smith (Baton Rouge, Louisiana, USA)

Roger Guilard (Dijon, Bourgogne, France)January, 2010

xiii

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Contributing Authors*

xv

Hasrat AliUniversit de SherbrookeSherbrooke, Qubec, CanadaChapter 16

Cristina AlonsoUniversity of HullKingston-upon-Hull, HU6 7RX, UKChapter 17

Edith AntunesRhodes UniversityGrahamstown, 6139, South AfricaChapter 34

Naoki ArataniKyoto UniversityKyoto 606-8502, [email protected] 1

Teodor Silviu BalabanKarlsruhe Institute of TechnologyD-76021 Karlsruhe, [email protected] 3

Alan L. BalchUniversity of California, DavisDavis, CA 95616, [email protected] 40

David P. BallouUniversity of MichiganAnn Arbor, MI 48109-5606, USAChapter 28

Faye BowlesUniversity of California, DavisDavis, CA 95616, USAChapter 40

Ross W. BoyleUniversity of HullKingston-upon-Hull, HU6 7RX, [email protected] 17

Ozguncem BozkulakChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Martin BrringTechnische Universitt Carolo-

Wilhelmina zu BraunschweigHagenring 30, Braunschweig, [email protected] 41

Kevin BurgessTexas A&M UniversityCollege Station, TX 77842, [email protected] 37

*Full contact information for authors can be found on the title page of each chapter.

b867_Vol-06_FM.qxd 6/10/2010 4:44 PM Page xv

Jos A.S. CavaleiroUniversity of Aveiro3810-193 Aveiro, [email protected] 9

Sung ChoYonsei UniversitySeoul, 120-747, KoreaChapter 5

Daniel P. CollinsUniversity of South CarolinaColumbia, SC 29208, USAChapter 28

John H. DawsonUniversity of South CarolinaColumbia, SC 29208, [email protected] 28

Ilia G. DenisovThe University of IllinoisUrbana, IL 61801, [email protected] 26

Charles Michael DrainHunter College of The City University of

New YorkNew York, NY 10065, [email protected] 15

Francis DSouzaWichita State UniversityWichita, KS 67260-0051, [email protected] 4

Florence DuclairoirInstitut Nanosciences et Cryognie38054 Grenoble cedex 9, FranceChapter 47

Manivannan EthirajanRoswell Park Cancer InstituteBuffalo, NY 14263, USAChapter 19

Alessandro FeisUniversity of FlorenceI-50019 Sesto Fiorentino, ItalyChapter 31

Angela FerrarioChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Kimberly B. FieldsUniversity of South FloridaTampa, FL 33620, USAChapters 13, 43

Takamitsu FukudaOsaka UniversityToyonaka 560-0043, [email protected] 42

Shunichi FukuzumiOsaka UniversitySuita, Osaka 565-0871, [email protected] Chapter 46

Hiroyuki FurutaKyushu UniversityFukuoka 819-0395, [email protected] 10

xvi Contributing Authors

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Jean-Paul GisselbrechtUniversit de Strasbourg67000 Strasbourg, [email protected] 14

Charles J. GomerUniversity of Southern CaliforniaLos Angeles, CA 90027, USAChapter 22

Bruno GrimmFriedrich-Alexander-University Erlangen-

Nuremberg91058 Erlangen, GermanyChapter 2

Dirk M. GuldiFriedrich-Alexander-University Erlangen-

Nuremberg91058 Erlangen, [email protected] 2

Anita HausmannFriedrich-Alexander-University Erlangen-


Takashi HayashiOsaka UniversitySuita 565-0871, [email protected] 23

Petra HellwigUniversit de Strasbourg67000 Strasbourg, [email protected] 36

Yoshio HisaedaKyushu UniversityFukuoka 819-0395, [email protected] 48

Barry D. HowesUniversity of FlorenceI-50019 Sesto Fiorentino, ItalyChapter 31

Akira IkezakiToho UniversityOta-ku, Tokyo 143-8540, JapanChapter 32

Osamu ItoTohoku UniversitySendai, 981-3215, JapanChapter 4

Anabella IvancichCentre Nationale de la Recherche

Scientifique (URA 2096)F-91191 Gif-sur-Yvette, FranceChapter 31

Christophe JeandonUniversit de Strasbourg67000 Strasbourg, [email protected] 14

Norbert JuxUniversitt Erlangen-Nrnberg91054 Erlangen, [email protected] 20

Contributing Authors xvii

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Axel KahntFriedrich-Alexander-University Erlangen-


David KesselWayne State University School of

MedicineDetroit, MI 48201, [email protected] 21

Dongho KimYonsei UniversitySeoul, 120-747, [email protected] 5, 6

Kil Suk KimYonsei UniversitySeoul, 120-747, KoreaChapter 6

Nagao KobayashiTohoku UniversitySendai 980-8578, [email protected] 33, 42

Lechosl/aw Latos-Graz.ynskiUniversity of WrocawWrocaw 50 383, [email protected] 8

Genxi LiNanjing UniversityNanjing 210093, PR [email protected] 27

Jong Min LimYonsei UniversitySeoul, 120-747, KoreaChapter 6

Aurore LoudetTexas A&M UniversityCollege Station, TX 77842, USAChapter 37

Evgeny A. LukyanetsOrganic Intermediates and Dyes InstituteMoscow, 123995, [email protected] 11

Marian LunaChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Hiromitsu MaedaRitsumeikan UniversityKusatsu 525-8577, [email protected] 38

Jean-Claude MarchonInstitut Nanosciences et Cryognie38054 Grenoble cedex 9, [email protected] 47

M. Victoria Martnez-Daz Universidad Autnoma de Madrid28049-Madrid, SpainChapter 45

xviii Contributing Authors

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Frederic MelinUniversit de Strasbourg67000 Strasbourg, FranceChapter 36

Shingo NaganoTottori UniversityTottori 680-8552, JapanChapter 25

Mikio NakamuraToho UniversityOta-ku, Tokyo 143-8540, [email protected] 32

Wonwoo NamEwha Womans UniversitySeoul 120-750, South [email protected] 44

Victor N. NemykinUniversity of Minnesota DuluthDuluth, MN 55812, [email protected] 11

Maria G.P.M.S. NevesUniversity of Aveiro3810-193 Aveiro, PortugalChapter 9

Tebello NyokongRhodes UniversityGrahamstown, 6139, South [email protected] 34

Yoshiki OhgoToho UniversityOta-ku, Tokyo 143-8540, JapanChapter 32

Tetsuo OkujimaEhime UniversityMatsuyama 790-8577, JapanChapter 7

Noboru OnoEhime UniversityMatsuyama 790-8577, [email protected] 7

Atsuhiro OsukaKyoto UniversityKyoto 606-8502, [email protected] 1

Ravindra K. PandeyRoswell Park Cancer InstituteBuffalo, NY 14263, [email protected] 19

Nayan J. PatelRoswell Park Cancer InstituteBuffalo, NY 14263, USAChapter 19

Mil/osz PawlickiUniversity of WrocawWrocaw 50 383, PolandChapter 8

Contributing Authors xix

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Sbastien RicheterUniversit Montpellier 234095 Montpellier Cedex 5, [email protected] 14

Beate RderHumboldt-Universitt zu Berlin12489 Berlin, [email protected] 20

Natalie RuckerChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Joshua V. RuppelUniversity of South FloridaTampa, FL 33620, USAChapters 13, 43

Romain RuppertUniversit de Strasbourg67000 Strasbourg, [email protected] 14

Aoife RyanTrinity College DublinDublin 2, IrelandChapter 12

Wolfgang SeitzFriedrich-Alexander-University

Erlangen-Nuremberg91058 Erlangen, GermanyChapter 2

Mathias O. SengeTrinity College DublinDublin 2, [email protected] 12

Natalia N. SergeevaTrinity College DublinDublin 2, IrelandChapter 12

Hisashi ShimakoshiKyushu UniversityFukuoka 819-0395, JapanChapter 48

Jae-Yoon ShinYonsei UniversitySeoul, 120-747, KoreaChapter 6

Yoshitsugu ShiroHarima InstituteHyogo 679-5148, [email protected] 24, 25

Martha Sibrian-VazquezPortland State UniversityPortland, OR 97201, USAChapter 18

Sunaina SinghHunter College of The City University of

New YorkNew York, NY 10065, USAChapter 15

xx Contributing Authors

b867_Vol-06_FM.qxd 6/10/2010 4:44 PM Page xx

Stephen G. SligarThe University of IllinoisUrbana, IL 61801, [email protected] 26

Giulietta SmulevichUniversity of FlorenceI-50019 Sesto Fiorentino, [email protected] 31

Nicole L. SnyderHamilton CollegeClinton, NY 13323, USAChapters 13, 43

Fabian SpnigFriedrich-Alexander-University Erlangen-


Tatyana SpolitakUniversity of MichiganAnn Arbor, MI 48109-5606, USAChapter 28

Hiroshi SugimotoHarima InstituteHyogo 679-5148, JapanChapter 24

Osamu TakikawaNational Center for Geriatrics and

GerontologyObu, Aichi 474-8522, JapanChapter 24

Alison ThompsonDalhousie UniversityHalifax, Nova Scotia, [email protected] 39

Motoki ToganohKyushu UniversityFukuoka 819-0395, JapanChapter 10

Augusto C. TomUniversity of Aveiro3810-193 Aveiro, PortugalChapter 9

Tomas TorresUniversidad Autnoma de Madrid28049-Madrid, [email protected] 45

Paola TuranoUniversity of FlorenceI-50019 Sesto Fiorentino, [email protected] 30

Md. Imam UddinDalhousie UniversityHalifax, Nova Scotia, CanadaChapter 39

Johan E. van LierUniversit de SherbrookeSherbrooke, Qubec, [email protected] 16

Contributing Authors xxi

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Maria da Graa H. VicenteLouisiana State UniversityBaton Rouge, LA 70803, [email protected] 18

Sam P. de VisserThe University of ManchesterManchester M1 7DN, [email protected] 44

F. Ann WalkerUniversity of ArizonaTucson, AZ 85721-0041, [email protected] 29

Jacek WalukPolish Academy of Sciences01-224 Warsaw, [email protected] 35

Sam WongChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Tabitha E. WoodDalhousie UniversityHalifax, Nova Scotia, CanadaChapter 39

Frank XuChildrens Hospital Los AngelesLos Angeles, CA 90027, USAChapter 22

Hiroko YamadaEhime UniversityMatsuyama 790-8577, JapanChapter 7

Jaesung YangYonsei UniversitySeoul, 120-747, KoreaChapter 5

Hyejin YooYonsei UniversitySeoul, 120-747, KoreaChapter 5

Min-Chul YoonYonsei UniversitySeoul, 120-747, KoreaChapter 6

Zin Seok YoonYonsei UniversitySeoul, 120-747, KoreaChapter 5

X. Peter ZhangUniversity of South FloridaTampa, FL 33620, [email protected] 13, 43

xxii Contributing Authors

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xxiii

Contents of Volumes 110

Volume 1 Supramolecular Chemistry

1. Synthetic Strategies Toward Multiporphyrinic ArchitecturesNaoki Aratani and Atsuhiro Osuka

2. Charge Transfer Between Porphyrins/Phthalocyanines and CarbonNanostructuresBruno Grimm, Anita Hausmann, Axel Kahnt, Wolfgang Seitz,Fabian Spnig and Dirk M. Guldi

3. Self-Assembling Porphyrins and Chlorins as Synthetic Mimics of theChlorosomal BacteriochlorophyllsTeodor Silviu Balaban

4. TetrapyrroleNanocarbon Hybrids: Self-Assembly and PhotoinducedElectron TransferFrancis DSouza and Osamu Ito

5. Photophysical Properties of Various Directly Linked Porphyrin ArraysZin Seok Yoon, Jaesung Yang, Hyejin Yoo, Sung Cho and Dongho Kim

6. Photophysics and Photochemistry of Various Expanded PorphyrinsJong Min Lim, Min-Chul Yoon, Kil Suk Kim, Jae-Yoon Shinand Dongho Kim

Volume 2 Synthesis and Coordination Chemistry

7. Synthesis of Porphyrins Fused with Aromatic RingsNoboru Ono, Hiroko Yamada and Tetsuo Okujima

8. Carbaporphyrinoids Synthesis and Coordination PropertiesMil/osz Pawlicki and Lechosl/aw Latos-Graz.ynski

9. meso-Tetraarylporphyrin Derivatives: New Synthetic MethodologiesJos A.S. Cavaleiro, Augusto C. Tom and Maria G.P.M.S. Neves

10. Synthesis and Metal Coordination of N-Confused and N-FusedPorphyrinoidsMotoki Toganoh and Hiroyuki Furuta

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Volume 3 Synthetic Methodology

11. The Key Role of Peripheral Substituents in the Chemistryof PhthalocyaninesVictor N. Nemykin and Evgeny A. Lukyanets

12. Organometallic CC Coupling Reactions for PorphyrinsNatalia N. Sergeeva, Mathias O. Senge and Aoife Ryan

13. Porphyrin Functionalization via Palladium-Catalyzed CarbonHeteroatomCross-Coupling ReactionsKimberly B. Fields, Joshua V. Ruppel, Nicole L. Snyderand X. Peter Zhang

14. Peripherally Metalated Porphyrin Derivatives: Synthetic Approachesand PropertiesSbastien Richeter, Christophe Jeandon, Jean-Paul Gisselbrechtand Romain Ruppert

15. Combinatorial Libraries of Porphyrins: Chemistry and ApplicationsCharles Michael Drain and Sunaina Singh

Volume 4 Phototherapy, Radioimmunotherapy and Imaging

16. Porphyrins and Phthalocyanines as Photosensitizers and RadiosensitizersHasrat Ali and Johan E. van Lier

17. Bioconjugates of Porphyrins and Related Molecules for PhotodynamicTherapyCristina Alonso and Ross W. Boyle

18. Syntheses of Boronated Porphyrins and Their Application in BNCTMaria da Graa H. Vicente and Martha Sibrian-Vazquez

19. Porphyrin-Based Multifunctional Agents for Tumor-Imaging andPhotodynamic Therapy (PDT)Manivannan Ethirajan, Nayan J. Patel and Ravindra K. Pandey

20. Targeting Strategies for Tetrapyrrole-Based Photodynamic Therapy ofTumorsNorbert Jux and Beate Rder

21. Mechanisms of Cell Death in Photodynamic TherapyDavid Kessel

22. Photodynamic Therapy and the Tumor MicroenvironmentCharles J. Gomer, Angela Ferrario, Marian Luna, Natalie Rucker,Sam Wong, Ozguncem Bozkulak and Frank Xu

xxiv Contents of Volumes 110

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Volume 5 Heme Proteins

23. Hemoproteins Reconstituted with Artificially Created HemesTakashi Hayashi

24. Tryptophan Catabolism by Heme DioxygenasesHiroshi Sugimoto, Osamu Takikawa and Yoshitsugu Shiro

25. NO Chemistry by Heme-EnzymesYoshitsugu Shiro and Shingo Nagano

26. Cytochrome P450 EnzymesIlia G. Denisov and Stephen G. Sligar

27. Heme Protein-Based Electrochemical BiosensorsGenxi Li

28. The Generation and Characterization of the Compounds I and ES States ofCytochrome P450 Using Rapid Mixing MethodsDaniel P. Collins, Tatyana Spolitak, David P. Ballou and John H. Dawson

Volume 6 NMR and EPR Techniques

29. NMR and EPR Spectroscopy of Paramagnetic Metalloporphyrinsand Heme ProteinsF. Ann Walker

30. Heme Acquisition by Hemophores: A Lesson from NMRPaola Turano

31. StructureFunction Relationships Among Heme Peroxidases: New Insightsfrom Electronic Absorption, Resonance Raman and MultifrequencyElectron Paramagnetic Resonance SpectroscopiesGiulietta Smulevich, Alessandro Feis, Barry D. Howesand Anabella Ivancich

Volume 7 Physicochemical Characterization

32. Electronic and Magnetic Structures of Iron Porphyrin ComplexesMikio Nakamura, Yoshiki Ohgo and Akira Ikezaki

33. Optically Active Porphyrin Systems Analyzed by Circular DichroismNagao Kobayashi

34. Photochemical and Photophysical Properties of MetallophthalocyaninesTebello Nyokong and Edith Antunes

35. Structure, Spectroscopy, Photophysics, and Tautomerism of Free-BasePorphycenes and Other Porphyrin IsomersJacek Waluk

Contents of Volumes 110 xxv

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36. Recent Applications of Infrared Spectroscopy and Microscopyin Chemistry, Biology and MedicinePetra Hellwig and Frdric Melin

Volume 8 Open-Chain Oligopyrrole Systems

37. BODIPY Dyes and Their Derivatives: Syntheses and SpectroscopicPropertiesAurore Loudet and Kevin Burgess

38. Supramolecular Chemistry of Pyrrole-Based -Conjugated AcyclicAnion ReceptorsHiromitsu Maeda

39. The Synthesis and Properties of DipyrrinsTabitha E. Wood, Md. Imam Uddin and Alison Thompson

40. Coordination Chemistry of Verdohemes and Open-Chain OligopyrroleSystems Involved in Heme Oxidation and Porphyrin DestructionAlan L. Balch and Faye L. Bowles

41. Beyond Dipyrrins: Coordination Interactions and TemplatedMacrocyclizations of Open-Chain OligopyrrolesMartin Brring

Volume 9 Electronic Absorption Spectra Phthalocyanines

42. UV-Visible Absorption Spectroscopic Properties of Phthalocyaninesand Related MacrocyclesTakamitsu Fukuda and Nagao Kobayashi

Volume 10 Catalysis and Bio-Inspired Systems Part I

43. Metalloporphyrin-Catalyzed Asymmetric Atom/Group Transfer ReactionsJoshua V. Ruppel, Kimberly B. Fields, Nicole L. Snyderand X. Peter Zhang

44. High-Valent Iron-Oxo Porphyrins in Oxygenation ReactionsSam P. de Visser and Wonwoo Nam

45. On the Significance of Phthalocyanines in Solar CellsM. Victoria Martnez-Daz and Toms Torres

46. Artificial Photosynthetic Systems Composed of Porphyrinsand PhthalocyaninesShunichi Fukuzumi

xxvi Contents of Volumes 110

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47. Anchoring of Porphyrins and Phthalocyanines on Conductorsand Semiconductors for Use in Hybrid ElectronicsFlorence Duclairoir and Jean-Claude Marchon

48. Bioinspired Catalysts with B12 Enzyme FunctionsYoshio Hisaeda and Hisashi Shimakoshi

Contents of Volumes 110 xxvii

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NMR and EPR Spectroscopy ofParamagnetic Metalloporphyrinsand Heme Proteins

F. Ann Walker

Department of Chemistry and Biochemistry, University of Arizona,1306 E. University B1, Tucson, AZ 85721-0041, USA

I. Introduction and Background 7A. Structures and Electron Configurations of Metalloporphyrins 10

II. Principles 19A. Proton Chemical Shifts 19

1. Contact Shifts 202. Pseudocontact (Dipolar) Shifts 23

a. Pseudocontact Shifts of Metalloporphyrin Substituents 23b. Measurement of Magnetic Susceptibility Anisotropies

of Ferriheme Proteins 26c. Residual Dipolar Couplings of Proteins for Structure

Determination 293. Temperature Dependence of Contact

and Pseudocontact Shifts 31B. Nuclear Relaxation and Linewidths 33

1. Chemical Exchange Line Broadening and EXSYCross Peaks 33

2. Proton T1 and T2 Relaxation Times, as Controlledby Electron Spin Relaxation Times, T1e 34a. Electron Spin Relaxation Times, T1e or s 34b. Nuclear Spin-Lattice Relaxation Times, T1 34c. Nuclear SpinSpin Relaxation Times, T2 38

C. Spin Density and Bonding: Mechanisms of SpinDelocalization 391. The Metal Ion 392. The Porphyrin Ring 40

1

29

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3. The Effect of Axial Ligand Plane Orientation on theCombined Contact and Pseudocontact Shifts of Low-SpinFerriheme Proteins and Synthetic Hemins withHindered Axial Ligand Rotation 50

4. Mechanisms of Spin Delocalization throughChemical Bonds, and Strategies for Separationof Contact and Pseudocontact Shifts 55

D. Methods of Assignment of the 1H NMR Spectraof Paramagnetic MetalIoporphyrins 571. Substitution of H by CH3 or Other Substituent 572. Deuteration of Specific Groups 583. 2D 13C Natural Abundance HMQC Spectra and

Specific or Complete 13C Labeling of Protoheminfor the Assignment of Heme Resonances in Proteins 59

4. Saturation Transfer NMR Experiments 625. NOE Difference Spectroscopy 636. Two-Dimensional NMR Techniques 64

III. Spectral Analysis 69A. Resolution and Assignment 69B. Analysis of Shifts 75

1. Curvature in the Curie Plot over the Temperature Rangeof the Measurement 77a. Zero-Field Splitting Contributions to the

Pseudocontact Shift 79b. Nonzero Intercepts of the Curie Plot 79

2. Empirical Methods 803. g-Tensor Anisotropy 83

IV. Effect of Metal Ion and Spin State on Bonding 83A. Iron Porphyrins 83

1. Iron(I) Porphyrins 832. Diamagnetic Iron(II) Porphyrins 84

a. Six-Coordinate Diamagnetic Complexes 84b. Five-Coordinate Diamagnetic Complexes 85

3. Intermediate-Spin Iron(II) Porphyrins: Observed Shiftsand the Mechanism of Spin Delocalization 85

4. Five-Coordinate High-Spin Iron(II) Porphyrins: ObservedShifts and the Mechanism of Spin Delocalization 93a. Models of Deoxyhemoglobin and Deoxymyoglobin 93b. Models for the Reduced States of Cytochrome P450

and Chloroperoxidase 96

2 Walker


c. Models for the Heme a3-CuB and Heme-NonhemeFe Centers of Cytochrome Oxidase and NOReductase 96

d. Hydroxide or Fluoride Complexes 103e. N-Alkyl (aryl) Porphyrin Complexes 103f. Nitrene Complexes 104g. Verdoheme Analogs: Iron(II) Complexes

of Octaethyloxaporphyrin, OEOP 104h. N-confused or N-inverted Iron(II) Porphyrins

and Related N-modified Macrocycle Complexes 1055. Possible Iron(II) Porphyrin -Cation Radicals 1066. High-Spin Iron(III) Porphyrins: Observed Shifts

and the Mechanism of Spin Delocalization 107a. Five-Coordinate, Monomeric Iron(III) Porphyrin

Complexes 107b. Six-Coordinate Monomeric High-Spin Iron(III)

Porphyrin Complexes 112c. Monomeric Complexes of Reduced or Oxidized

Ferrihemes 117i. Iron(III) Sulfhemins 117

ii. Iron(III) Octaethyl- and Tetraphenylchlorin 117iii. Two Iron(III) Octaethylisobacteriochlorin Isomers 121iv. An Iron(III) Monooxochlorin Complex 122v. Two Iron(III) Dioxooctaethylisobacteriochlorin

Complexes 123vi. The Iron(III) Complex of Tetraphenyl-21-

Oxaporphyrin 123vii. The High-Spin Iron(III) Complexes of

Mono-meso-octaethyloxaporphyrin andMono-meso-octaethylazaporphyrin 124

d. Bridged Dimeric Complexes of High-Spin Iron(III)Porphyrins and Chlorins 125

7. Intermediate-Spin Iron(III) Porphyrins: Observed Shiftsand the Mechanism of Spin Delocalization 128

8. Low-Spin Iron(III) Porphyrins 132a. Griffiths Three-Orbital Theory and Experimental

EPR Data for Low-Spin Iron(III) Porphyrinsand Related Macrocycles 134

b. NMR Studies of Low-Spin Iron(III) Porphyrins Havingthe (dxy)2(dxz,dyz)3 Ground State 147

29/NMR and EPR Spectroscopy 3


i. Effect of Porphyrin Substituents on the Patternof Spin Delocalization 147

ii. The Shifts of Coordinated Imidazole Ligandsand the Effect of Imidazole Deprotonationon the Pattern and Extent of Spin Delocalization 151(a) Neutral Imidazole Ligands 151(b) Imidazolate Ligands 152

iii. Effect of Imidazole Plane Orientation on theParamagnetic Shifts of Low-Spin Iron(III)Porphyrins 152

iv. Bis-Ammine, Amino Ester and PhosphineComplexes 159

v. Mixed-Ligand Complexes 160c. Observed Shifts and the Mechanism of Spin

Delocalization for the (dxz,dyz)4(dxy)1 Ground State 161d. The Mixed Ground State Behavior of Bis-Cyanide

Complexes of Low-Spin Ferrihemes: Observed Shiftsand the Mechanism of Spin Delocalization 164

e. The Mixed Ground State Behavior of Bis-(pyridine)Complexes of Low-Spin Ferrihemes: Observed ShiftTrends and the Mechanism of Spin Delocalization 167

f. The Mixed Ground State Behavior of Bis-(pyridine)Complexes of Low-Spin Iron(III) Complexesof Oxophlorins and Meso-Amino Porphyrins 174

g. Low-Spin FeIII Complexes of MesoMeso-Linked5,5-Bis(10,15,20-Triphenylporphyrin) 178

h. Five-Coordinate Low-Spin Iron(III) Porphyrinsand a Porphycene 179

i. Low-Spin Iron(III) Complexes of Reduced Hemes 182j. Low-Spin Iron(III) Complexes of N-Alkylporphyrins 186k. Thermodynamics of Axial Ligation of Iron(III)

Porphyrins 186l. Kinetics of Axial Ligand Exchange 187

9. Electron Exchange Between Low-Spin Iron(III)and Low-Spin Iron(II) Porphyrins 188

10. 1H and 13C NMR Spectroscopy of High- and Low-SpinFerriheme Proteins: The Nitrophorins and HemeOxygenases 190a. NMR Spectroscopy of the Nitrophorins 190

4 Walker


i. NMR Investigations of the High-Spin Formsof the Nitrophorins from Rhodnius prolixus 196

ii. pH Titration of the High-Spin Nitrophorins fromRhodnius prolixus 204

iii. NMR Investigations of the Low-Spin Formsof the Nitrophorins from Rhodnius prolixus,and Comparison to Other Heme Proteins 207

iv. Heme Ruffling of the Nitrophorins andComparison to Other Heme Proteins 217

v. Nitrite Reductase Activity of Nitrophorin 7 221vi. Dimerization of NP4 222

vii. NMR Spectroscopy of Apo-Nitrophorin 2 223b. NMR Spectroscopy of the Hemin-Containing Heme

Oxygenases 225i. NMR Study of High- and Low-Spin Mammalian

Heme Oxygenases 227ii. NMR Studies of Bacterial Heme Oxygenases 230

(a) Heme Propionate-Polypeptide InteractionsDictate Regioselectivity in HOs 231

(b) NMR Studies of Heme Electronic Structureand its Potential Implications to the Mechanismof Heme Oxidation 236

(c) NMR Spectroscopic Studies of DynamicReactivity Relationships 243

c. NMR Spectroscopy of Miscellaneous OtherHeme Proteins 249

11. Iron(III) Macrocycle -Cation Radicals 251a. High-Spin Iron(III) Porphyrin -Cation Radicals 251b. Spin-Admixed and Intermediate-Spin Iron(III)

Porphyrin -Cation Radicals 253c. Low-Spin Iron(III) Porphyrin -Cation Radicals 254d. Iron(III) -Cation Radicals of Oxophlorins 254e. Iron(III) Corrole -Radicals 256

12. Iron(IV) Porphyrins 258a. Six-Coordinate, Bis-Methoxide Iron(IV) Porphyrins 259b. Five- and Six-Coordinate Ferryl, (Fe=O)2+, Porphyrin

Complexes 259c. Five-Coordinate Iron(IV) Phenyl Porphyrins 260d. Comparison of Iron(IV) Porphyrins and Iron(III)

Porphyrin -Radicals 261



13. Iron(IV) Porphyrin -Radicals 26114. Iron(V) Porphyrins 266

B. Ruthenium and Osmium Porphyrins 266C. Cobalt Porphyrins 268

1. Low-Spin Cobalt(II) Porphyrins 268a. Observed Shifts and the Pseudocontact Interaction 268b. Oxidation of Cobalt(II) Porphyrins to Produce

-Radical Dimers 270c. Low-Spin Cobalt(II) Oxaporphyrins and

Porphodimethenes 271d. High-Spin Cobalt(II) N-Alkylporphyrins and Alkoxy

Adducts of Oxaporphyrins 2722. High-Spin Cobalt(II) Complexes of a Weak-Field

Porphyrin Ligand 2733. Alkylcobalt(III) Porphyrins: Agostic Interactions

or Paramagnetic Excited States? 2734. A Cobalt(III) Porphyrin -Cation Radical 275

D. Rhodium Porphyrins 275E. Manganese Porphyrins 278

1. High-Spin Manganese(II) Porphyrins 2792. High-Spin Manganese(III) Porphyrins 2793. Low-Spin Manganese(III) Porphyrins 2804. Manganese(III) Corrole at Low Temperatures =

Manganese(II) Corrole -Cation Radical at AmbientTemperatures 280

5. Manganese(III) Porphyrin -Radicals and TheirTransformation to Dichloromanganese(IV) Porphyrins 283

6. Oxomanganese(IV) Porphyrins 284F. Nickel Porphyrins 284G. Lanthanide Porphyrins 292H. Miscellaneous Metalloporphyrins which Have Extremely

Broad Lines 2941. Copper(II) and Silver(II) Porphyrins 2942. Vanadium(IV) Porphyrins 2973. Chromium Porphyrins 298

I. Summary of Paramagnetic Shifts and Mechanisms of SpinDelocalization for the Metalloporphyrins 299

V. Acknowledgments 303VI. References 304

6 Walker


I. Introduction and Background

Magnetic resonance techniques, both nuclear magnetic resonance (NMR) andelectron paramagnetic resonance (EPR) spectroscopies are excellent techniquesfor investigating the structure, bonding, dynamics, electron configurations andmagnetism of transition metal complexes, and this certainly includes metallopor-phyrins. Of course, EPR techniques can only be used when there are odd numbersof unpaired electrons present, either on the metal itself or in some cases, on theligand(s). The most common number of unpaired electrons present in systemswhich are studied by EPR spectroscopy is one, although five is another fairly com-mon number, and three is also possible. The development of pulsed EPR tech-niques, which make possible the study of all magnetic nuclei that are within 35 of the site of the unpaired electron (usually the metal, although there are anincreasing number of cases of macrocycle -cation or -anion radicals that havebeen studied). The types of magnetic resonance techniques, both those belongingto the EPR and to the NMR family, that are commonly used to study metallopor-phyrin complexes are summarized and briefly described in Table 1. Although theresults of pulsed EPR experiments are mentioned, where appropriate, the experi-ments and the detailed description of the results is beyond the scope of this chap-ter. Some of these may be found in a review published in Inorganic Chemistry.1

While EPR spectroscopy requires the complex to have at least one unpairedelectron or multiple unpaired electrons, NMR techniques can be utilized whetherthe complex is diamagnetic or paramagnetic, and a large number of the metallicelements and/or their compounds have paramagnetic states (S 1/2). In fact, anumber of the transition metals have several oxidation states that are reasonablystable and can be studied by NMR spectroscopy, and for a given oxidation statethere are often several different spin states that are possible. However, not all ofthese oxidation/spin state possibilities have the proper relaxation properties tobe easily studied by NMR spectroscopy, as we will discuss later in Section II.B.This author has been involved in the writing of the previous chapters on NMR ofparamagnetic metalloporphyrins,24 and within the 30 year time span covered upto the present, some of the systems previously thought to be impossible to studyby NMR spectroscopy have been reported, including chromium(III), man-ganese(IV), copper(II) and silver(II) porphyrins. In most of these cases it has been2H NMR spectroscopic investigations, where the chemical shifts are extremelysimilar to those of the corresponding 1H complexes, but the linewidths of the res-onances can be as much as a factor of 42 sharper, which have made possible thedetection of signals previously thought to be entirely too broad to observe.Discussion of such systems will be found in Sections II.B.2, IV.B.7 and IV.H.1,IV.H.3.



8 Walker

Table 1. Common NMR and EPR experiments used for investigating paramagneticmetalloporphyrins.

NMR experiment Information Obtained

1D 1H, 2H or 13C Chemical shift isotropic or paramagnetic shift mechanism ofspin delocalization; from temperature dependence information about excited states.

1D NOE difference Proximity of one type of proton to another, up to a distance ofspectroscopy about 5 . A spectrum is obtained in which a particular

resonance is irradiated. Then a reference, off-resonanceirradiation dataset is acquired and the reference is subtractedfrom the first. The irradiated peak appears as a strong negativepeak, and those peaks that are due to protons in close proximityappear as either weak negative or positive peaks, depending onthe size of the molecule.

1D saturation transfer Identifies resonances that are involved in chemical exchange. Thedifference experiment is the same as that used for 1D NOE differencespectroscopy (also spectroscopy. Both irradiated peak and that (those) connected tocalled STD) it by chemical exchange are of negative phase if the reference

spectrum is subtracted from each peak irradiated.

2D COSY; TOCSY Correlation SpectroscopY; Total Correlation SpectroscopY.J-coupling information is obtained; helps identify components ofa spin system such as an ethyl or propionate group.

2D HETCOR; HMQC HETeronuclear CORrelation spectroscopy, a 13C-detected 2Dheteronuclear experiment; Heteronuclear Multiple QuantumCorrelation spectroscopy, a 1H-detected heteronuclear 2Dexperiment. C-H J-coupling information is obtained in bothcases; both identify which proton(s) are bound to which carbon.HMQC has a much higher signal-to-noise (at least a factor of 4)ratio than HETCOR.

2D HMBC Heteronuclear Multiple Bond Correlation spectroscopy, amodification of the HMQC experiment which allows detectionof long-range H-C J-couplings. Very useful for assigning thespectra of unsymmetrical metalloporphyrins.

2D NOESY; EXSY; Nuclear Overhauser and Exchange SpectroscopY (sensitive to bothROESY through-space (NOE) and chemical exchange processes; NOEs

may be zero for intermediate-sized molecules, i.e., manymetalloporphyrins); Exchange SpectroscopY (the sameexperiment), which identifies protons in chemical exchangewith each other; Rotating frame Overhauser and ExchangeSpectroscopY (also sensitive to both through-space andchemical exchange, but shows NOEs the opposite phase andchemical exchange peaks the same phase as the diagonal, nomatter what the size of the molecule).

Continuous wave EPR g-values orbital of the unpaired electron; superhyperfinecouplings, a or A, due to nuclei that are strongly coupled to theunpaired electron.

(Continued )


NMR spectroscopy is particularly valuable for studying paramagnetic metal-loporphyrins because the unpaired electrons illuminate the heme or other met-alloporphyrin center by causing paramagnetic shifts of the protons (or carbons) ofthe macrocycle, the axial ligand(s) of the metal and, in the case of heme proteins,nearby protein residues, that are exquisitely sensitive to heme substituents, axialligand effects such as histidine imidazole NH hydrogen-bonding5,6 or deprotona-tion,5 axial ligand plane orientation,5,7,8 methionine-SCH3 chirality,9 and cyanide


Table 1. (Continued )

NMR Experiment Information Obtained

ESEEM Electron Spin Echo Envelope Modulation signals due to allnuclei that are so weakly coupled to the unpaired electron thatthey show no superhyperfine couplings in the continuous waveEPR spectrum. Magnetic field (g-value dependence of theintensity and frequency of double quantum proton peaks neartwice the Larmor frequency orientation of the g-tensor;similar analysis of the 14N and/or 2H peaks also provides veryuseful bonding and structural information. Intensities of ESEEMpeaks are usually readily fit quantitatively to theoretical models.

HYSCORE 2D Hyperfine Sublevel CORrElation spectroscopy: A twodimensional ESEEM experiment, /2 /2 T1 T2 /2 echo, in which correlation is produced between nuclearfrequencies in the spin manifolds for an electronic transition.In a HYSCORE experiment, the time between the second /2and pulse is varied in one dimension and the time betweenthe and third /2 pulse is varied in a second dimension. Atwo- dimensional FT gives the 2D spectra which can beanalyzed in terms of the frequencies of the nuclei involved. Asin an ESEEM experiment, the peaks observed are essentially anNMR spectrum of nuclei that are coupled to the electron.However, the 2D technique allows one to separate overlappingpeaks. Peaks appearing in the upper right and lower leftquadrants typically arise from nuclei in which the hyperfinecoupling is less than the Larmor frequency. They appear at theLarmor frequency of the nucleus, separated by the hyperfinecoupling. Peaks from nuclei in which the hyperfine interaction isgreater than the Larmor frequency of the nucleus appear in theupper left and lower right quadrants. The HYSCORE spectrumcan become very complicated for nuclei with a spin I greaterthan 1/2, with many peaks arising from the additionalcomplication of addition nuclear Zeeman and quadrupolelevels. Even with the complexity of the spectra, HYSCORE oncomplicated systems with multiple nuclei can make ESEEMspectra that would be difficult or impossible to interpret muchmore manageable.


off-axis tilting (in protein complexes such as cyanometmyoglobin),10,11 as well asthe effect of nonbonded protein substituents,5,12 on the electronic state of the heme.The chemical shifts of the heme macrocycle of heme proteins, also known as para-magnetic or hyperfine shifts if the metal is paramagnetic, are extremely sensitiveto all of these factors, as well as to others that will be enumerated in later sectionsof this chapter, and much can be learned by carrying out a detailed study of a para-magnetic heme protein. Because of this extreme sensitivity, it is sometimes diffi-cult to determine the relative importance of all the factors that might affect thechemical shifts. For this reason, a number of investigators have carried outdetailed NMR investigations of appropriately designed model hemes or othermetal tetrapyrroles having relatively high molecular symmetry (as compared tonaturally-occurring hemes), in order to probe the importance of such factors assubstituent effects, electronic asymmetry due to unsymmetrical substitution, axialligand basicity and steric effects, axial ligand plane orientation, thermodynamicsand kinetics of axial ligand binding, exchange and rotation, and hydrogen-bondingeffects of these ligands on the NMR spectra of metalloporphyrins. The results ofa large number of these investigations have been summarized previously in at leasta dozen important chapters or books.25,1320 However, the focus in all but four ofthese24,15 has been on heme proteins, where the metal ion was either iron(II) oriron(III). For that reason, this chapter will focus on the proton (and deuteron)NMR spectroscopy of metalloporphyrins of all known oxidation and spin statesthat have occurred since the literature search for the publication of the previouschapters in The Porphyrins2 and The Porphyrin Handbook,4 with only occasionaldetailed recapitulation of earlier topics at points where a background must be builtup before the recent work can be discussed. The 13C chemical shifts of a numberof metalloporphyrins and heme proteins, and occasionally, the 19F chemical shiftswill also be discussed. Parts of this chapter will cover some of the same topics asThe Porphyrin Handbook chapter4 published about ten years ago, which is neces-sary for completeness. The NMR spectra of the heme centers of heme proteins willbe discussed where the results of NMR studies of paramagnetic metalloporphyrinshave direct relevance to interpretations of the heme protein data, or vice versa. Inparticular, a section on NMR spectroscopy of the nitrophorins and heme oxyge-nases, and a brief update on other heme proteins, is included (Section IV.A.10).

A. Structures and Electron Configurations of Metalloporphyrins

Structural formulas 13 show the general structures of porphyrins. The structuresof closely related ring systems, including chlorins, isobacteriochlorins, dioxo-isobacteriochlorins and corroles are shown in Structures 47. Examples of the sub-stituents present on commonly investigated metal macrocycles are summarized in

10 Walker



N N

NN

CH3

CH3

CH2

CH2

CH2

CH2

H3C

H3C

R2

H

H

R4

H

COO-COO-

H

1

2 3

4

5

67

8

I II

IIIIV

1

HH

N N

NNR12

R7

R17 R13

R18

R2

R3

R8

H15

H20

3 H5

H10H

H

N N

NN

R'2'

R17

R'7' R3

R8

R12

R13

R18

R5

R10

5 R15

R20

R7R'8'

R2

R'3'

HH

N N

NN

R12

R7

R17R13

R18

R2

R3

R8

H15

7 H5

H10H

H

N N

NN

R12

R7

R13

R18

R'2O

O

6

R17

R'7

R2

H

H

N N

NNR5

R17

R12

R'2' R18

R3

R7

R8

R13

R20

4 R10

R15

R2R'3'

HH

N N

NN

R5 H

H

H

HR15H

H

H

H

R20

2

R10H

H


Tables 25. Porphyrin macrocycles can be classified as derivatives of natural por-phyrins, of which deuteroporphyrin IX is considered the parent. The arrangementof the peripheral substituents of natural porphyrin derivatives is shown inStructure 1, with the 18 and numbering system used in much of the NMRliterature, and the nature of the variable substituents, the common names of themacrocycle and the symbols used in this chapter, are listed in Table 2. For casesin which the propionic acid side chains in Structure 1 are esterified, for example,as dimethyl esters, the symbol will have DME appended to the abbreviation. Two

12 Walker

Table 2. Natural porphyrin dianion derivatives.a

2,4-Substituent Name Symbol

2,4-H Deuteroporphyrin IX DP2

2,4-Divinyl Protoporphyrin IX PP2

2,4-Diethyl Mesoporphyrin IX MP2

2,4-Dibromo Dibromodeuteroporphyrin Br2DP2

2,4-Diacetyl Diacetyldeuteroporphyrin Ac2DP2

2-Acetyl, 4-H 2-Monoacetyldeuteroporphyrin 2-AcDP2

2-H, 4-Acetyl 4-Monoacetyldeuteroporphyrin 4-AcDP2

aSee Structure 1 for structural formula of the parent free-base porphyrin.If the propionates are esterifed, DME is appended to symbolize dimethyl ester.aSee Structure 2 for structural formula of the free base porphyrin.

Table 3. Meso-substituted synthetic porphyrin dianion derivatives.a

R5, R10, R15, R20 Name Symbol

H Porphine Po2

Phenyl Tetraphenylporphyrin TPP2

p-Tolyl Tetra-p-tolylporphyrin p-TTP2

m-Tolyl Tetra-m-tolylporphyrin m-TTP2

o-Tolyl Tetra-o-tolylporphyrin o-TTP2

p-Chlorophenyl Tetra-p-chlorophenylporphyrin (p-Cl)4TPP2

Mesityl Tetramesitylporphyrin TMP2

N-Methyl-4-Pyridyl Tetra-N-methyl-4-pyridylporphyrin (p-MePy)4P 2+ or T(p-MePy)P2+

p-SO3-Phenyl Tetra-p-sulfonatophenylporphyrin TPPS6

5-(m-nitrophenyl)-Mono-m-NO2-Phenyl, 10,15,20-tris-(m-fluorophenyl)- (m-NO2)(m-F)3TPP2

tris-m-F-Phenyl porphyrinMethyl Tetra-methylporphyrin TMeP2

n-Propyl Tetra-n-propylporphyrin TnPrP2

iso-Propyl Tetra-i-propylporphyrin TiPrP2

Cyclohexyl Tetra-cyclohexylporphyrin TCxP2

aSee Structure 2 for structural formula of the free base porphyrin.


29/NM

R and EPR

Spectroscopy13

Table 4. Pyrrole-substituted synthetic porphyrin dianion derivatives.a

R2 R3 R7 R8 R12 R13 R17 R18 mesob Name Symbol

H H H H H H H H H Porphine Po2

Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl H Octaethylporphyrin OEP 2

Ethyl Methyl Ethyl Methyl Ethyl Methyl Ethyl Methyl H Etioporphyrin I EP-I2

Ethyl Methyl Methyl Ethyl Ethyl Methyl Methyl Ethyl H Etioporphyrin II EP-II2

Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Ethyl Phenyl Octaethyltetraphenylporphyrin OETPP2

N=CHCH=N H H H H H H Phenyl Quinoxalinotetraphenylporphyrin QTPP2

aSee Structure 3 for structural formula of the free base porphyrin.bmeso = 5,10,15,20 positions; the OETPP and QTPP dianions are hybrids of Structures 2 and 3.


14W

alker

Table 5. Structures of well-known chlorin, isobacteriochlorin, dioxoisobacteriochlorin and corrole derivatives.

R2 R3 R7 R8 R12 R13 R17, R17 R18, R18 mesob Name Symbol

Chlorins, Structure 4

H H H H H H H,H H,H Phenyl Tetraphenylchlorin TPC2

Et Et Et Et Et, Et Et,H Et,H H Octaethylchlorin OEC2

PrOc Me Me V Me V Me,OH OH, PrOc H Heme d NoneMe V Me V Me C=O H,MePropionate H,Me 5, 10, 20-H, Pyropheophorbide a None

| 15CH2C=OCH215meso connected to

13CMe V Me Et CO2Me H,MePropionate 5,10,20-H, Chlorin e6 None

15-CH2CO2Me

R2 R3 R7 R8 R12, R12 R13 R13 R17, R17 R18, R18 mesob Name Symbol

Isobacteriochlorins, Structure 5

Et Et Et Et Et, H Et, H Et, H Et, H H Octaethylisobac- OEiBC2

teriochlorinAcOc PrOc PrOc AcOc H,PrOc H,PrOc H,PrOc H,PrOc H Siroheme None

(Continued )


29/NM

R and EPR

Spectroscopy15

Table 5. (Continued )

R2R2 R7R7 R12 R13 R17 R18 mesob Name Symbol

Dioxoisobacteriochlorins, Structure 6

Me,AcOc Me,AcOc Me PrOc AcrOc Me H Heme d1 None

R2 R3 R7 R8 R12 R13 R17 R18 mesob Name Symbol

Corroles, a Structure 7Me Me Me Me Me Me Me H Octamethylcorrole OMCor3 or

Me8Cor3

Et Me Et Et Et Et Me Et H 7,13-Dimethylhexa- 7,13-ethlcorrole Me2Et6Cor3

aSee Structure 4 for structural formula of the free base chlorin, Structure 5 for that of the free base isobacteriochlorin, Structure 6 for that of thefree base dioxoisobacteriochlorin, and Structure 7 for that of the corrole.bmeso.5,10,15,20 positions for all but the corrole (5,10,15).cAcO = acetate; PrO = propionate; AcrO = acrylate.


types of synthetic porphyrins, those having either meso or -pyrrole substituents,are shown in Structures 2 and 3, respectively, and common substituents, IUPACnumbering system, ligand names and abbreviations are given in Tables 3 and 4,respectively.

Chlorins, isobacteriochlorins, 2,7-dioxoisobacteriochlorins and corrolemacrocycles are shown in Structures 47, respectively, and examples of high sym-metry macrocycles of each type, with common substituents, ligand names andabbreviations are summarized in Table 5. Chang and coworkers have been instru-mental in proving the structures of several of the so-called green hemes, includ-ing heme d (actually a chlorin, 5,6-dihydroxy protochlorin IX21) and heme d1(actually a porphinedione or 2,7-dioxoisobacteriochlorin22). Other related ringstructures that are involved in the stepwise oxidation of the heme to verdohemeand beyond are shown in Structures 813, including 5-hydroxyporphyrins (oxophlo-rins), Structures 8, oxaporphyrins (verdohemes), Structures 9, azaporphyrins, 10,formylbiliverdins, 11, dioxodipyrromethanes, 12 and dialkyldipyrromethanes, 13.The structures of other related macrocycles, including etioporphycene, por-phyrazines (meso-tetraazaporphyrins), phthalocyanines and texaphyrin, are shownin Structures 1417, respectively. An example of how saturated -pyrrole bonds cancreate an optically active macrocycle is shown in Structure 18 for the 5-coordinatehigh-spin state of chloroiron trans,trans-octaethylchlorin.

16 Walker

N N

NNR12

R7

R17 R13

R18

R2

R3

R8

H

H

8OH

HN N

NNR12

R7

R17 R13

R18

R2

R3

R8

H

H

O

HHH

H H H

N

O+

N

NN

R12

R7

R17 R13

R18

R2

R3

R8

H

H

9

HH

H N

N

N

NN

R12

R7

R17 R13

R18

R2

R3

R8

10

H

H



N

O

N

NN

R12

R7

R17 R13

R18

R2

R3

R8

H

H

11

HH

H

O

H

N N

NN

R12

R7

R17 R13

R18

R2

R3

R8

13

H

R15

H

R5 H

H

N N

NNMe

Et

Et Et

Me

Me

Et

Me

14

HH

N

N

N

N

N

N

NN

16

H

HNH

N

N

N

N

17

R8

H

R12 R13

HR18

R19

R20

R21H

R2R3

H

R7

N

N

N

N

N

NNN

R12

R7

R17 R13

R18

R2

R3

R8

15

H

H

N N

NN

O

R12

R7

R17 R13

R18

R2

R3

R8

12 O

H

H


Coordination numbers possible for synthetic and natural metalloporphyrinsinclude 4-coordinate (no axial ligands), 5-coordinate (one axial ligand), 6-coordinate(two axial ligands) and 7- to 9-coordinate (one bidentate ligand and up to threemonodentate ligands, as observed for certain lanthanide porphyrins) geometries.The number of axial ligands, and their nature, have dramatic influences on the spinstate, optical spectra, EPR g-values and in some cases superhyperfine couplingconstants, NMR paramagnetic shifts, Mssbauer isomer shifts, quadrupole split-tings and hyperfine coupling constants, as well as reduction potentials and reac-tivity towards reagents such as molecular oxygen, carbon monoxide, nitric oxide,alkyl halides, alkenes, alkynes and many others.

The molecular and electronic structures of metalloporphyrins may, in certaincases, depend on solvent, anion and degree of aggregation. Deuterated solventsare almost invariably used, except when deuterated porphyrin 2H signals are beingobserved. Some solvents, including water (D2O), dimethylsulfoxide (DMSO-d6),dimethylformamide (DMF-d7), methanol (CD3OD), pyridine-d5, and tetrahydrofu-ran (THF-d8) may coordinate to the metal, or may simply change the dielectricconstant of the medium, if strongly-binded ligands are separately provided for themetalloporphyrin. Water and alcohols can also be hydrogen-bond donors to theaxial ligands. Chlorinated hydrocarbons, including chloroform (CDCl3), methyl-ene chloride (CD2Cl2) and dichloroethane (C2D4Cl2), are typically good at dis-solving metalloporphyrins, although they may contain traces of acid (DCl) thatcould react with the metalloporphyrin. Aromatic hydrocarbons, such as toluene-d8,can interact with the -system of the porphyrin, both to help dissolve the complexand also to change the -acidity or -basicity of the macrocycle, as in the case ofCoII porphyrins. Carbon disulfide, CS2, is also good at dissolving uncharged por-phyrins. Anions can stabilize either high-spin states (halides, alkoxy, aryloxy,polyoxo anions) or low-spin states (cyanide, aryl, alkyl anions) if they remaincoordinated to the metalloporphyrin when dissolved in the chosen solvent.

18 Walker

N N

NN

Et

H

Et Et

Et

Et

Et

H

18

Fe

Cl

EtEt

N N

NN

Et

H

Et Et

Et

EtEt

H

Fe

ClEt

Et

3

2

5 78

10

12

131517

18

20


The known or suspected oxidation states of iron porphyrins, the most com-monly studied metalloporphyrins, range from FeI (d7) to (possibly) FeV (d3).Among these, the possible spin states include high-, intermediate- and low-spinstates of FeII (d6) and FeIII (d5), high- and low-spin FeI (d7) and FeIV (d4), althoughthere have been no reports of high-spin FeI or high-spin FeIV. In comparison, forthe metals of the first transition series on either side of iron, cobalt porphyrins,both CoII (d7) and CoIII (d6) complexes have been studied, with reports of bothhigh- and low-spin CoII complexes as a function of porphyrin substituents, butonly low-spin (diamagnetic) CoIII porphyrins. For manganese porphyrins, MnII(d5), MnIII (d4), MnIV (d3), and possibly MnV (d2), the latter two as oxoman-ganese(IV) and oxomanganese(V), have been studied by NMR spectroscopy;MnII exhibits only the high-spin state while MnIII exhibits both high- and low-spinstates, and the two highest oxidation states both appear to be high-spin.Chromium porphyrins have been studied as CrII (d4) and CrIII (d3), the latter ofwhich has extremely broad lines. Although nickel macrocycle complexes areknown for NiI (d9), NiII (d8) and NiIII (d7), only the NiII (d8) systems have beenstudied by NMR spectroscopy; the 5- and 6-coordinate complexes are paramag-netic, while the 4-coordinate complexes are diamagnetic. Copper porphyrins areknown only as CuII (d9), which have very broad lines. For most of these metals,the corrole ring is said to stabilize a higher apparent metal oxidation state than dothe porphyrin or reduced porphyrin rings, although it often turns out that the elec-tron is missing from the macrocycle rather than the metal. The only diamagneticmembers of the extensive series of possible oxidation and spin states are low-spind6 6- (and sometimes 5-) coordinate FeII, RuII, OsII, 6-coordinate CoIII, RhIII, and4-coordinate NiII, PdII and PtII. The NMR spectra observed for representativeexamples of each of the possible oxidation and spin states will be discussed inSection IV.

II. Principles

A. Proton Chemical Shifts

The chemical shifts which are observed for protons in paramagnetic molecules arethe result of a combination of diamagnetic and paramagnetic contributions. Thesecontributions are additive:

obs = dia + para. (1)

The diamagnetic contribution, dia, is the chemical shift which would have beenobserved if the molecule had no unpaired electron(s). It is directly evaluated by



recording the NMR spectrum of an appropriate diamagnetic analog of the moleculeof interest, usually one containing a diamagnetic metal such as Zn(II), Ni(II), orCd(II). The metal-free (free base) porphyrin, H2P is sometimes used if no dia-magnetic metal complex is available; the chemical shifts of the protons of ametal-free porphyrin are usually within 0.2 ppm of those of a diamagnetic metalcomplex.

The paramagnetic contribution, para, is often called the hyperfine shift, hf,since it arises from the hyperfine interaction (scalar coupling) of an unpairedelectron with the proton nucleus of interest, or the isotropic shift, iso, sinceit is typically observed for molecules or ions in homogeneous solution,where electron spin relaxation and molecular rotation are both generallyrapid, and thus yield an isotropic spectrum. The paramagnetic shift consistsof two terms, the contact and the pseudocontact (or electron-nuclear dipolar)contributions:

para = iso = hf = obsdia = con + pc. (2)

The contact (con) and pseudocontact (pc) terms arise because spin delocalizationfrom the unpaired electron, usually located on the metal ion, to the protons at theperiphery of the molecule, can occur either through chemical bonds or throughspace (or a combination of the two, as in the case of high- and low-spin FeIII por-phyrins, see also Sections IV.A.6 and IV.A.8). The sign convention which will beused throughout this chapter is the one commonly used by all chemists: obs is pos-itive if the resonance is at higher frequency (lower shielding) than that of tetram-ethylsilane (TMS), and negative if the resonance is at lower frequency (highershielding) than that of TMS. Hence, para is positive when the resonance is atlower, and negative when it is at higher shielding than that of the diamagnetic ref-erence compound. This is the reverse of the sign convention that was used in theearlier literature for paramagnetic complexes, including several of the earlierreviews.2,13 The signs of all contact and pseudocontact (dipolar) shifts reported inearlier publications have been changed herein to conform to the now-acceptedsign convention.

1. Contact Shifts

Contact shifts of protons, deuteriums, carbons and other nuclei in paramagneticmolecules originate from scalar coupling between electron spins and individualnuclei. In the most general case, the contact shift depends on the principal com-ponents of the electronic g-tensor of the paramagnetic center (gii), the magneticsusceptibility tensor of the molecule (ii) and the Fermi hyperfine (contact) coupling

20 Walker


constant (Ah) for coupling the spin of the electron to the spin of the nucleus ofinterest:23

(3)

where B is the Bohr magneton, ii are the molecular magnetic susceptibilities,and gii are the g-values along each of the principal axes of the molecule. If asingle spin state with an isotropic g-tensor is populated, and to the extent that theCurie law is valid, Eq. 3 reduces to a simpler form that is usually applicable tometalloporphyrins:24,25

(4)

where S is the total electron spin quantum number, g is the average (isotropic)g-value, B is the Bohr magneton, N is the magnetogyric ratio of the nucleus inquestion, and T is the absolute temperature. Thus, in simple, well-behaved systems,contact shifts are expected to vary linearly with 1/T and to extrapolate to zero atinfinite temperature (1/T = 0). However, in cases where a thermally-accessibleexcited state exists, as is typically the case with low-spin FeIII porphyrins andheme proteins, as well as many other cases, the contact shifts may not extrapolateto zero at infinite temperature, and an expanded form of the Curie law must beemployed,25 as will be discussed later in Section II.A.3.

The interpretation of the contact contributions to the paramagnetiic shifts ofprotons or deuteriums of paramagnetic molecules in terms of the covalency ofmetal-ligand bonds relies to a large extent on the McConnell equation:26

(5)

where AH is the hyperfine coupling constant for each individual proton, Q is aconstant for a given type of proton and C is the unpaired spin density of the elec-tron at the carbon to which the proton is attached; the same holds for deuteriumnuclei, which have almost identically the same chemical shifts, contact shifts andpseudocontact shifts as protons. For the case of spin delocalization to a orbitalon the carbon, QH = 63 MHz, and the contact shifts are negative (to lower fre-quency or higher shielding). If the proton directly bound to a carbon which hasspin density in a orbital is replaced by a methyl or other aliphatic group, QCH2Ris positive and somewhat variable, because it depends on the rate of rotation

A QSHC=

r2

,

dm

gconh B

N

A g S SkT

=+( )

,

13

dg m

c c ccon

h

N B

xx

xx

yy

yy

zz

zz

Ag g g

=

+ +

3 ,



(or preferred orientation, if rotation is slow) of the methyl or other aliphatic group;values of +70 to +75 MHz have been used.27,28 For alkyl groups larger than CH3,preferred orientations are the rule, and Q can vary from nearly zero to +100 MHz,but follows the general expression:

(6)

where B0 and B2 are positive numbers, and is the angle between the CCHplane and the pz orbital axis on the aromatic carbon. B2 is usually small, and, sincecos2 is positive for all angles , QCH2R is always positive. Therefore, if spindelocalization occurs to a particular carbon atom, a proton directly bound to thatcarbon will have a contact shift to lower frequency (higher shielding, con nega-tive), while replacement of the proton at that carbon position with a methyl orother aliphatic group will produce a contact shift to higher frequency (lowershielding, con positive). Such reversal in the sign of paramagnetic shifts for H andCH3 or CH2R or CHR2 at a given position is a clear sign of dominance of the para-magnetic shifts by spin delocalization, as will be seen in many parts of this chap-ter. For spin delocalization to a -symmetry orbital on the carbon, Q is positiveand depends on the number of bonds through which the spin is delocalized. Forthe electron in the 1s orbital of the hydrogen atom, AH is 1419 MHz, a positivequantity.29 For protons attached to carbons in metalloporphyrins, where metalelectrons are delocalized through either or orbitals of the carbons, the amountof spin density at a given proton on the periphery of the molecule is miniscule incomparison to the hydrogen atom, and is not directly comparable to 1419 MHzbecause the hydrogens in chemical compounds are involved in chemical bonds.

For spin delocalization to protons connected to a carbon framework, Q isalso positive, and the spin density, C, is attenuated sharply as the number of bonds between the unpaired electron and the proton increases. In any case, thepositive sign of Q means that the contact shifts are positive (to high frequency orlow shielding) when spin delocalization occurs to the carbon to which the protonis attached. Thus, if the contact contribution to the paramagnetic shifts of protonsin molecules such as metalloporphyrins can be separated from the pseudocontact(dipolar) contribution, the pattern of shifts to lower and higher frequencies, asdirectly-bound protons are replaced by methyl groups at the same symmetry posi-tions on the ring, for example, readily reveals the mechanism(s) of spin delocal-ization to each symmetry position. This will be discussed further after thepseudocontact (dipolar) shift has been introduced, and after the general strategiesfor separation of the contact and pseudocontact contributions to the paramagneticshift have been summarized.

AQ

SB B

SCH RCH R C C

22

2 20 2

2= =

+r j r( cos ),

22 Walker


2. Pseudocontact (Dipolar) Shifts

a. Pseudocontact shifts of metalloporphyrin substituents

The electron-nuclear dipolar contribution to the paramagnetic shift is now usuallycalled the pseudocontact contribution. This is actually a misnomer, since the electron-nuclear dipolar shift contains no contribution from scalar coupling of the electronspin with the spin of the nucleus of interest, that is, no Fermi contact term. Rather,the electron-nuclear dipolar shift results from through-space dipolar coupling ofthe electronic and nuclear magnetic moments which arise from either the magneticanisotropy of the metal ion or from zero-field contributions (in cases where thetotal spin of the ion is greater than one-half). Nevertheless, the use of the termpseudocontact to describe the dipolar shift is well entrenched in the literature ofheme proteins, as well as throughout the high-resolution NMR community, and wewill thus use this term throughout this chapter. However, one must be aware of thefact that it represents the through-space dipolar interaction between the unpairedelectron(s) and the nucleus of interest, which should more c

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