ContentsI. Introduction 705 II. General Principles 706 A. One Electron Oxidized Amino Acids Thus Far Identified in Proteins 706 B. Biosynthesis of (Modified) Amino Acid Radicals 706 C. Emerging General Chemical Properties of Enzymes Utilizing Protein Radicals for Catalysis 708 D. Methods To Examine Radical Dependent Reactions 710 III. Background on Ribonucleotide Reductases 710 IV. Class I Ribonucleotide Reductases 712 A. Formation of the Tyrosyl Radical 713 B. Function of the Tyrosyl Radical 714 C. Thiyl Radical Involvement in Catalysis 716 D. Evidence for Enzyme-Mediated Radical Chemistry Using Nucleotide Analogues 717 E. Structure 719 V. Class II Ribonucleotide Reductases 719 A. Exchange Reaction: Detection of the Elusive Thiyl Radical 719 B. Role of the Thiyl Radical in Catalysis 721 VI. Pyruvate Formate Lyase 722 A. Characterization of the Glycyl Radical 723 B. Formation of the Glycyl Radical 723 C. Catalytic Mechanism 724 D. Enzyme-Mediated Radical Chemistry with Pyruvate Analogues 727 VII. Anaerobic Ribonucleotide Reductase 728 A. Background 728 B. Requirement for a Glycyl Radical 728 C. Mechanism of Nucleotide Reduction 729 VIII. Cytochrome c Peroxidase 730 A. Formation of the Ferryl Heme/Tryptophan Cation Radical 730 B. Proposed Function of the Tryptophan Radical in Electron Transfer 731 IX. Prostaglandin H Synthase 733 A. Structure: A Tyrosine Residue Revealed 733 B. Proposed Role of the Tyrosyl Radical in Catalysis 734 X. Photosynthetic Oxygen Evolution 736 A. Background 736 B. Proposed Roles of Tyrosyl Radicals Y Z • and Y D • 737 C. Spectroscopic Studies on Y Z • and Y D • 738 D. Proposed Mechanisms for Water Oxidation and O 2 Evolution 739 XI. Galactose Oxidase 741 A. Different Redox States of Galactose Oxidase 741 B. Structure: A Modified Tyrosine Residue Revealed 742 C. Is the Oxidized Amino Acid Associated with Apo Oxidized GAO the Novel Ortho-Thiol-Substituted Tyrosine? 743 D. Catalytic Mechanism 743 XII. Quinoproteins 744 A. Copper-Dependent Amine Oxidases 744 B. Methylamine Dehydrogenase 749 XIII. Other Systems in Which Protein-Based Radicals Have Been Proposed or Detected 751 A. Bovine Liver Catalase 751 B. DNA Photolyase 751 C. Dopamine β Monooxygenase 752 XIV. Summary and Outlook 754 XV. Abbreviations 754 XVI. Acknowledgments 755 XVII. References 755 JoAnne Stubbe is the Novartis professor of Chemistry and Biology at the Massachusetts Institute of Technology. She received her undergraduate degree from the University of Pennsylvania working with Ed Thornton and did NSF-sponsored undergraduate research with Ed Trachtenberg at Clark University. These mentors played a strong role in her interest in physical organic chemistry. She received her Ph.D.

The R2 subunit of Escherichia coli ribonucleotide reductase (RNR) contains a stable tyrosyl radical (•Y122) diferric cluster cofactor. Earlier studies on the cofactor assembly reaction detected a paramagnetic intermediate, X, that was found to be kinetically competent to oxidize Y122. Studies using rapid freeze-quench (RFQ) Mössbauer and EPR spectroscopies led to the proposal that X is comprised of two high spin ferric ions and a S = 1/2 ligand radical, mutually spin coupled to give a S = 1/2 ground state (Ravi, N.; Bollinger, J. M., Jr.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8007−8014). An extension of RFQ methodology to Q-band ENDOR spectroscopy using 57Fe has shown that one of the irons has a very nearly isotropic hyperfine tensor (A(FeA) = −[74.2(2), 72.2(2), 73.2(2)] MHz) as expected for FeIII, but that the other iron site displays considerable anisotropy (A(FeB) = +[27.5(2), 36.8(2), 36.8(2)] MHz), indicative of substantial FeIV character. Reanalysis of the Mössbauer data using these results leads to isomer shifts of δ(FeA) = 0.56(3) mm/s and δ(FeB) = 0.26(4) mm/s. Based on the hyperfine anisotropy of FeB plus the reduced isomer shift, X is now best described as a spin-coupled FeIII/FeIV center without a radical, but with significant spin delocalization onto the oxygen ligand(s).

The class Ib ribonucleotide reductase of Escherichia coli can initiate reduction of nucleotides to deoxynucleotides with either a MnIII2-tyrosyl radical (Y•) or a FeIII2-Y• cofactor in the NrdF subunit. Whereas FeIII2-Y• can self-assemble from FeII2-NrdF and O2, activation of MnII2-NrdF requires a reduced flavoprotein, NrdI, proposed to form the oxidant for cofactor assembly by reduction of O2. The crystal structures reported here of E. coli MnII2-NrdF and FeII2-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of MnII2-NrdF in complex with reduced and oxidized NrdI, a continuous channel connects the NrdI flavin cofactor to the NrdF MnII2 active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of MnIII2-Y• cofactor assembly.