Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase Jeffrey Baldwin, ‡ Walter C. Voegtli, § Nelly Khidekel, § Pierre Moe 1 nne-Loccoz, || Carsten Krebs, ⊥ Alice S. Pereira, ⊥,² Brenda A. Ley, ‡ Boi Hanh Huynh, ⊥ Thomas M. Loehr, || Pamela J. Riggs-Gelasco, # Amy C. Rosenzweig, § and J. Martin Bollinger, Jr.* ,‡ Contribution from the Department of Biochemistry and Molecular Biology, PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802, Department of Biochemistry, Molecular Biology, and Cell Biology and Department of Chemistry, Northwestern UniVersity, EVanston, Illinois 60208, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97006-8921, Department of Physics, Emory UniVersity, Atlanta, Georgia 30322, and Department of Chemistry and Biochemistry, College of Charleston, Charleston, South Carolina 29424 ReceiVed June 12, 2000 Abstract: The outcome of O 2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122 • ) 1 production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine) 2 - (carboxylate) 4 ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a µ-1,2-peroxodiiron(III) intermediate, 2,3 which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H peroxo ) in O 2 activation by MMOH. 4 In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the “extra” electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. 5-7 Decay of the µ-1,2- peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 • and which converts very slowly (t 1/2 ∼ 7 h) upon incubation at 0 °C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone ǫ-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with 16 O 2 or 18 O 2 show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O 2 . Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mo ¨ssbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(III)-phenolate species is ascribed to a ligand rearrangement in which µ-O 2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little ǫ-hydroxyphenylalanine is formed and pathways leading to Y122 • formation predominate in both R2-D84E and R2-W48F. 2,7 Structurally characterized members of the diiron-carboxylate family of oxidases and oxygenases include the R2 subunit of ribonucleotide reductase (R2, from Escherichia coli 8,9 and mouse 10 ), the hydroxylase component of soluble methane monooxygenase (MMOH, from Methylococcus capsulatus Bath 11,12 and Methylosinus trichosporium OB3b 13 ), and stearoyl acyl carrier protein ∆ 9 -desaturase (∆9D, from castor seed 14 ). These proteins have similar tertiary structures 15,16 and contain nearly identical (histidine) 2 (carboxylate) 4 -coordinated dinuclear iron clusters, which function in reductive activation of O 2 . 17-21 Despite these similarities, the outcomes of their reactions with O 2 range from alkane hydroxylation in MMOH, 18 to fatty acid * Address correspondence to this author. E-mail: jmb21@psu.edu ‡ Pennsylvania State University. § Northwestern University. | Oregon Graduate Institute of Science and Technology. ⊥ Emory University. ² Present address: Departamento de Quı ´mica, Faculdade de Cie ˆncias e Tecnologia, Universidade Nova de Lisboa, 2825 Monte de Caparica, Portugal. # College of Charleston. (1) Abbreviations used: R2, R2 subunit of ribonucleotide reductase; Y122 • , neutral tyrosyl radical in R2 derived from one-electron oxidation of tyrosine number 122; MMOH, hydroxylase component of methane mo- nooxygenase; P, the putative peroxodiiron(III) complex that accumulates during O 2 activation by MMOH; X, the formally Fe(III)Fe(IV) cluster that accumulates during oxygen activation by R2; RR, resonance Raman; EXAFS, extended X-ray absorption fine structure; ∆9D, stearoyl acyl carrier protein ∆ 9 desaturase; wt, wild-type; equiv, equivalents; HEPES, 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid; EMTS, ethyl mercurithiosal- icylic acid; MES, 2-(N-morpholino)ethanesulfonic acid; PEG, poly(ethylene glycol); δ, Mo ¨ssbauer isomer shift; ∆EQ, quadrupole splitting parameter; GC/MS, gas chromatography/mass spectrometry; W +• , tryptophan cation radical; Q, the formally diiron(IV) intermediate that accumulates during O2 activation by MMOH and is thought to hydroxylate methane. (2) Bollinger, J. M., Jr.; Krebs, C.; Vicol, A.; Chen, S.; Ley, B. A.; Edmondson, D. E.; Huynh, B. H. J. Am. Chem. Soc. 1998, 120, 1094- 1095. 7017 J. Am. Chem. Soc. 2001, 123, 7017-7030 10.1021/ja002114g CCC: $20.00 © 2001 American Chemical Society Published on Web 06/29/2001