Reversible, Long-Range Radical Transfer in E. coli Class Ia Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDPs or NTPs where N = C, U, G, or A) to 2′-deoxynucleotides (dNDPs or dNTPs)[superscript 1] and are responsible for controlling the relative ratios and absolute concentrations of cellular dNTP pools. For this reason, RNRs play a major role in ensuring the fidelity of DNA replication and repair. RNRs are found in all organisms and are classified based on the metallocofactor used to initiate catalysis,[superscript 1] with the class Ia RNRs requiring a diferric-tyrosyl radical (Y•) cofactor.National Institutes of Health (U.S.) (GM47274)National Institutes of Health (U.S.) (GM29595

Redox-Linked Changes to the Hydrogen-Bonding Network of Ribonucleotide Reductase β2

Ribonucleotide reductase (RNR) catalyzes conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleotides, a critical step in DNA replication and repair in all organisms. Class-Ia RNRs, found in aerobic bacteria and all eukaryotes, are a complex of two subunits: α2 and β2. The β2 subunit contains an essential diferric–tyrosyl radical (Y122O•) cofactor that is needed to initiate reduction of NDPs in the α2 subunit. In this work, we investigated the Y122O• reduction mechanism in Escherichia coli β2 by hydroxyurea (HU), a radical scavenger and cancer therapeutic agent. We tested the hypothesis that Y122OH redox reactions cause structural changes in the diferric cluster. Reduction of Y122O• was studied using reaction-induced FT-IR spectroscopy and [[superscript 13]C]aspartate-labeled β2. These Y122O• minus Y122OH difference spectra provide evidence that the Y122OH redox reaction is associated with a frequency change to the asymmetric vibration of D84, a unidentate ligand to the diferric cluster. The results are consistent with a redox-induced shift in H-bonding between Y122OH and D84 that may regulate proton-transfer reactions on the HU-mediated inactivation pathway in isolated β2.National Institutes of Health (U.S.) (Grant GM29595

Mechanistic studies of PCET in aminotyrosine- and fluorotyrosine- substituted class Ia RNR

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to 2'- deoxynucleotides in all organisms. The class Ia RNR from Escherichia coli is active as an a2p2 complex and utilizes an unprecedented mechanism of reversible proton-coupled electron transfer (PCET) to propagate a stable tyrosyl radical (Yi22-) in P2 over a distance of >35 A to an active site cysteine (C4 3 9) in a2 on each turnover. Generation of the cysteinyl radical (C4 3 9-) initiates active site nucleotide reduction. Radical propagation over 35 A by a pure tunneling mechanism would be too slow to support the observed turnover number. Instead, long-range, reversible PCET is proposed to occur by radical hopping along a specific pathway of redox-active amino acids: ... The details of this mechanism are kinetically masked in wild-type RNR, and mutation of any of these residues to another native amino acid inactivates the enzyme. Recent development of technology for the in vivo, site-specific incorporation of unnatural amino acids into proteins has provided the opportunity to systematically perturb the native PCET pathway by introduction of tyrosine analogues with modified redox potentials and/or pKas. This thesis focuses on 3-aminotyrosine (NH2Y) and fluorotyrosines (FnYs). NH2Y has a lower reduction potential than Y and, when incorporated at the three sites of transient Ye formation, generates a thermodynamic minimum and reduces kcat sufficiently to allow characterization of NH2Y. intermediates. A kinetic model for catalysis by NH2Y-RNRs has been proposed from the mechanistic studies described herein. Furthermore, the ability to generate NH2Y* on the pathway has afforded the first characterization of a kinetically stable c2p2 complex. FnYs span a wide range of solution pKas and reduction potentials and thus may be used to investigate both PT and ET events. The evolution of an orthogonal, polyspecific tRNA/tRNA synthetase pair for FnYs is reported. FnYs at positions 356, 730, and 731 have been used to measure the pH dependence of RNR activity, whereas FnY-s at position 122 of $2 have been used as radical initiators to begin mapping the relative thermodynamic landscape of the PCET pathway.by Ellen Catherine Minnihan.Ph.D

Use of 3-Aminotyrosine To Examine the Pathway Dependence of Radical Propagation in Escherichia coli Ribonucleotide Reductase

Escherichia coli ribonucleotide reductase (RNR), an α2β2 complex, catalyzes the conversion of nucleoside 5′-diphosphate substrates (S) to 2′-deoxynucleoside 5′-diphosphates. α2 houses the active site for nucleotide reduction and the binding sites for allosteric effectors (E). β2 contains the essential diferric tyrosyl radical (Y[subscript 122]•) cofactor which, in the presence of S and E, oxidizes C[subscript 439] in α to a thiyl radical, C[subscript 439]•, to initiate nucleotide reduction. This oxidation occurs over 35 Å and is proposed to involve a specific pathway: Y[subscript 122]• --> W[subscript 48 --> Y[subscript 356] in β2 to Y[subscript 731] --> Y[subscript 730] --> C[subscript 439] in α2. 3-Aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH[subscript 2]Y•) has been examined by stopped-flow (SF) UV−vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double mutant complexes Y[subscript 356]F-β2:Y[subscript 731]NH[subscript 2]Y-α2, Y[subscript 356]F-β2:Y[subscript 730]NH[subscript 2]Y-α2, and wt-β2:Y[subscript 731]F/Y[subscript 730]NH[subscript 2]Y-α2, in which the nonoxidizable F acts as a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH[subscript 2]Y• was detected. To study off-pathway oxidation, Y[subscript 413], located 5 Å from Y[subscript 730] and Y[subscript 731] but not implicated in long-range oxidation, was examined. Evidence for NH[subscript 2]Y[subscript 413]• was sought in three complexes: wt-β2:Y[subscript 413]NH[subscript 2]Y-α2 (a), wt-β2:Y[subscript 731]F/Y[subscript 413]NH[subscript 2]Y-α2 (b), and Y[subscript 356]F-β2:Y[subscript 413]NH[subscript 2]Y-α2 (c). With (a), NH[subscript 2]Y• was formed with a rate constant that was 25−30% and an amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH[subscript 2]Y• formation was 0.2−0.3% of that observed at 731 and 730, and with (c), no NH[subscript 2]Y• was observed. These studies suggest the evolution of an optimized pathway of conserved Ys in the oxidation of C[subscript 439].National Institutes of Health (U.S.) (Grant GM29595)David H. Koch Institute for Integrative Cancer Research at MIT (Koch Graduate Fellowship

Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5′-diphosphates (NDPs) to 2′-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C[subscript 439]) in α2 by a stable diferric tyrosyl radical (Y[subscript 122]•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y[subscript 122]•→ W[subscript 48]→ Y[subscript 356] in β2 to Y[subscript 731]→ Y[subscript 730]→ C[subscript 439] in α2. To study the details of this process, 3-aminotyrosine (NH[subscript 2]Y) has been site-specifically incorporated in place of Y[subscript 356] of β. The resulting protein, Y[subscript 356]NH[subscript 2]Y-β2, and the previously generated proteins Y[subscript 731]NH[subscript 2]Y-α2 and Y[subscript 730]NH[subscript 2]Y-α2 (NH[subscript 2]Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2–0.7 s[superscript –1]. Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH[subscript 2]Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH[subscript 2]Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH[subscript 2]Y• formation is biphasic (k[subscript fast] of 9–46 s[superscript –1] and k[subscript slow] of 1.5–5.0 s[subscript –1]) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH[subscript 2]Y• formation, while the k[subscript cat] of 0.5 s[superscript –1] is proposed to be associated with rate-limiting oxidation by NH[subscript 2]Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y[subscript 730]NH[subscript 2]Y-α2 and Y[subscript 731]NH[subscript 2]Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.National Institutes of Health (U.S.) (GM29595