Mechanistic studies of proton-coupled electron transfer in aminotyrosine- and fluorotyrosine- substituted class Ia Ribonucleotide reductase

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2012

Author dc:creator

• Minnihan, Ellen Catherine

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/73369

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/73369