Photoinitiated proton-coupled electron transfer and radical transport kinetics in class la ribonucleotide reductase

Proton-coupled electron transfer (PCET) is a critical mechanism in biology, underpinning key processes such as radical transport, energy transduction, and enzymatic substrate activation. Ribonucleotide reductases (RNRs) rely on PCET to mediate the rate-limiting step in the synthesis of DNA precursors. E. coli class Ia RNR consists of two dimeric subunits: [alpha]₂ contains the active site, while [beta]₂ contains a stable diferric-tyrosyl radical cofactor. During turnover, transport occurs over 35 Ȧ, from Y₁₂₂ in [beta]₂ to C₄₃₉ in [alpha]₂) where an active-site thiyl radical mediates turnover. Radical transport is proposed to occur over a series of highly conserved redox-active amino acids, including Y₃₅₆ in [beta]₂,and Y₇₃₁ and Y₇₃₀ in [alpha]₂ . This thesis examines three subject areas of PCET that pertain to RNR: Small-molecule model systems provide insights into tyrosine oxidation and radical generation. Under physiological conditions, tyrosine oxidation is accompanied by deprotonation and occurs by PCET. A critical factor in PCET reactions is the nature ofthe proton acceptor and the presence ofhydrogen bonding. In a modular model system, pyridyl-amino acid-methyl esters are appended to rhenium(I) tricarbonyl phenanthroline to yield rhenium-amino acid complexes. In dichloromethane solution, bases coordinate to tyrosine by hydrogen bonding. Emission kinetics reveal base-dependent oxidation by PCET. A photopeptide composed of the 19 C-terminal residues of [beta]₂, fluorinated tyrosine in place of Y₃₅₆, and a rhenium(I) bipyridine photooxidant enables photoinitated radical transport into [alpha]₂. Transient absorption kinetics show rapid radical transport (10⁵ s-¹) that is only observed when both Y₇₃₁ and Y₇₃₀, are present, suggesting a critical role for the Y₇₃₁-Y₇₃₀, dyad for radical transport in RNR. An intact, photochemical [beta]₂ enables studies of an [alpha]₂:[beta]₂ complex. A bromomethylpyridine rhenium(I) phenanthroline photooxidant labels a single surface-cysteine mutant of [beta]₂ at position 355 to yield [Re]- [beta]₂. Under flash-quench conditions, transient absorption reveals a tyrosine radical. [Re] -[beta]₂ binds [alpha]₂ and is capable of light-initiated substrate turnover. Transient emission quenching experiments reveal Y₃₅₆ oxidation that is dependent on the presence of Y₇₃₁ in [alpha]₂. This result suggests that a Y₃₅₆-Y₇₃₁-Y₇₃₀ triad mediates radical transport across the subunit interface and into [alpha]₂.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2013

Author dc:creator

• Pizano, Arturo A. (Arturo Alejandro)

Advisor dc:contributor.advisor

• Daniel G. Nocera.

• 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/1721.1/84377

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/84377