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

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.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.by Arturo A. Pizano.Ph.D

Photochemical tyrosine oxidation with a hydrogen-bonded proton acceptor by bidirectional proton-coupled electron transfer

Amino acid radical generation and transport are fundamentally important to numerous essential biological processes to which small molecule models lend valuable mechanistic insights. Pyridyl-amino acid-methyl esters are appended to a rhenium(I) tricarbonyl 1,10-phenanthroline core to yield rhenium–amino acid complexes with tyrosine ([Re]–Y–OH) and phenylalanine ([Re]–F). The emission from the [Re] center is more significantly quenched for [Re]–Y–OH upon addition of base. Time-resolved studies establish that excited-state quenching occurs by a combination of static and dynamic mechanisms. The degree of quenching depends on the strength of the base, consistent with a proton-coupled electron transfer (PCET) quenching mechanism. Comparative studies of [Re]–Y–OH and [Re]–F enable a detailed mechanistic analysis of a bidirectional PCET process.Chemistry and Chemical Biolog

Modulation of Y356 Photooxidation in E. coli Class Ia Ribonucleotide Reductase by Y731 Across the α2:β2 Interface

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 A, which occurs by a multi-step proton-coupled electron transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface—Y356F(β) and Y731F(α)—this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.Chemistry and Chemical Biolog

Modulation of Y [subscript 356] Photooxidation in E. Coli Class Ia Ribonucleotide Reductase by Y [subscript 731] Across the α [subscript 2] :β [subscript 2] Interface

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β[subscript 2] subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α[subscript 2]:β[subscript 2] complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y[subscript 356]F(β) and Y[subscript 731]F(α), this oxidation is identified to be localized on Y[subscript 356]. The rate of Y[subscript 356] oxidation depends on the presence of Y[subscript 731] across the interface. This observation supports the proposal that unidirectional PCET across the Y[subscript 356](β)–Y[subscript 731](α)–Y[subscript 730](α) triad is crucial to radical transport in RNR.National Institutes of Health (U.S.) (Postdoctoral Fellowship GM 087034)National Science Foundation (U.S.). Graduate Research Fellowship ProgramNational Institutes of Health (U.S.) (GM 29595

Direct Interfacial Y731 Oxidation in α2 by a Photoβ2 Subunit of E. Coli Class Ia Ribonucleotide Reductase

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β2 and α2) involving multiple PCET steps and the conserved tyrosine triad [Y356(β2)–Y731(α2)–Y730(α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y356C-(β2) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F3Y) is incorporated in place of Y731 in α2. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re356]-β2:2,3,5-F3Y731-α2 in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α2 from β2 in the absence of the interfacial Y356 residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y731(α2), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y356 and Y731, at the subunit interface.Chemistry and Chemical Biolog

Direct interfacial Y731 oxidation in α2 by a photoβ2 subunit of E. coli class Ia ribonucleotide reductase

Proton-coupled electron transfer (PCET) is a fundamental mechanism important in a wide range of biological processes including the universal reaction catalysed by ribonucleotide reductases (RNRs) in making de novo, the building blocks required for DNA replication and repair. These enzymes catalyse the conversion of nucleoside diphosphates (NDPs) to deoxynucleoside diphosphates (dNDPs). In the class Ia RNRs, NDP reduction involves a tyrosyl radical mediated oxidation occurring over 35 Å across the interface of the two required subunits (β[subscript 2] and α[subscript 2]) involving multiple PCET steps and the conserved tyrosine triad [Y[subscript 356](β[subscript 2])–Y[subscript 731](α[subscript 2])–Y[subscript 730](α2)]. We report the synthesis of an active photochemical RNR (photoRNR) complex in which a Re(I)-tricarbonyl phenanthroline ([Re]) photooxidant is attached site-specifically to the Cys in the Y[subscript 356]C-(β[subscript 2]) subunit and an ionizable, 2,3,5-trifluorotyrosine (2,3,5-F[subscript 3]Y) is incorporated in place of Y[subscript 731] in α[subscript 2]. This intersubunit PCET pathway is investigated by ns laser spectroscopy on [Re[subscript 35]6]-β[subscript 2]:2,3,5-F[subscript 3]Y[subscript 731]-α[subscript 2] in the presence of substrate, CDP, and effector, ATP. This experiment has allowed analysis of the photoinjection of a radical into α[subscript 2] from β[subscript 2] in the absence of the interfacial Y[subscript 356] residue. The system is competent for light-dependent substrate turnover. Time-resolved emission experiments reveal an intimate dependence of the rate of radical injection on the protonation state at position Y[subscript 731](α[subscript 2]), which in turn highlights the importance of a well-coordinated proton exit channel involving the key residues, Y[subscript 356] and Y[subscript 731], at the subunit interface.National Science Foundation (U.S.) (Division of Graduate Education Grant DGE-1144152)National Science Foundation (U.S.) (Post-Doctoral Fellowship (GM087034))National Institutes of Health (U.S.) (grant GM047274)National Institutes of Health (U.S.) (NIH grant GM029595

Modulation of Y₃₅₆ Photooxidation in E. coli Class Ia Ribonucleotide Reductase by Y₇₃₁ Across the α₂:β₂ Interface

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled β₂ subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α₂:β₂ complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y₃₅₆F­(β) and Y₇₃₁F­(α), this oxidation is identified to be localized on Y₃₅₆. The rate of Y₃₅₆ oxidation depends on the presence of Y₇₃₁ across the interface. This observation supports the proposal that unidirectional PCET across the Y₃₅₆(β)–Y₇₃₁(α)–Y₇₃₀(α) triad is crucial to radical transport in RNR

Deciphering Radical Transport in the Large Subunit of Class I Ribonucleotide Reductase

Incorporation of 2,3,6-trifluorotyrosine (F₃Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the <i>C</i>-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F₃Y radical from the [Re]–F₃Y−βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y–Y established, radical transport occurs with a rate constant of 3 × 10⁵ s^–1. Point mutations that disrupt the Y–Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme

Deciphering Radical Transport in the Large Subunit of Class I Ribonucleotide Reductase

Incorporation of 2,3,6-trifluorotyrosine (F₃Y) and a rhenium bipyridine ([Re]) photooxidant into a peptide corresponding to the <i>C</i>-terminus of the β protein (βC19) of Escherichia coli ribonucleotide reductase (RNR) allows for the temporal monitoring of radical transport into the α2 subunit of RNR. Injection of the photogenerated F₃Y radical from the [Re]–F₃Y−βC19 peptide into the surface accessible Y731 of the α2 subunit is only possible when the second Y730 is present. With the Y–Y established, radical transport occurs with a rate constant of 3 × 10⁵ s^–1. Point mutations that disrupt the Y–Y dyad shut down radical transport. The ability to obviate radical transport by disrupting the hydrogen bonding network of the amino acids composing the colinear proton-coupled electron transfer pathway in α2 suggests a finely tuned evolutionary adaptation of RNR to control the transport of radicals in this enzyme

Pregnant women infected with pandemic H1N1pdm2009 influenza virus displayed overproduction of peripheral blood CD69+ lymphocytes and increased levels of serum cytokines.

The first pandemic of the 21st century occurred in 2009 and was caused by the H1N1pdm influenza A virus. Severe cases of H1N1pdm infection in adults are characterized by sustained immune activation, whereas pregnant women are prone to more severe forms of influenza, with increased morbi-mortality. During the H1N1pdm09 pandemic, few studies assessed the immune status of infected pregnant women. The objective of this study was to evaluate the behavior of several immune markers in 13 H1N1pdm2009 virus-infected pregnant (PH1N1) women, in comparison to pregnant women with an influenza-like illness (ILI), healthy pregnant women (HP) and healthy non-pregnant women (HW). The blood leukocyte phenotypes and the serological cytokine and chemokine concentrations of the blood leukocytes, as measured by flow cytometry, showed that the CD69+ cell counts in the T and B-lymphocytes were significantly higher in the PH1N1 group. We found that pro-inflammatory (TNF-α, IL-1β, IL-6) and anti-inflammatory (IL-10) cytokines and some chemokines (CXCL8, CXCL10), which are typically at lower levels during pregnancy, were substantially increased in the women in the ILI group. Our findings suggest that CD69 overexpression in blood lymphocytes and elevated levels of serum cytokines might be potential markers for the discrimination of H1N1 disease from other influenza-like illnesses in pregnant women