Dataset Information

Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking.

ABSTRACT: Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion exchange chromatography and digested with trypsin. The peptides were purified by reverse phase chromatography, identified by scintillation counting and analyzed by Edman sequencing. Three [(14)C]-labeled peptides were identified: two contained a peptide in beta to which the BP was attached. The third contained the same beta peptide and a peptide in alpha found in its alphaD helix. These results provide direct support for the proposed docking model of alpha2beta2.

SUBMITTER: Hassan AQ

PROVIDER: S-EPMC2602823 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking.

Hassan A Quamrul AQ Stubbe JoAnne J

Bioorganic & medicinal chemistry letters 20080819 22

Escherichia coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates and is a 1:1 complex of two homodimeric subunits: alpha2 and beta2. As a first step towards mapping the subunit interface, beta2 (V365C) was labeled with [(14)C]-benzophenone (BP) iodoacetamide. The resulting [(14)C]-BP-beta2 (V365C) was complexed with alpha2 and irradiated at 365nm for 30min at 4 degrees C. The cross-linked mixture was purified by anion excha ...[more]

PMID: 18762419

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Project description:Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multistep proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ∼35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask examination of the kinetics of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [Re(I)] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET), and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively "tight" subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.

Dataset Information

Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking.

Publications

Mapping the subunit interface of ribonucleotide reductase (RNR) using photo cross-linking.

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