Dataset Information

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

ABSTRACT: Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the ?2 subunit (Y122•) necessary for nucleotide reductase activity. Upon binding the cognate ?2 subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active ?2?2 complex from the Y122• site in ?2 to the substrate activating cysteine residue (C439) in ?2 via a pathway of redox active amino acids (Y122[?] ? W48[?]? ? Y356[?] ? Y731[?] ? Y730[?] ? C439[?]) spanning >35 Å. Ionizable residues at the ?2?2 interface are essential in mediating RT, and therefore control activity. One of these mutations, E350X (where X = A, D, Q) in ?2, obviates all RT, though the mechanism of control by which E350 mediates RT remains unclear. Herein, we utilize an E350Q-photo?2 construct to photochemically rescue RNR activity from an otherwise inactive construct, wherein the initial RT event (Y122• ? Y356) is replaced by direct photochemical radical generation of Y356•. These data present compelling evidence that E350 conveys allosteric information between the ?2 and ?2 subunits facilitating conformational gating of RT that specifically targets Y122• reduction, while the fidelity of the remainder of the RT pathway is retained.

SUBMITTER: Greene BL

PROVIDER: S-EPMC6249109 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Greene Brandon L BL Stubbe JoAnne J Nocera Daniel G DG

Journal of the American Chemical Society 20181112 46

Class Ia ribonucleotide reductase (RNR) of Escherichia coli contains an unusually stable tyrosyl radical cofactor in the β2 subunit (Y122•) necessary for nucleotide reductase activity. Upon binding the cognate α2 subunit, loaded with nucleoside diphosphate substrate and an allosteric/activity effector, a rate determining conformational change(s) enables rapid radical transfer (RT) within the active α2β2 complex from the Y122< ...[more]

PMID: 30347141

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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.

Project description:A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli. The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T. acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the T. acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme. Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B12 nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry.

Dataset Information

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Publications

Photochemical Rescue of a Conformationally Inactivated Ribonucleotide Reductase.

Similar Datasets

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