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 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^• site in β₂ to the substrate activating cysteine residue (C₄₃₉) in α₂ via a pathway of redox active amino acids (Y₁₂₂[β] ↔ W₄₈[β]? ↔ Y₃₅₆[β] ↔ Y₇₃₁[α] ↔ Y₇₃₀[α] ↔ C₄₃₉[α]) spanning >35 Å. Ionizable residues at the α₂β₂ interface are essential in mediating RT, and therefore control activity. One of these mutations, E₃₅₀X (where X = A, D, Q) in β₂, obviates all RT, though the mechanism of control by which E₃₅₀ mediates RT remains unclear. Herein, we utilize an E₃₅₀Q-photoβ₂ 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 α₂ and β₂ subunits facilitating conformational gating of RT that specifically targets Y₁₂₂^• reduction, while the fidelity of the remainder of the RT pathway is retained.