Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C₄₃₉•), and the β subunit houses the diferric-tyrosyl radical (Y₁₂₂•) that is essential for C₄₃₉• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y₁₂₂•[β] ↔ W₄₈?[β] ↔ Y₃₅₆[β] ↔ Y₇₃₁[α] ↔ Y₇₃₀[α] ↔ C₄₃₉[α]. In a recent cryo-EM structure, Y₃₅₆[β] was revealed for the first time and it, along with Y₇₃₁[α], spans the asymmetric α/β interface. An E₅₂[β] residue, which is essential for Y₃₅₆ oxidation, allows access to the interface and resides at the head of a polar region comprising R₃₃₁[α], E₃₂₆[α], and E₃₂₆[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y₃₅₆• was photochemically generated using a photosensitizer covalently attached adjacent to Y₃₅₆[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E₅₂[β], R₃₃₁[α], E₃₂₆[α], and E₃₂₆[α'] network plays the essential role of shuttling protons associated with Y₃₅₆ oxidation from the interface to bulk solvent.