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

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 (C 439 •), and the β subunit houses the diferric-tyrosyl radical (Y 122 •) that is essential for C 439 • formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y 122 •[β] ↔ W 48 ?[β] ↔ Y 356 [β] ↔ Y 731 [α] ↔ Y 730 [α] ↔ C 439 [α]. In a recent cryo-EM structure, Y 356 [β] was revealed for the first time and it, along with Y 731 [α], spans the asymmetric α/β interface. An E 52 [β] residue, which is essential for Y 356 oxidation, allows access to the interface and resides at the head of a polar region comprising R 331 [α], E 326 [α], and E 326 [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, Y 356 • was photochemically generated using a photosensitizer covalently attached adjacent to Y 356 [β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E 52 [β], R 331 [α], E 326 [α], and E 326 [α'] network plays the essential role of shuttling protons associated with Y 356 oxidation from the interface to bulk solvent.