Gated Proton Release during Radical Transfer at the Subunit Interface of Ribonucleotide Reductase.

The class Ia ribonucleotide reductase of requires strict regulation of long-range radical transfer between two subunits, α and β, through a series of redox-active amino acids (Y•[β] ↔ W?[β] ↔ Y[β] ↔ Y[α] ↔ Y[α] ↔ C[α]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y is regulated by proton release involving a specific residue, E[β], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent. An EQ variant is incapable of Y oxidation via the native radical transfer pathway or non-native photochemical oxidation, following photosensitization by covalent attachment of a photo-oxidant at position 355[β]. Substitution of Y for various FY analogues in an EQ-photoβ, where the side chain remains deprotonated, recovered photochemical enzymatic turnover. Transient absorption and emission data support the conclusion that Y oxidation requires E for proton management, suggesting its essential role in gating radical transport across the protein-protein interface.