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

The class Ia ribonucleotide reductase of Escherichia coli requires strict regulation of long-range radical transfer between two subunits, α and β, through a series of redox-active amino acids (Y₁₂₂•[β] ↔ W₄₈?[β] ↔ Y₃₅₆[β] ↔ Y₇₃₁[α] ↔ Y₇₃₀[α] ↔ C₄₃₉[α]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y₃₅₆ is regulated by proton release involving a specific residue, E₅₂[β], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent. An E₅₂Q variant is incapable of Y₃₅₆ 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 Y₃₅₆ for various F_nY analogues in an E₅₂Q-photoβ₂, where the side chain remains deprotonated, recovered photochemical enzymatic turnover. Transient absorption and emission data support the conclusion that Y₃₅₆ oxidation requires E₅₂ for proton management, suggesting its essential role in gating radical transport across the protein-protein interface.