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, alpha and beta through a series of redox-active amino acids (Y-122.[beta] <-> W-48?[beta] <-> Y-356[beta] <-> Y-731[alpha] <-> Y-730[alpha] <-> C-439[alpha]). Nowhere is this more precarious than at the subunit interface. Here, we show that the oxidation of Y-356 is regulated by proton release involving a specific residue, E-52[beta], which is part of a water channel at the subunit interface for rapid proton transfer to the bulk solvent. An E(52)Q variant is incapable of Y-356 oxidation via the native radical transfer pathway or non-native photochemical oxidation, following photosensitization by covalent attachment of a photo-oxidant at position 355[beta]. Substitution of Y-336 for various FnY analogues in an E-52 Q-photofi 2 , where the side chain remains deprotonated, recovered photochemical enzymatic turnover. Transient absorption and emission data support the conclusion that Y-356 oxidation requires E-52 for proton management, suggesting its essential role in gating radical transport across the protein-protein interface.