Ribonucleotide reductase (RNR) enzymes catalyze the conversion of ribonucleotides to 2'-deoxyribonucleotides, providing the building blocks required for DNA synthesis and repair. Class Id RNRs, which are found exclusively in prokaryotes, utilize an oxidized di-manganese metallocofactor in the beta subunit to generate a catalytic cysteine thiyl radical in the alpha subunit via a long-range radical translocation pathway. Unlike the class Ib RNRs, which employ a similar di-Mn(II) precursor but require an accessory protein to generate superoxide from oxygen, Id-beta uses a uniquely simple mode of activation: direct scavenge of superoxide. Superoxide capture is enabled by a distortion in a core helix that introduces a solvent-exposed cavity over the metal binding site and the presence of the cationic side chain of a strictly conserved second sphere lysine residue adjacent to the metal site. Although the tyrosine residue that provides the initial radical in the class Ia and Ib enzymes is conserved and necessary for activity, in Id-beta it is held away from the cofactor via a hydrogen bond with a subclass-specific threonine. As a result, Id-beta does not stabilize appreciable Y* and instead uses the oxidized manganese cluster directly for radical initiation. In this work we investigate the structural features that influence Mn₂^III/IVassembly in four different Id RNR homologs. Each homolog reacts with Mn^II and superoxide to form an EPR-distinguishable active Mn₂^III/IV cluster. We also interrogate the roles of conserved second-sphere residues in the cofactor assembly process and demonstrate a clear role for the cofactor proximal Lys in superoxide capture. Comparison of structures of reduced and oxidized beta subunits provides a rationale for how Id RNRs stabilize a high-valent metallocofactor in a cofactor site that is initially highly solvent-exposed.

Jonathan Jung

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