A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y•) in its R2 subunit for reversible generation of a 3′-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis (Ct) uses a MnIV/FeIII cofactor (specifically, the MnIV ion) in place of the Y• for radical initiation. Ct R2 is activated when its MnII/FeII form reacts with O2 to generate a MnIV/FeIV intermediate, which decays by reduction of the FeIV site to the active MnIV/FeIII state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the MnIV/FeIV intermediate by ∼10-fold and diminishes the ability of ascorbate to accelerate the decay by ∼65-fold but has no detectable effect on the catalytic activity of the MnIV/FeIII-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 ⇔ R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli (Ec) R2], has almost no effect on decay of the MnIV/FeIV intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 ⇔ R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the MnIV/Fe IV intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y•-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.
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