Abstract
A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y•) or a MnIV/FeIII cluster in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit, generating a thiyl radical that abstracts hydrogen (H•) from the substrate. With either oxidant, the inter-subunit “hole-transfer” or “radical-translocation” (RT) process is thought to occur by a “hopping” mechanism involving multiple tyrosyl (and perhaps one tryptophanyl) radical intermediates along a specific pathway. The hopping intermediates have never been directly detected in a Mn/Fe-dependent (class Ic) RNR nor in any wild-type (wt) RNR. The MnIV/FeIII cofactor of Chlamydia trachomatis RNR assembles via a MnIV/FeIV intermediate. Here we show that this cofactor-assembly intermediate can propagate a hole into the RT pathway when α is present, accumulating radicals with EPR spectra characteristic of Y•’s. The dependence of Y• accumulation on the presence of substrate suggests that RT within this “super-oxidized” enzyme form is gated by the protein, and the failure of a β variant having the subunit-interfacial pathway Y substituted by phenylalanine to support radical accumulation implies that the Y•(s) in the wt enzyme reside(s) within the RT pathway. Remarkably, two variant β proteins having pathway substitutions rendering them inactive in their MnIV/FeIII states can generate the pathway Y•’s in their MnIV/FeIV states and also effect nucleotide reduction. Thus, the use of the more oxidized cofactor permits the accumulation of hopping intermediates and the “hurdling” of engineered defects in the RT pathway.
