Radical-translocation intermediates and hurdling of pathway defects in "super-oxidized" (Mn^IV/Fe^IV) Chlamydia trachomatis ribonucleotide reductase

A class I ribonucleotide reductase (RNR) uses either a tyrosyl radical (Y^•) or a Mn^IV/Fe^III 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 Mn^IV/Fe^III cofactor of Chlamydia trachomatis RNR assembles via a Mn^IV/Fe^IV 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 Mn^IV/Fe^III states can generate the pathway Y^•'s in their Mn^IV/Fe^IV 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.