Radical-Translocation Intermediates and Hurdling of Pathway Defects in 'Super-oxidized' (Mn~Ⅳ/Fe~Ⅳ) Chlamydia trachomatis Ribonucleotide Reductase

摘要

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 ? away in its a 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 a 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 nudeotide 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.