Kinetically Divergent Dimanganese-Tyrosyl Radical Cofactor Assembly in Listeria monocytogenes Class I Ribonucleotide...

Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to deoxynucleotides in all organisms. Class I RNRs comprise two subunits, α2 (containing the active site) and β2 (containing the radical cofactor essential for initiating catalysis). The association of α2 and β2 is well known to regulate RNR activity, but a role for α2 in kinetically controlling cofactor assembly in β2 has not been reported. The class I RNR from the important human intracellular pathogen, Listeria monocytogenes (Lmo) is a member of the largest cluster of β subunit sequences (the "2RCC cluster") remaining to be biochemically characterized. This protein was previously suggested to be a class Ia enzyme, active with a diferric-tyrosyl radical (Y•) cofactor. Here, we demonstrate that the Lmo class I RNR, in fact, is most active with a dimanganese-Y• cofactor, assembled with the help of the flavoprotein, NrdI. While superficially similar to previously characterized manganese-dependent class Ib RNRs, the Lmo RNR features a number of unusual properties. Most strikingly, the MnIIIMnIV intermediate formed from reaction of the dimanganese(II) cluster, NrdI, and O2 converts extremely slowly (t1/2 = 11 min) to an active dimanganese(III)-Y• cofactor. However, in the presence of α2, cofactor assembly is accelerated by >10-fold (t1/2 = 0.6 min). Therefore, α2-driven conformational changes in Lmo β2 are likely required physiologically for not only catalysis but also activation. The properties of Lmo's distinctive class Ib RNR expand the known mechanisms of RNR activity regulation and may have implications for the observed sensitivity of L. monocytogenes to iron excess during infection.