Evidence that the β subunit of chlamydia trachomatis ribonucleotide reductase is active with the manganese ion of its manganese(IV)/iron(III) cofactor in site 1

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the β subunit oxidizes a cysteine residue ∼35 Å away in the α subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the Mn ^IV ion of a Mn ^IV/Fe ^III cluster, which assembles in a reaction between O ₂ and the Mn ^II/Fe ^II complex of β. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the Mn ^IV ion. Because site 1 is closer to the conserved location of the cysteine-oxidizing tyrosyl radical of class Ia and Ib RNRs, we suggested that the Mn ^IV ion most likely resides in this site (i.e., ¹Mn ^IV/ ²Fe ^III), but a subsequent computational study favored its occupation of site 2 ( ¹Fe ^III/ ²Mn ^IV). In this work, we have sought to resolve the location of the Mn ^IV ion in Ct RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted in vitro by two different procedures. In samples containing primarily Mn ^IV/Fe ^III clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both ¹Mn ^II/ ²Fe ^II and ¹Fe ^II/ ²Mn ^II complexes are competent to react with O ₂ to produce the corresponding oxidized states. However, with diminished Mn ^II loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these "low-Mn" samples on a per-Mn basis implies that the ¹Mn ^IV/ ²Fe ^III-β is at least the more active of the two oxidized forms and may be the only active form.