Evidence That the β Subunit of <i>Chlamydia trachomatis</i> 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 <i>Chlamydia trachomatis</i> (<i>Ct</i>), the cysteine oxidant is the Mn^IV ion of a Mn^IV/Fe^III cluster, which assembles in a reaction between O₂ 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 <i>Ct</i> RNR-β by correlating X-ray crystallographic anomalous scattering intensities with catalytic activity for samples of the protein reconstituted <i>in vitro</i> 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 O₂ 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