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.
ASJC Scopus subject areas
- Colloid and Surface Chemistry