Evidence That the β Subunit of Chlamydia trachomatis Ribonucleotide Reductase Is Active with the Manganese Ion of Its Manganese(Ⅳ)/lron(Ⅲ) Cofactor in Site 1

摘要

The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the β subunit oxidizes a cysteine residue ～35 A away in the a 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 MnⅡ/FeⅡ 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 la 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.