文摘
The reaction of a class I ribonucleotide reductase (RNR) begins when a cofactor in the 尾 subunit oxidizes a cysteine residue 35 脜 away in the 伪 subunit, generating a thiyl radical. In the class Ic enzyme from Chlamydia trachomatis (Ct), the cysteine oxidant is the MnIV ion of a MnIV/FeIII cluster, which assembles in a reaction between O2 and the MnII/FeII complex of 尾. The heterodinuclear nature of the cofactor raises the question of which site, 1 or 2, contains the MnIV 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 MnIV ion most likely resides in this site (i.e., 1MnIV/2FeIII), but a subsequent computational study favored its occupation of site 2 (1FeIII/2MnIV). In this work, we have sought to resolve the location of the MnIV ion in Ct RNR-尾 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 MnIV/FeIII clusters, Mn preferentially occupies site 1, but some anomalous scattering from site 2 is observed, implying that both 1MnII/2FeII and 1FeII/2MnII complexes are competent to react with O2 to produce the corresponding oxidized states. However, with diminished MnII loading in the reconstitution, there is no evidence for Mn occupancy of site 2, and the greater activity of these 鈥渓ow-Mn鈥?samples on a per-Mn basis implies that the 1MnIV/2FeIII-尾 is at least the more active of the two oxidized forms and may be the only active form.