We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis (Ct) uses a MnIV/Fe III cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The MnIV site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [Högbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gräslund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (MnII/FeII, Mn III/FeIII, and MnIV/FeIII) toward hydrogen peroxide. Not only is the activity of the MnIV/Fe III-R2 intermediate stable to prolonged (> 1 h) incubations with as much as 5 mM H2O2, but both the fully reduced (Mn II/FeII) and one-electron-reduced (MnIII/ FeIII) forms of the protein are also efficiently activated by H 2O2. The MnIII/FeIII-R2 species reacts with a second-order rate constant of 8 ± 1 M-1 s _1 to yield the MnIV/FeIV-R2 intermediate previously observed in the reaction of MnII/FeII-R2 with O2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active MnIV/Fe III-R2 complex. The reaction of the MnII/Fe II-R2 species with H2O2 proceeds in three resolved steps: sequential oxidation to MnIII/FeIII-R2 (k = 1.7 ± 0.3 mM-1 s-1) and MnIV/Fe IV-R2, followed by decay of the intermediate to the active Mn IV/FeIII-R2 product. The efficient reaction of both reduced forms with H2O2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe2II/II) to the "met" (Fe2III/III) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe2 III/III-Y•) form [Sahlin, M., Sjöberg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].
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