Rapid and Quantitative Activation of Chlamydia trachomatis Ribonucleotide Reductase by Hydrogen Peroxide

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• 作者：Wei Jiang ; Jiajia Xie ; Hanne Nø ; rgaard ; J. Martin Bollinger Jr. ; Carsten Krebs
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：April 15, 2008
• 年：2008
• 卷：47
• 期：15
• 页码：4477 - 4483
• 全文大小：881K
• 年卷期：v.47,no.15(April 15, 2008)
• ISSN：1520-4995

文摘

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis (Ct) uses a Mn^IV/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 Mn^IV 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 (Mn^II/Fe^II, Mn^III/Fe^III, and Mn^IV/Fe^III) toward hydrogen peroxide. Not only is the activity of the Mn^IV/Fe^III−R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H₂O₂, but both the fully reduced (Mn^II/Fe^II) and one-electron-reduced (Mn^III/Fe^III) forms of the protein are also efficiently activated by H₂O₂. The Mn^III/Fe^III−R2 species reacts with a second-order rate constant of 8 ± 1 M⁻¹ s⁻¹ to yield the Mn^IV/Fe^IV−R2 intermediate previously observed in the reaction of Mn^II/Fe^II−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 Mn^IV/Fe^III−R2 complex. The reaction of the Mn^II/Fe^II−R2 species with H₂O₂ proceeds in three resolved steps: sequential oxidation to Mn^III/Fe^III−R2 (k = 1.7 ± 0.3 mM⁻¹ s⁻¹) and Mn^IV/Fe^IV−R2, followed by decay of the intermediate to the active Mn^IV/Fe^III−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 (Fe₂^II/II) to the “met” (Fe₂^III/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].