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

Abstract

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 H 2O 2, 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 H 2O 2. 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 O 2 [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 H 2O 2 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 H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (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 (Fe 2 (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].

Redox Intermediates of the Mn-Fe Site in Subunit R2 of Chlamydia trachomatis Ribonucleotide Reductase

“…Features-EPR signals were detected of Mn(II)Fe(II) and Mn(III)Fe(III), as previously observed (27,32,71). The Mn(IV)Fe(IV) state attributed to a precursor of Mn(III)Fe(III) in Refs.…”

Metal use in ribonucleotide reductase R2, di-iron, di-manganese and heterodinuclear—an intricate bioinorganic workaround to use different metals for the same reaction

“…The tyrosyl radical is believed to be a target for direct reactions with radical scavengers, such as NO, and the underlying reason for avoiding the tyrosyl radical may be to produce a system that is more resistant to radical scavengers. 58,107 In both cases, it appears that Nature has devised a truly 21 (42) group, establishes R2-like proteins as di-metal substrate-converting catalysts with interesting implications for both academy and industry.…”

Function and Regulation of Class I Ribonucleotide Reductase-Encoding Genes in Mycobacteria

“…The recent identification and characterization of the R2 subclass found in Chlamydia (27) suggested that the class Ic RNR may represent an adaptation of the enzyme that confers increased resistance to poisoning by reactive nitrogen and oxygen intermediates, such as NO (21,30), to which intracellular pathogens are exposed during the course of infection. The class Ic R2 is the only small RNR subunit found in Chlamydia and other organisms, and it associates with a class Ia-type large subunit (NrdA) to form a functional enzyme (27).…”

“…(16). The biochemical characteristics of the chlamydial class Ic RNR (30) and the predicted lifestyle of M. tuberculosis suggest that NrdB may serve a specialist function in dNTP supply under conditions of nitrosative stress. The class II RNR-encoding gene nrdZ belongs to the regulon of "dormancy" genes controlled by the DosRST two-component regulator system and induced by hypoxia and low-dose NO (51,63) but is dispensable for growth and survival in mice (16).…”