6 research outputs found

    An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase

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    Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5′-diphosphates to deoxynucleoside 5′-diphosphates and is expressed under iron-limited and oxidative stress conditions. This RNR is composed of two homodimeric subunits: α2 (NrdE), where nucleotide reduction occurs, and β2 (NrdF), which contains an unidentified metallocofactor that initiates nucleotide reduction. nrdE and nrdF are found in an operon with nrdI, which encodes an unusual flavodoxin proposed to be involved in metallocofactor biosynthesis and/or maintenance. Ni affinity chromatography of a mixture of E. coli (His)[subscript 6-]NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with Mn[superscript II] and incubated with fully reduced NrdI (NrdI[subscript hq]) and O[subscript 2]. Active RNR was rapidly produced with 0.25 ± 0.03 tyrosyl radical (Y·) per β2 and a specific activity of 600 units/mg. EPR and biochemical studies of the reconstituted cofactor suggest it is Mn[superscript III][subscript 2-]Y·, which we propose is generated by Mn[superscript II][subscript 2-]NrdF reacting with two equivalents of HO[subscript 2]−, produced by reduction of O[subscript 2] by NrdF-bound NrdI[subscript hq.] In the absence of NrdI[subscript hq], with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with Fe[superscript II] and incubated with O[subscript 2] in the presence or absence of NrdI[subscript hq] gave 0.2 and 0.7 Y·/β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdI[subscript hq] hinders Fe[superscript III][subscript 2-]Y· cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the Mn[superscript III][subscript 2-]Y· cofactor, not the diferric-Y· one, is the active metallocofactor in vivo.National Institutes of Health (U.S.) (Grant number GM81393)National Defense Science and Engineering Graduate Fellowshi

    Superoxide Dismutases and Superoxide Reductases

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    The Family Corynebacteriaceae

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