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

Joseph A. Cotruvo, Joanne Stubbe

Research output: Contribution to journalArticlepeer-review

95 Scopus citations

Abstract

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)6-NrdI and NrdF demonstrated tight association between these proteins. To explore the function of NrdI and identify the metallocofactor, apoNrdF was loaded with MnII and incubated with fully reduced NrdI (NrdIhq) and O2. 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 MnIII 2-Y ·, which we propose is generated by MnII2-NrdF reacting with two equivalents of HO2-, produced by reduction of O 2 by NrdF-bound NrdIhq. In the absence of NrdI hq, with a variety of oxidants, no active RNR was generated. By contrast, a similar experiment with apoNrdF loaded with FeII and incubated with O2 in the presence or absence of NrdIhq gave 0.2 and 0.7 Y · /β2 with specific activities of 80 and 300 units/mg, respectively. Thus NrdIhq hinders FeIII2-Y · cofactor assembly in vitro. We propose that NrdI is an essential player in E. coli class Ib RNR cluster assembly and that the MnIII 2-Y · cofactor, not the diferric-Y · one, is the active metallocofactor in vivo.

Original languageEnglish (US)
Pages (from-to)1297-1309
Number of pages13
JournalBiochemistry
Volume49
Issue number6
DOIs
StatePublished - Feb 16 2010

All Science Journal Classification (ASJC) codes

  • Biochemistry

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