An active dimanganese(III)-tyrosyl radical cofactor in Escherichia coli class Ib ribonucleotide reductase
Name
Stubbe_An active.pdf
Size
902.51 KB
Format
Adobe PDF
Checksum (MD5)
66b1a6b680e0397f17b971197e40a0f2
Author(s)
Cotruvo, Joseph A.
Stubbe, JoAnne
Date Issued
January 2010
Journal
Biochemistry
Publisher
American Chemical Society (ACS)
Citation
Cotruvo, Joseph A., and JoAnne Stubbe. “An Active Dimanganese(III)−Tyrosyl Radical Cofactor in Escherichia Coli Class Ib Ribonucleotide Reductase.” Biochemistry 49.6 (2010): 1297–1309.
Version
Author's final manuscript
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)[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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
Terms of Use
Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use.
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1021/bi902106n
