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J Biol Chem, Vol. 275, Issue 17, 12367-12373, April 28, 2000
From the Department of Biophysics, Arrhenius Laboratories,
Stockholm University, S-106 91 Stockholm, Sweden
We report an EPR study of the iron-sulfur enzyme,
anaerobic ribonucleotide reductase activase from Lactococcus
lactis. The activase (nrdG gene) together with
S-adenosyl-L-methionine (AdoMet) give rise to a
glycyl radical in the NrdD component. A semi-reduced [4Fe-4S]+ cluster with an axially symmetric EPR signal
was produced upon photochemical reduction of the activase. Air exposure
of the reduced enzyme gave a [3Fe-4S]+ cluster. The
Fe3S4 cluster was convertible to the EPR-active [4Fe-4S]+ cluster by renewed treatment with reducing
agents, demonstrating a reversible
[3Fe-4S]+- to-[4Fe-4S]+ cluster
conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of
dithionite also resulted in a semi-reduced [4Fe-4S]+
cluster. Prolonged reduction gave an EPR-silent fully reduced state,
which was enzymatically inactive. Both reduced states gave the
[3Fe-4S]+ EPR signal after air exposure. The iron-sulfur
cluster interconversion was also studied in the presence of AdoMet. The
EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was
independent of which reductant was applied, whereas the EPR signal of
the [3Fe-4S]+ cluster after air exposure was unchanged.
The results indicate that an AdoMet-mediated [4Fe-4S]+
center is the native active species that induces the formation of a
glycyl radical in the NrdD component.
Electron Paramagnetic Resonance Evidence for a Novel
Interconversion of [3Fe-4S]+ and [4Fe-4S]+
Clusters with Endogenous Iron and Sulfide in Anaerobic Ribonucleotide
Reductase Activase in Vitro*,
and
*
This work was supported in part by research grants from the
Swedish Natural Science Research Council and the Carl Trygger Foundation (to A. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. 1S and 2S, showing
simulated EPR spectra and a distorted Fe3S4 EPR signal.
Supported by an Axel Wenner-Gren Foundation fellowship and
National Natural Science Foundation of China Grant 29301004. On leave
from the Dept. of Chemistry, Xiamen University, Xiamen 361005, People's Republic of China. Present address: Dept. of Chemistry, University of Minnesota, Minneapolis, MN 55455.
§
To whom correspondence should be addressed. Tel.: 46-8-162450; Fax:
46-8-155597; E-mail: astrid@biophys.su.se.
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