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Biochemistry (Moscow)

, Volume 77, Issue 2, pp 135–142 | Cite as

Biochemical characterization of iron-sulfur cluster assembly in the scaffold IscU of Escherichia coli

  • Genfu WuEmail author
  • Lingfei Li
Accelerated Publication

Abstract

Iron-sulfur cluster is one of the most common prosthetic groups, and it functions in numerous biological processes. However, little is currently known about the mechanisms of iron-sulfur cluster biosynthesis. In this study, we cloned and purified iron-sulfur cluster assembly proteins from Escherichia coli and assembled the cluster in vitro. The results showed that the assembly of iron-sulfur cluster is completed in about 20 min. Although iron or sulfur binds with IscU equivalently, 2-fold amount of iron or cysteine compared with that of IscU is better for the cluster formation, while high concentrations of IscS (IscS/IscU > 1: 10) do not facilitate the cluster formation. Environmental pH plays an important role in iron-sulfur cluster assembly; the cluster was well assembled at pH 7.6–8.0, but was inhibited at pH less than 7.4. On supply of a catalytic amount of IscS (1/50 of IscU) and excess of other substrates, with increasing each of IscU, iron, or cysteine concentration, the iron-sulfur cluster assembly process developed from first order reaction, mixed order reaction to zero order reaction, and up to 64% of apo-IscU was converted to the [2Fe-2S] cluster-bound IscU under the optimal laboratory conditions.

Key words

iron-sulfur cluster IscS IscU IscA-Fe iron cysteine 

Abbreviations

DTT

dithiothreitol

FPLC

fast protein liquid chromatography

Isc

iron-sulfur cluster

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Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  1. 1.College of Life ScienceZhejiang UniversityHangzhouChina

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