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Author Notes:

Corresponding author: mkj@uga.edu; Fax: 706-542-9454; Tel: 706-542-9378

B.Z. and S.B. contributed equally to this work


Research Funding:

This work was supported by grants from the National Institutes of Health (GM62524 to M.K.J. and GM47295 to B.H.H.) and the Agence Nationale de la Recherche (2010BLAN1616 to N.R. and J.C.)

Monothiol Glutaredoxins Can Bind Linear [Fe 3 S 4 ] + and [Fe 4 S 4 ] 2+ Clusters in Addition to [Fe 2 S 2 ] 2+ Clusters: Spectroscopic Characterization and Functional Implications


Journal Title:

Journal of the American Chemical Society


Volume 135, Number 40


, Pages 15153-15164

Type of Work:

Article | Post-print: After Peer Review


Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has been shown to function in biological [Fe2S2]2+ cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear [Fe3S4]+ cluster. The excited state electronic properties and ground state electronic and vibrational properties of the linear [Fe3S4]+ cluster have been characterized using UV-visible absorption/CD/MCD, EPR, Mössbauer and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear [Fe3S4]+ cluster with properties similar to those reported for synthetic linear [Fe3S4]+ clusters and the linear [Fe3S4]+ clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of [Fe2S2]2+ clusters, rather than linear [Fe3S4]+ clusters or mixtures of linear [Fe3S4]+ and [Fe2S2]2+ clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one [Fe4S4]2+ cluster that competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear [Fe3S4]+ and [Fe4S4]2+ clusters in Grx5 has been assessed by spectroscopic, mutational and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear [Fe3S4]+ clusters released during protein unfolding under oxidative stress conditions and in maturation of [Fe4S4]2+ cluster-containing proteins are discussed in light of these results.

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© 2013 American Chemical Society

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