title: Monothiol glutaredoxins can bind linear [Fe3S4]+ and [Fe4S4]2+ clusters in addition to [Fe2S2]2+ clusters: spectroscopic characterization and functional implications.
abstract: 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-vis 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 is 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.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia , Athens, Georgia 30602, United States.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.forename: Sibali
authors.initials: S
authors.lastname: Bandyopadhyay
authors.forename: Priyanka
authors.initials: P
authors.lastname: Shakamuri
authors.forename: Sunil G
authors.initials: SG
authors.lastname: Naik
authors.forename: Boi Hanh
authors.initials: BH
authors.lastname: Huynh
authors.forename: Jérémy
authors.initials: J
authors.lastname: Couturier
authors.forename: Nicolas
authors.initials: N
authors.lastname: Rouhier
authors.forename: Michael K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D001051:Apoenzymes / Q000378:metabolism; D004789:Enzyme Activation; D054477:Glutaredoxins / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013057:Spectrum Analysis; D013455:Sulfur / Q000378:metabolism*
chemical_list: D001051:Apoenzymes; D054477:Glutaredoxins; C528773:Grx5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013455:Sulfur; D007501:Iron; D000154:Aconitate Hydratase; D005978:Glutathione
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