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-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.

Resonance Raman spectroscopy of Fe–S proteins and their redox properties

“…In combination with other spectroscopic techniques, RR has revealed the details about the cluster formation, type and coordination along these processes. The studied systems include proteins that are stress-responsive transcriptional regulators, and/or participate in iron metabolism, such as BolA proteins and homologues [8, 39]. Among the latter is Fra2, which plays a key role in regulating the iron homeostasis in yeasts.…”

“…After “Introduction”, we will first briefly describe the basics of RR spectroscopy of Fe–S proteins, together with the most commonly used approaches for determination of their redox properties. Then we will focus on the major contributions of RR spectroscopy in the studies of diverse Fe–S proteins, such as those that participate in ET [1, 13–21], DNA repair [11], biogenesis of Fe–S clusters [3, 22–30] and heme cofactors [31], substrate binding and activation, S-donation and catalysis [32–35], and regulation of gene expression [5, 8, 10, 36–39]. We will conclude with several examples of recent RR studies of enzymes carrying complex polychromophoric clusters [7, 40, 41].…”

“…Furthermore, they led to remarkable studies of dynamic processes that involve cluster inter-conversion, biogenesis, disassembly and catalysis; a particularly large body of work on these transient systems comes from Johnson’s group and co-workers [3, 5, 8, 10, 13, 22–28, 30, 32–34, 36–39, 44]. …”

The mitochondrial monothiol glutaredoxin S15 is essential for iron-sulfur protein maturation in Arabidopsis thaliana

“…Another important difference is that yeast Grx5p contains an additional cysteine residue (C 90 or C 117 including the targeting sequence) implicated in intramolecular disulfide exchange reactions (15). Whereas genetic analyses indicated that this cysteine does not seem essential for ISC biogenesis (25), Zhang et al showed in mutational studies that this cysteine is required for the assembly of Fe 4 S 4 cluster and that the Fe 4 S 4 cluster-bound form of Grx5p is competent for restoring the activity of recombinant ACO in vitro (17). Although this cysteine is present in most algal isoforms, it is replaced by a serine in orthologs from terrestrial plants, except Selaginella moellendorffii (26).…”

“…In this case, the visible part of the absorption spectrum of the reconstituted protein presented a prominent absorbance peak at approximately 420 nm compared with the apoprotein (Fig. 4A) + clusters (17). Next, we evaluated the capacity of GRXS15 to transfer its ISC to an acceptor protein.…”