Spectroscopic and functional characterization of monothiol glutaredoxins and the fumarate nitrate reduction regulatory protein
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The objectives of this work were to investigate the properties and functions of iron-sulfur (Fe-S) cluster-bound forms of monothiol glutaredoxins with CGFS active-site (CGFS-Grxs), a ubiquitous family of proteins involved in Fe-S cluster biogenesis and Fe homeostasis, and to elucidate the oxygen-sensing mechanism of the E. coli fumarate and nitrate reduction transcriptional regulatory protein (FNR). The approach involved using a combination of biophysical techniques to elucidate the nature and properties of the Fe-S centers under steady-state conditions and/or during in vitro cluster transfer. Our results show that the recombinant S. cerevisiae mitochondrial Grx5 is a versatile Fe-S protein that is able to accommodate [2Fe-2S], linear [3Fe-4S], and [4Fe-4S] clusters in vitro depending on the reconstitution conditions and that the [4Fe-4S] cluster-bound Sc Grx5 is competent for in vitro activation of apo aconitase at physiological relevant rates. These results suggested potential roles of Sc Grx5 in scavenging linear [3Fe-4S] clusters released during protein unfolding under oxidative stress conditions and in the maturation of [4Fe-4S] cluster-containing proteins. Functional studies of [2Fe-2S] cluster-bound CGFS- Grxs were carried out using A. vinelandii Grx5 and Grx-nif by investigating in vitro cluster exchange of these two proteins with physiologically relevant partners from the ISC and NIF cluster assembly pathways, respectively. The results show that CGFS-Grxs can efficiently mediate [2Fe-2S] cluster trafficking from U-type primary scaffolds to apo acceptor proteins via intact cluster transfer. Moreover, Av Grx5 and Grx-nif have the potential to accept [2Fe-2S] clusters from [4Fe-4S] cluster-containing scaffolds and/or carrier proteins. These results provide further support for the proposed Fe-S cluster trafficking and storage functions of CGFS- Grxs. Spectroscopic and mass spectrometry investigation of the oxygen-induced [4Fe-4S]-to-[2Fe- 2S] cluster conversion in FNR revealed formation of atypical [2Fe-2S] clusters with one or two cysteine persulfide ligands that result from sulfur-based oxidation and retention of bridging sulfides. The cluster transformation can be reversed under anaerobic conditions upon incubation with DTT and Fe2+ ion. The observation of analogous oxygen-induced [4Fe-4S]-to-[2Fe-2S] cluster conversion in enzymes with oxygen-sensitive [4Fe-4S] clusters suggests that this novel type of cluster interconversion may represent a new mechanism for the assembly and repair of biological [4Fe-4S] clusters.