Characterization of the roles of A-type proteins and monothiol CGFS glutaredoxins in iron-sulfur cluster biogenesis
Mapolelo, Daphne Thusang
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Iron-sulfur (Fe–S) clusters constitute a ubiquitous class of protein cofactors with diverse biological functions. The objectives of this work were to elucidate the roles of A-type Fe−S cluster assembly proteins and monothiol CGFS glutaredoxins in Fe–S cluster biogenesis and Fe homeostasis. The approach involved in vitro characterization of the properties of Fe- and Fe−S cluster-bound forms of Azotobacter vinelandii NifIscA using UV−visible absorption, CD and VTMCD, EPR, resonance Raman, and Mössbauer spectroscopies, coupled with in vitro cluster transfer studies to physiologically relevant acceptor proteins and from monothiol CGFS glutaredoxins and physiologically relevant U-type Fe−S cluster scaffold proteins. The results show that NifIscA can bind one mononuclear high-spin (S = 2) Fe(II) or intermediate-spin (S = 3/2) Fe(III) per dimer. Both have the potential to function as Fe donors for U-type scaffold proteins, based on facile cysteine-mediated Fe release, and the redox potential for the Fe(III/II) couple (+36 mV versus NHE) which indicates that the Fe(II)-bound form is more likely to be present under physiological conditions. [2Fe-2S]2+ clusters assembled on NifIscA were shown to undergo dithiol-mediated reductive coupling to form [4Fe-4S]2+ clusters and oxidative cleavage to yield the original [2Fe-2S]2+ clusters. The [4Fe-4S]2+ cluster-bound NifIscA is competent for in vitro maturation of apo-nitrogenase Fe protein and apo-NifIscA was shown to accept clusters from NifU. These results indicate that NifIscA can function under aerobic or oxidative stress conditions as an alternative scaffold for the assembly of [4Fe-4S]2+ clusters or as a cluster carrier for the delivery of clusters assembled on NifU. Yeast cytosolic monothiol glutaredoxins were shown to interact with Fra2 to form a [2Fe-2S]2+ cluster-bound heterodimeric complex involved in Fe sensing and Fe regulation. Spectroscopic characterization of Grx3/4 homodimer revealed an all-cysteinyl-ligated [2Fe-2S]2+ cluster while the Fra2-Grx3 heterodimeric cluster is ligated by two cysteines, one histidine. The [2Fe-2S]2+ cluster-bound Fra2-Grx3 heterodimer can be formed by binding Fra2 to cluster-bound Grx3 or by intact cluster transfer from NifIscA to apo-Fra2-Grx3. [2Fe-2S]2+ clusters on monothiol glutaredoxins are rapidly and quantitatively transferred to apo AvNifIscA indicating that these two classes of proteins function together in Fe–S cluster trafficking.
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