Monothiol Glutaredoxins Function in Storing and Transporting [Fe₂S₂] Clusters Assembled on IscU Scaffold Proteins

Abstract

In the bacterial ISC system for iron–sulfur cluster assembly, IscU acts as a primary scaffold protein, and the molecular co-chaperones HscA and HscB specifically interact with IscU to facilitate ATP-driven cluster transfer. In this work, cluster transfer from Azotobacter vinelandii [Fe2S2]2+ cluster-bound IscU to apo-Grx5, a general purpose monothiol glutaredoxin in A. vinelandii, was monitored by circular dichroism spectroscopy, in the absence and in the presence of HscA/HscB/Mg-ATP. The results indicate a 700-fold enhancement in the rate of [Fe2S2]2+ cluster transfer in the presence of the co-chaperones and Mg-ATP, yielding a second-order rate constant of 20 000 M–1 min–1 at 23 °C. Thus, HscA and HscB are required for efficient ATP-dependent [Fe2S2]2+ cluster transfer from IscU to Grx5. The results support a role for monothiol Grx’s in storing and transporting [Fe2S2]2+ clusters assembled on IscU and illustrate the limitations of interpreting in vitro cluster transfer studies involving [Fe2S2]-IscU in the absence of the dedicated HscA/HscB co-chaperone system.

The Role of Mitochondria in Cellular Iron-Sulfur Protein Biogenesis: Mechanisms, Connected Processes, and Diseases

“…Grx5 possibly serves as an Fe/S cluster transfer protein by transiently coordinating the Isu1-derived [2Fe-2S] cluster between two protein subunits and two protein-bound glutathione molecules (Bandyopadhyay et al 2008b;Shakamuri et al 2012). Recently, Grx5 was shown to specifically associate with Ssq1 at a site different from the chaperone peptide-binding pocket (Uzarska et al 2013).…”

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli

“…Two heat shock cognate proteins, HscB and HscA, interact with the scaffold protein IscU and regulate the iron-sulfur cluster transfer from IscU to target proteins (10,13,14). Specific glutaredoxins are also involved in storing and transporting iron-sulfur clusters from scaffold proteins to target proteins (15)(16)(17). Nevertheless, the iron donor for iron-sulfur cluster biogenesis remains elusive.…”

Iron–sulfur cluster biogenesis and trafficking in mitochondria

“…2) (27,65,67). In turn, that reaction is believed to labilize the [2Fe-2S] cluster on Isu1 providing a facile relay to Grx5.…”

“…Here, we primarily use the corresponding yeast nomenclature to describe the highly conserved protein machinery and biochemical reactions (24). Key to recent progress in the untangling of the molecular mechanisms involved has been the development of in vitro reconstitution assays that employ spectroscopic techniques to follow Fe/S cluster synthesis (25)(26)(27)(28), labeling methods (29), or the use of isolated mitochondria (30). Structural and biophysical work on Fe/S cluster biogenesis factors from bacteria to humans has stimulated and complemented these assays, providing molecular details into the ISC proteins and the modes of transient Fe/S cluster coordination by these factors.…”

“…Grx5 is the only known [2Fe-2S] cluster-binding protein able to directly receive clusters from Isu1 by physically interacting with Ssq1 at a nonsubstrate-binding site (70). Notably, the suggested role of the chaperone system in [2Fe-2S] cluster trafficking has been reconstituted in vitro only in the bacterial ISC system, where the chaperones HscA and HscB stimulated up to 700-fold the [2Fe-2S] cluster transfer from the IscU scaffold to bacterial GrxD (27).…”