Oligomeric Yeast Frataxin Drives Assembly of Core Machinery for Mitochondrial Iron-Sulfur Cluster Synthesis

Abstract

Mitochondrial biosynthesis of iron-sulfur clusters (ISCs) is a vital process involving the delivery of elemental iron and sulfur to a scaffold protein via molecular interactions that are still poorly defined. Analysis of highly conserved components of the yeast ISC assembly machinery shows that the iron-chaperone, Yfh1, and the sulfur-donor complex, Nfs1-Isd11, directly bind to each other. This interaction is mediated by direct Yfh1-Isd11 contacts. Moreover, both Yfh1 and Nfs1-Isd11 can directly bind to the scaffold, Isu1. Binding of Yfh1 to Nfs1-Isd11 or Isu1 requires oligomerization of Yfh1 and can occur in an iron-independent manner. However, more stable contacts are formed when Yfh1 oligomerization is normally coupled with the binding and oxidation of Fe 2؉ . Our observations challenge the view that iron delivery for ISC synthesis is mediated by Fe 2؉ -loaded monomeric Yfh1. Rather, we find that the iron oxidation-driven oligomerization of Yfh1 promotes the assembly of stable multicomponent complexes in which the iron donor and the sulfur donor simultaneously interact with each other as well as with the scaffold. Moreover, the ability to store ferric iron enables oligomeric Yfh1 to adjust iron release depending on the presence of Isu1 and the availability of elemental sulfur and reducing equivalents. In contrast, the use of anaerobic conditions that prevent Yfh1 oligomerization results in inhibition of ISC assembly on Isu1. These findings suggest that iron-dependent oligomerization is a mechanism by which the iron donor promotes assembly of the core machinery for mitochondrial ISC synthesis.

Architecture of the Yeast Mitochondrial Iron-Sulfur Cluster Assembly Machinery

“…By uncoupling oligomerization of Yfh1 from iron binding and oxidation, the Y73A mutation makes Yfh1 similar to human frataxin, which has an iron-independent mechanism of oligomerization involving its N-terminal region (9). However, the Y73A mutation has no effects on the function of Yfh1, including an ability to bind, store, and deliver iron for Fe-S cluster synthesis in the presence of Isu1, [Nfs1]⅐[Isd11], and L-cysteine (8).…”

“…Similar to the yeast system, overexpression of wild type Yfh1 in E. coli yields monomeric protein that can oligomerize in an iron-dependent manner in vitro as described above. In contrast, overexpression of the Yfh1 Y73A variant in E. coli yields trimer as well as smaller levels of Yfh1 Y73A 24-mer that form as a result of increasing trimer concentrations during protein overexpression (8,25).…”

“…When purified from E. coli, Yfh1 Y73A trimer contains no measurable iron (25) and Yfh1 Y73A 24-mer contains only traces of iron (8). The availability of these iron-free functional oligomers has enabled structural studies that together have revealed the importance of the N-terminal region for Yfh1 oligomerization (25,30).…”

“…Thus, in both bacteria and mitochondria, Fe-S cluster assembly is centered around IscU-type protein scaffolds (bacterial IscU/yeast Isu1) upon which new Fe-S clusters are assembled using the following proteins: (i) a pyridoxal phosphate-dependent cysteine desulfurase (bacterial IscS/yeast Nfs1) with a stabilizing binding partner in eukaryotes (yeast Isd11) that serve as the sulfur donor (1); (ii) the ironbinding protein frataxin (bacterial CyaY/yeast Yfh1) that serves as the iron donor (7)(8)(9)(10) as well as a regulator of the cysteine desulfurase activity (11,12); and (iii) the electron donor chain formed by ferredoxin reductase and ferredoxin (13).…”

Missense Mutations Linked to Friedreich Ataxia Have Different but Synergistic Effects on Mitochondrial Frataxin Isoforms

“…To measure the effects of wild type and mutant FXN isoforms on NFS1 activity, the NFS1-ISD11 complex was incubated with 3 mol eq of ISCU in the presence or absence of 3 mol eq of the indicated FXN isoform at 37°C for 30 min before adding L-cysteine to the reaction mixture. An Fe-S cluster assembly assay were performed as described in detail previously (15,30).…”