Characterization of Iron-Sulfur Protein Assembly in Isolated Mitochondria

Abstract

To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from ⌬yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from ⌬nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation.Proteins with iron-sulfur (Fe/S) 1 cluster cofactors are ubiquitous in both eukaryotic and prokaryotic organisms (1). They play central roles in various cellular processes that include redox reactions, metabolic catalysis, and the sensing of iron and ambient oxygen levels (2-4). The process of Fe/S protein biogenesis is highly conserved in nature and is mediated by a set of key components that, in bacteria, are frequently encoded by the so-called isc (iron-sulfur cluster assembly) operon (5, 6). Homologues of several of these Isc components are found in eukaryotes (7,8) and in the nif gene cluster of nitrogen-fixing bacteria, where they participate in the formation of the Fe/S cluster cofactors of nitrogenase (9, 10). Other homologues are part of the suf operon, which is thought to be involved in iron and sulfur metabolism in bacteria and intracellular parasites (11,12). Fe/S protein assembly is initiated by the abstraction of sulfan sulfur from cysteine, a reaction catalyzed by a cysteine desulfurase such as IscS or NifS (13-15). A similar activity of the eukaryotic homologue Nfs1 is likely but hitherto has not been shown. In vitro, cysteine desulfurase is sufficient to mediate the assembly of functional Fe/S proteins from cysteine and ferrous iron. However, in biological systems, the reaction is far more complex and involves proteins such as IscU (yeast Isu proteins) or IscA (yeast Isa proteins) that function as initial iron-and sulfur-binding proteins and as scaffolds for the assembly of a transient Fe/S cluster (16 -25). Moreover, specific chaperones...

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