The Interaction of Mitochondrial Iron with Manganese Superoxide Dismutase

Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p.Metal-containing enzymes are generally quite specific for their cognate cofactor. Misincorporation of the wrong metal ion can be deleterious and tends to be a rare occurrence in biology. A prime example of metal ion selectivity is illustrated by the family of manganese- and iron-containing superoxide dismutases (SODs)³. This large family of enzymes utilizes either manganese or iron as cofactors to scavenge superoxide anion. The iron- and manganese-containing forms are highly homologous to one another at primary, secondary, and tertiary levels and have virtually identical metal binding and catalytic sites (1–3). Despite this extensive homology, Mn- and Fe-SODs are only active with their cognate metal. Misincorporation of iron into Mn-SOD or vice versa alters the redox potential of the enzyme''s active site and prohibits superoxide disproportionation (4, 5). Nevertheless, misincorporation of iron into Mn-SOD does occur in vivo (6, 7). The isolated Mn-SOD from Escherichia coli is found as a mixture of manganese- and iron-bound forms (7); binding of manganese is favored under oxidative stress, whereas iron binding is increased under anaerobic conditions (3, 8). It has been proposed that changes in bioavailability of manganese versus iron determine the metal selectivity of Mn-SOD in bacterial cells (3, 8). But is this also true for Fe-SOD? Currently, there is no documentation of manganese misincorporation into Fe-SOD in vivo.Unlike bacteria that co-express Mn- and Fe-SOD molecules in the same cell, eukaryotic mitochondria generally harbor only one member of the Fe/Mn-SOD family, a tetrameric Mn-SOD typically known as SOD2 (9). In some organisms, SOD2 is essential for survival (10–12), and mitochondria have therefore evolved to prevent iron-SOD2 interactions despite high levels of mitochondrial iron relative to manganese. Using a yeast model system, we have shown previously that metal ion mis-incorporation can occur with Saccharomyces cerevisiae Sod2p (7). Specifically, iron binds and inactivates yeast Sod2p when cells are either starved for manganese or have certain disruptions in mitochondrial iron homeostasis. These disruptions include mutations in MTM1, a mitochondrial carrier protein that functions in iron metabolism (7, 13), and mutations in GRX5 or SSQ1, involved in iron-sulfur biogenesis (14). We proposed that these disruptions lead to expansion of a mitochondrial pool of so-called SOD2-reactive iron (7). Currently, it is unknown whether SOD2-reactive iron represents a major shift in the chemistry of bulk mitochondrial iron or whether it is just a small pool of the metal emerging from one or more specific sites.The grx5 and ssq1 mutants that promote iron-SOD2 interactions encode just two of many components of a complex pathway for iron-sulfur biogenesis (15, 16). One of the key components is a well conserved iron-sulfur scaffold protein originally described for bacteria as IscU, also known as mammalian ISCU and S. cerevisiae Isu1p and Isu2p, referred collectively herein as “Isu proteins” (17–22). The iron-sulfur clusters on Isu proteins are labile and can be transferred to target iron-sulfur proteins through the aid of mitochondrial factors including Grx5p and Ssq1p (15, 16). It is not clear whether disruption of the iron-sulfur pathway per se is sufficient to promote iron interactions with yeast Sod2p or whether this effect is specific to grx5, ssq1, and mtm1 mutants.In the current study, we explore the nature of mitochondrial iron that can interact with Sod2p. We find that the changes in mitochondrial metal homeostasis that shift metal binding in yeast Sod2p likewise alter metal cofactor selection in a heterologously expressed Mn-SOD, but not in a Fe-SOD molecule. Through x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) analyses of mitochondrial iron, we detected no major change in bulk mitochondrial iron under conditions that promote iron-SOD2 interactions. SOD2-reactive iron appears to represent a small pool of the metal, and we provide evidence that the iron-sulfur scaffold Isu1p can act as an important source of this reactive iron.