Iron use for haeme synthesis is under control of the yeast frataxin homologue (Yfh1)

Abstract

The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In Deltayfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in Deltayfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of Deltayfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies showed that Deltayfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in Deltayfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe-S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.

Metal Ion availability in mitochondria

“…Thus, metallochaperones for Fe(II) and Mn(II) may be important in controlling the reactivity of these ions together with ensuring their bioavailability and specific delivery. Iron donation to ferrochelatase and Isu1 for formation of heme and Fe-S cluster biogenesis, respectively, may involve the matrix protein frataxin (Lesuisse et al 2003;Muhlenhoff et al 2002). Consistent with these roles, Yfh1 interacts with both the Hem15 ferrochelatase and Isu1.…”

“…Consistent with these roles, Yfh1 interacts with both the Hem15 ferrochelatase and Isu1. Cells lacking the yeast frataxin (Yfh1) are heme deficient (Lesuisse et al 2003) and compromised in Fe-S cluster biogenesis (Muhlenhoff et al 2002). However, frataxin is not essential for donating iron to ferrochelatase, as limited heme formation occurs in yfh1Δ cells.…”

“…The loss of Yeast lacking Yfh1 accumulate Zn-protoporphyrin IX (Lesuisse et al 2003). The accumulation of Zn-protoporphyrin IX is also seen in Fe-deficient cells, suggesting that ferrochelatase can access Zn(II) pools within the mitochondrial matrix (Camadro and Labbe 1982;Labbe 1991;Zhang et al 2005b).…”

“…In contrast, cells lacking Yfh1 accumulate high levels of mitochondrial iron, yet this pool does not result in Sod2 inactivation. This second pool is less bioavailable, and preliminary evidence suggests that iron is predominantly insoluble Fe(III) (Lesuisse et al 2003). Factors controlling the distribution of iron into these two pools are unknown, but one factor may be the presence of ligands that stabilize Fe(II) levels.…”

Yeast Frataxin Solution Structure, Iron Binding, and Ferrochelatase Interaction,

“…A cellular frataxin deficiency causes mitochondrial iron overload and impairment of both heme and Fe-S cluster biosynthesis (1,2,8). Cellular heme production is accomplished by the enzyme ferrochelatase, which utilizes iron and protoporphyrin as substrates to produce heme, and recent reports indicate frataxin binds to ferrochelatase with a nanomolar binding affinity (5,9). Frataxin has also been shown to donate iron to ferrochelatase for the completion of in vitro heme synthesis (9,10).…”