The Structure of the Complex between Yeast Frataxin and Ferrochelatase

Abstract

Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe 2؉ and Co 2؉ , led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe 2؉ -to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe 2؉ delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe 2؉ to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.In an oxidative environment, like that of the mitochondrial matrix (1), free Fe 2ϩ is rapidly oxidized to Fe 3ϩ with the subsequent formation of insoluble Fe(OH) 3 . This type of Fe 2ϩ oxidation generally produces oxygen radicals. In addition, through the Fenton reaction in which Fe 2ϩ reacts with hydrogen peroxide, highly toxic hydroxyl radicals are produced. Organisms have evolved mechanisms for the control of iron uptake, delivery, storage, and detoxification, including those for Fe 2ϩ handling within mitochondria. Frataxin, a major mitochondrial protein player in this process, has a central role in iron detoxification and iron delivery in heme and iron-sulfur cluster (ISC) 5 synthesis (2-5) as well as in aconitase repair (6). Low levels of frataxin in humans are responsible for the progressive neurodegenerative disease Friedreich's ataxia, caused by trinucleotide repeat expansions in the first intron of the frataxin gene and the consequent gene silencing. Frataxin deficiency results in aberrations in cellular iron homeostasis, progressive accumulation of iron in mitochondria, high levels of oxidative stress, and deficiency in heme and ISC biosynthesis (4, 7-9).Numerous studies focusing on human and Saccharomyces cerevisiae (Yfh1) frataxin (FXN) and their Esch...

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“…The second one would be related to its role as an iron chaperone: frataxins would keep iron in a non-toxic, easy-deliverable form. Therefore, its deficiency would increase the presence of free or toxic iron forms [29] , [32] . Regarding the nature of these anomalous iron species and its capacity to activate Aft1, Seguin et al [30] , showed that Yfh1-deficient yeast cells accumulated iron in the mitochondria in the form of amorphous nanoparticles of ferric phosphate.…”

“…Nevertheless, the analysis of the effects of frataxin deficiency in several models indicates that frataxin is not essential for iron-sulfur biogenesis in vivo and suggests that frataxin may have additional functions [35] . In this regard, frataxin has also been related with heme biosynthesis [32] , iron storage and/or detoxification [29] , and modulation of iron regulatory protein-1 activation [9] . Frataxin may be also interacting with components of the OXPHOS system [12] and may confer oxidative stress protection [3] .…”

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“…It is known that FECH is involved in formation of various protein complexes with other proteins, for example, with frataxin [ 20 ] or with progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 [ 21 ]. However, these interactions involve FECH surface sites other than the contact region responsible for interaction of FECH monomer with each other.…”

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“…A study in which NMR spectroscopy was used to analyze the binding between both of the proteins suggested that ferrochelatase interacted with frataxin in a manner that included its iron-binding interface [ 37 ]. More recently, Söderberg and collaborators presented a model of the interaction of trimeric Yfh1 (yeast frataxin) and ferrochelatase in which one of the subunits of the Yfh1 trimer interacted with one subunit of the ferrochelatase dimer, whereas another trimer subunit was positioned for iron delivery [ 38 ]. These results support the hypothesis of frataxin acting as an iron donor in the biosynthesis of heme groups.…”