Mechanism of frataxin “bypass” in human iron–sulfur cluster biosynthesis with implications for Friedreich’s ataxia

Das, Deepika Sharma; Patra, Shachin; Bridwell‐Rabb, Jennifer; Barondeau, D.P.

doi:10.1074/jbc.ra119.007716

Cited by 21 publications

(16 citation statements)

References 41 publications

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“…Consistently, we also observe a correlation between the amount of dimeric IscS and the rate of decay of the Cys-quinonoid intermediate (Fig. 7A), which is dependent on the ability of the mobile S-transfer loop to function as a proton donor (45)(46)(47)…”

Section: Substitutions Have Varied Effects On the Dimeric Binding Consupporting

confidence: 83%

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Patra

Lin

Ghosh

et al. 2020

Preprint

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Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share ∼60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreich’s ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5’-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

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Section: Substitutions Have Varied Effects On the Dimeric Binding Consupporting

confidence: 83%

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Patra

Lin

Ghosh

et al. 2020

Preprint

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“…5, step I). This step was recently shown to be unaffected by FXN but accelerated for variants of ISCU2 (42). Interestingly, the FXN-dependent stimulation of [2Fe-2S] cluster synthesis and this FXN-independent acceleration of cluster transfer were shown to be additive, and both increase the overall Fe-S cluster flux through the pathway (42).…”

Section: Discussionmentioning

confidence: 92%

“…The truncated form, FXN Δ1-80 , has a modest binding affinity for iron (3 to 55 μM), which led to the proposal that FXN accelerates Fe-S cluster synthesis by functioning as a chaperone that donates iron (29)(30)(31)(32)(33)(34)(35)(36)(37)(38). More recently, multiple studies provide evidence that FXN enhances both the cysteine desulfurase activity (increasing the catalytic constant [k cat ] by ∼12-fold) and the subsequent Fe-S cluster assembly rate on the scaffold protein (5,22,(39)(40)(41)(42). Remarkably, both FXN and its prokaryotic homolog, CyaY, stimulate the in vitro Fe-S assembly reactions by the human Fe-S biosynthetic complex but inhibit the equivalent reaction by the prokaryotic system (43,44).…”

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confidence: 99%

Mechanism of activation of the human cysteine desulfurase complex by frataxin

Patra

Barondeau

2019

Proc. Natl. Acad. Sci. U.S.A.

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The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to the incurable neurodegenerative disease Friedreich’s ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondrial function. The structural and functional core of the Fe-S cluster assembly complex is a low-activity pyridoxal 5′-phosphate (PLP)–dependent cysteine desulfurase enzyme that consists of catalytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) subunits. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. Here, we develop a radiolabeling assay and use stopped-flow kinetics to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. Our results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. Our resulting architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.

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“…The most probable sources of the necessary iron are the mitochondrial labile iron pool and frataxin, which can bind iron through acidic regions within the protein. Frataxin interacts with the ISCU–NSF1–ISD11 complex, and has been hypothesized to mediate iron’s entry into the ISC biogenesis scaffold and/or act as a modulator for NFS1 that might facilitate persulfide transfer to ISCU [ 208 , 221 , 236 , 237 , 238 , 239 , 240 , 241 , 242 , 243 ]. Decreased expression of frataxin impairs ISC biogenesis and iron metabolism, as seen in Friedreich’s ataxia [ 77 , 244 ].…”

Section: Sites Of Mitochondrial Iron Utilizationmentioning

confidence: 99%

Down the Iron Path: Mitochondrial Iron Homeostasis and Beyond

Dietz

Fox

Khalimonchuk

2021

Cells

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Cellular iron homeostasis and mitochondrial iron homeostasis are interdependent. Mitochondria must import iron to form iron–sulfur clusters and heme, and to incorporate these cofactors along with iron ions into mitochondrial proteins that support essential functions, including cellular respiration. In turn, mitochondria supply the cell with heme and enable the biogenesis of cytosolic and nuclear proteins containing iron–sulfur clusters. Impairment in cellular or mitochondrial iron homeostasis is deleterious and can result in numerous human diseases. Due to its reactivity, iron is stored and trafficked through the body, intracellularly, and within mitochondria via carefully orchestrated processes. Here, we focus on describing the processes of and components involved in mitochondrial iron trafficking and storage, as well as mitochondrial iron–sulfur cluster biogenesis and heme biosynthesis. Recent findings and the most pressing topics for future research are highlighted.

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Mechanism of frataxin “bypass” in human iron–sulfur cluster biosynthesis with implications for Friedreich’s ataxia

Cited by 21 publications

References 41 publications

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

Mechanism of activation of the human cysteine desulfurase complex by frataxin

Down the Iron Path: Mitochondrial Iron Homeostasis and Beyond

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