Heterotrifunctional Chemical Cross-Linking Mass Spectrometry Confirms Physical Interaction between Human Frataxin and ISU

Watson, Heather M.; Gentry, Leslie E.; Asuru, Awuri P.; Wang, Yu; Marcus, Stevan; Busenlehner, Laura S.

doi:10.1021/bi300779f

Cited by 5 publications

(12 citation statements)

References 18 publications

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“…5 Some research suggests that frataxin is an iron-chaperone that delivers Fe 2+ to the Fe-S cluster complex. 6 In vitro , frataxin stimulates the rate of formation of [2Fe-2S] clusters on the surface of ISCU with externally supplied sulfide 7 and photo-activated chemical crosslinking 8 confirms the interaction between frataxin and ISCU. Other studies indicate that frataxin may regulate Fe-S cluster synthesis through its stimulation of NFS1 cysteine desulfurase activity as part of the ISCU/NFS1/ISD11 complex.…”

mentioning

confidence: 74%

“…8 Purification was performed in the presence of 2 mM EDTA in Chelex-treated, metal-free water. All glassware was rinsed with 10% nitric acid and metal-free water to remove trace metals.…”

Section: Methodsmentioning

confidence: 99%

“…H86A frataxin was expressed in BL21(DE3) pLysS and purified as for the wild-type protein. 8 Co 2+ and Fe 3+ titrations monitored by UV-visible spectroscopy of H86A frataxin were performed as for wild-type. The Fe 2+ titration of H86A frataxin in the presence of ferrozine chelator was also as described for the wild-type frataxin protein.…”

Section: Methodsmentioning

confidence: 99%

“…D37A ISCU was expressed in BL21(DE3) pLysS and purified as described for native ISCU. 8 D37A ISCU samples were anaerobically dialyzed into Chelex-treated 50 mM HEPES (pH 7.4), 150 mM NaCl with 10% glycerol. The concentration of ISCU was determined spectroscopically using ε 280nm = 9.97 mM −1 cm −1 .…”

Section: Methodsmentioning

confidence: 99%

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His86 from the N-Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

et al. 2013

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Human frataxin has a vital role in the biosynthesis of iron–sulfur (Fe-S) clusters in mitochondria and its deficiency causes the neurodegenerative disease Friedreich’s ataxia. Proposed functions for frataxin in the Fe-S pathway include iron donation to the Fe-S cluster machinery and regulation of cysteine desulfurase activity to control the rate of Fe-S production, although further molecular detail is required to distinguish these two possibilities. It is well established that frataxin can coordinate iron using glutamate and aspartate side chains on the protein surface; however, in this work we identify a new iron coordinating residue in the Nterminus of human frataxin using complementary spectroscopic and structural approaches. Further, we demonstrate that His86 in this N-terminal region is required for high affinity iron coordination and iron assembly of Fe-S clusters by ISCU as part of the Fe-S cluster biosynthetic complex. If a binding site that includes His86 is important for Fe-S cluster synthesis as part of its chaperone function, this raises the possibility that either iron binding at the acidic surface of frataxin may be spurious or that it is required for protein–protein interactions with the Fe-S biosynthetic quaternary complex. Our data suggest that iron coordination to frataxin may be significant to the Fe-S cluster biosynthesis pathway in mitochondria.

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confidence: 74%

“…8 Purification was performed in the presence of 2 mM EDTA in Chelex-treated, metal-free water. All glassware was rinsed with 10% nitric acid and metal-free water to remove trace metals.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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His86 from the N-Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

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“…It is known that chemical cross-linking can capture weak or transient protein–protein interactions. 22 …”

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confidence: 99%

[2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin

et al. 2013

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Escherichia coli [2Fe-2S]-ferredoxin (Fdx) is encoded by the isc operon along with other proteins involved in the ‘house-keeping’ mechanism of iron–sulfur cluster biogenesis. Although it has been proposed that Fdx supplies electrons to reduce sulfane sulfur (S0) produced by the cysteine desulfurase (IscS) to sulfide (S2–) as required for the assembly of Fe–S clusters on the scaffold protein (IscU), direct experimental evidence for the role of Fdx has been lacking. Here, we show that Fdx (in either oxidation state) interacts directly with IscS. The interaction face on Fdx was found to include residues close to its Fe–S cluster. In addition, C328 of IscS, the residue known to pick up sulfur from the active site of IscS and deliver it to the Cys residues of IscU, formed a disulfide bridge with Fdx in the presence of an oxidizing agent. Electrons from reduced Fdx were transferred to IscS only in the presence of l-cysteine, but not to the C328S variant. We found that Fdx, IscU, and CyaY (the bacterial frataxin) compete for overlapping binding sites on IscS. This mutual exclusion explains the mechanism by which CyaY inhibits Fe–S cluster biogenesis. These results (1) show that reduced Fdx supplies one electron to the IscS complex as S0 is produced by the enzymatic conversion of Cys to Ala and (2) explain the role of Fdx as a member of the isc operon.

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2013

Bioconjugate Techniques

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Heterotrifunctional Chemical Cross-Linking Mass Spectrometry Confirms Physical Interaction between Human Frataxin and ISU

Cited by 5 publications

References 18 publications

His86 from the N-Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

His86 from the N-Terminus of Frataxin Coordinates Iron and Is Required for Fe–S Cluster Synthesis

[2Fe-2S]-Ferredoxin Binds Directly to Cysteine Desulfurase and Supplies an Electron for Iron–Sulfur Cluster Assembly but Is Displaced by the Scaffold Protein or Bacterial Frataxin

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