Glutathione-complexed iron–sulfur clusters. Reaction intermediates and evidence for a template effect promoting assembly and stability

Qi, Wenbin; Li, Jingwei; Chain, Cecilia Yamil; Pasquevich, G. A.; Pasquevich, A. F.; Cowan, J. A.

doi:10.1039/c3cc43620a

Cited by 28 publications

(24 citation statements)

References 20 publications

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“…An MS signature peak for the intact cluster was observed at 1427.3 (Figure S6). Other spectral peaks were observed at 1413.3 and 1435.3 corresponding to intermediate species previously identified in mass spectra of [2Fe–2S](GS) 4 complex [44], and also similar to reaction intermediates observed in the cluster assembly product for the scaffold proteins (IscS-IscU) [45]. The cluster intermediate is consistent with previous GSH extraction reactions performed in our lab [20, 44] and can be attributed to loss of a sulfur during the ionization process of ESI–MS.…”

Section: Resultsmentioning

confidence: 85%

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

2016

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Human Nfu is an iron–sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron–sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe–2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe–2S] (GS)4 cluster complex, and extraction of the Nfu-bound iron–sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe–2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe–2S] cluster trafficking and delivery.

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Section: Resultsmentioning

confidence: 85%

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

2016

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“…6B), which is in the same range as the second-rate constant found for the native protein, indicating that dimerization has not influenced the ability to transfer a cluster out of G208C NFU1 to this target. Extraction of the cluster by GSH to form the [2Fe-2S](GS) 4 complex was confirmed by appearance of the cluster m/z peak of 1427.3 in ESI-MS, which corresponds to an adduct of the complex with one sodium [43, 46] (Fig. S5).…”

Section: Functional Impairment Of G208c Nfu1mentioning

confidence: 99%

“…This complex can be delivered to a number of iron-sulfur cluster proteins [43, 44] and is a viable substrate for the mitochondrial ABCB7 transporter [45, 46], suggesting a possible role for the [2Fe-2S](GS) 4 complex as a component of the labile iron pool. Native human NFU1 can take up a [2Fe-2S] cluster from the complex with a second-order rate constant of 1930 M −1 min −1 [1], as observed by circular dichroism (CD).…”

Section: Functional Impairment Of G208c Nfu1mentioning

confidence: 99%

Understanding the Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1)—Impact of a Disease-Causing Gly208Cys Substitution on Structure and Activity of NFU1 in the Fe/S Cluster Biosynthetic Pathway

Wachnowsky

Wesley

Fidai

et al. 2017

Journal of Molecular Biology

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Iron-sulfur (Fe/S) cluster-containing proteins constitute one of the largest protein classes, with varied functions that include electron transport, regulation of gene expression, substrate binding and activation, and radical generation. Consequently, the biosynthetic machinery for Fe/S clusters is evolutionarily conserved, and mutations in a variety of putative intermediate Fe/S cluster scaffold proteins can cause disease states, including multiple mitochondrial dysfunctions syndrome (MMDS), sideroblastic anemia and mitochondrial encephalomyopathy. Herein, we have characterized the impact of defects occurring in the MMDS1 disease state that result from a point mutation (Gly208Cys) near the active site of NFU1, an iron-sulfur scaffold protein, via an in vitro investigation into the structural and functional consequences. Analysis of protein stability and oligomeric state demonstrates that the mutant increases the propensity to dimerize and perturbs the secondary structure composition. These changes appear to underlie the severely decreased ability of mutant NFU1 to accept an iron-sulfur cluster from physiologically relevant sources. Therefore, the point mutation on NFU1 impairs downstream cluster trafficking and results in the disease phenotype, because there does not appear to be an alternative in vivo reconstitution path, most likely due to greater protein oligomerization from a minor structural change.

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“…ESI mass spectrometry, combined with studies of functional group modification suggest salt-bridges to be important in glutathione tetramer formation, which creates a macrocyclic ligand that accepts the [2Fe-2S] 2+ cluster core from the scaffold protein ISU. 18, 19 Functional studies of Atm1p have now been conducted with protein-embedded proteoliposomes and transport monitored by a novel application of both flow cytometry and tiron-ligated absorption assays. The flow cytometer was able to both detect proteoliposomes ~400 nm in diameter and quantitate the fluorescence signals of the content inside.…”

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confidence: 99%