X‐Ray Absorption Spectroscopy

Bencze, Krisztina Z.; Kondapalli, Kalyan C.; Stemmler, Timothy L.

doi:10.1002/9781119951438.eibc0305

Cited by 15 publications

(27 citation statements)

References 63 publications

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“…The EXAFS region of a XAS spectrum provides high resolution metrical details of the ligand coordination environment for metals bound to a protein (Bencze et al 2007). EXAFS was used to characterize the Fe(II) bound to wt-Isu1 to clarify details of the protein’s metal binding site.…”

Section: Resultsmentioning

confidence: 99%

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

et al. 2015

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Iron-sulfur (Fe-S) cluster containing proteins are utilized in almost every biochemical pathway. The unique redox and coordination chemistry associated with the cofactor allows these proteins to participate in a diverse set of reactions, including electron transfer, enzyme catalysis, DNA synthesis and signaling within several pathways. Due to the high reactivity of the metal, it is not surprising that biological Fe-S cluster assembly is tightly regulated within cells. In yeast, the major assembly pathway for Fe-S clusters is the mitochondrial ISC pathway. Yeast Fe-S cluster assembly is accomplished using the scaffold protein (Isu1) as the molecular foundation, with assistance from the cysteine desulfurase (Nfs1) to provide sulfur, the accessory protein (Isd11) to regulate Nfs1 activity, the yeast frataxin homologue (Yfh1) to regulate Nfs1 activity and participate in Isu1 Fe loading possibly as a chaperone, and the ferredoxin (Yah1) to provide reducing equivalents for assembly. In this report, we utilize calorimetric and spectroscopic methods to provide molecular insight into how wt-Isu1 from S. cerevisiae becomes loaded with iron. Isothermal Titration Calorimetry (ITC) and an iron competition binding assay were developed to characterize the energetics of protein Fe(II) binding. Differential Scanning Calorimetry (DSC) was used to identify thermodynamic characteristics of the protein in the apo state or under iron loaded conditions. Finally, X-ray Absorption Spectroscopy (XAS) was used to characterize the electronic and structural properties of Fe(II) bound to Isu1. Current data are compared to our previous characterization of the D37A Isu1 mutant, and these suggest that when Isu1 binds Fe(II) in a manner not perturbed by the D37A substitution, and that metal binding occurs at a site distinct from the cysteine rich active site in the protein.

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Section: Resultsmentioning

confidence: 99%

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

et al. 2015

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“…A scale factor (Sc) of 0.98% and an E 0 value of −3 eV for a Co-O/N bond, utilized during the protein simulations, were determined by fitting a crystallographically characterized hexaquacobalt (II) nitrate (53). Criteria for judging the best-fit simulation utilized both the lowest mean square deviation between data and fit (F′), corrected for the number of degrees of freedom (54), and a reasonable Debye-Waller factor (σ 2 < 0.006 Å 2 ).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Cobalt-Specific P_1B-ATPase

Zielazinski¹,

Cutsail²,

Hoffman

et al. 2012

Biochemistry

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The P1B-type ATPases are a ubiquitous family of P-type ATPases involved in the transport of transition metal ions. Divided into subclasses on the basis of sequence characteristics and substrate specificity, these integral membrane transporters play key roles in metal homeostasis, metal tolerance, and the biosynthesis of metalloproteins. The P1B-4-ATPases have the simplest architecture of the five P1B-ATPase families and have been suggested to play a role in Co2+ transport. A P1B-4-ATPase from Sulfitobacter sp. NAS-14.1, designated sCoaT, has been cloned, expressed, and purified. Activity assays indicate that sCoaT is specific for Co2+. A single Co2+ binding site is present, and optical, electron paramagnetic resonance (EPR), and X-ray absorption (XAS) spectroscopic data are consistent with tetrahedral coordination by oxygen and nitrogen ligands, including a histidine and likely a water. Surprisingly, there is no evidence for coordination by sulfur. Mutation of a conserved cysteine residue, Cys 327, in the signature transmembrane SPC metal binding motif does not abolish ATP hydrolysis activity or affect the spectroscopic analysis, establishing that this residue is not involved in the initial Co2+ binding by sCoaT. In contrast, replacements of conserved transmembrane residues Ser 325, His 657, Glu 658, and Thr 661 with alanine abolish ATP hydrolysis activity and Co2+ binding, indicating that these residues are necessary for Co2+ transport. These data represent the first in vitro characterization of a P1B-4-ATPase and its Co2+ binding site.

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“…A calibrated scale factor (Sc) of 0.95 and a threshold shift (ΔE 0 ) of −11.5 eV were used during protein data analysis and Sc and E 0 were not allowed to vary during the fitting analysis. Simulation protocols and criteria for judging the best fit were outlined previously (30). …”

Section: Methodsmentioning

confidence: 99%

Molecular Details of the Yeast Frataxin−Isu1 Interaction during Mitochondrial Fe−S Cluster Assembly

et al. 2010

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Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. KeywordsIron Chaperone; Frataxin; Yfh1; Isu1; Nfs1; NMR; ITC and Iron-Sulfur Cluster Assembly Iron-sulfur (Fe-S) clusters are central to life and found in nearly every class of organism (1). These ancient but conserved cofactors are bound to proteins involved in a diverse array of essential functions, ranging from DNA repair to respiration. Since Fe-S cofactors are essential for cell viability, it is no surprise proteins that produce these cofactors are tightly controlled and evolutionarily conserved (2-4). In eukaryotes, the major Fe-S cluster assembly machinery is found in the mitochondria. The process of Fe-S cluster synthesis involves the formation of an Fe-S cluster intermediate on a scaffold protein (ISCU in humans or Isu1 or Isu2 in yeast) and subsequent transfer of the cluster to recipient apo-* To whom correspondence should be sent: Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Telephone: 313-577-5712, Fax: 313-577-2765, tstemmle@med.wayne.edu NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 October 12. proteins. Formation of the Fe-S cluster by Isu1 requires a source of sulfur and a source of iron. The sulfur originates from cysteine via the activity of the cysteine desulfurase Nfs1, which is coordinated with the essential accessory protein Isd11. The source of the iron for Fe-S clusters has remained a mystery. Frataxin (Y...

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X‐Ray Absorption Spectroscopy

Cited by 15 publications

References 63 publications

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

Characterization of a Cobalt-Specific P_1B-ATPase

Molecular Details of the Yeast Frataxin−Isu1 Interaction during Mitochondrial Fe−S Cluster Assembly

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X‐Ray Absorption Spectroscopy

Cited by 15 publications

References 63 publications

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

Iron loading site on the Fe–S cluster assembly scaffold protein is distinct from the active site

Characterization of a Cobalt-Specific P1B-ATPase

Molecular Details of the Yeast Frataxin−Isu1 Interaction during Mitochondrial Fe−S Cluster Assembly

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Characterization of a Cobalt-Specific P_1B-ATPase