• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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• pp.86-90
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• 2020

Iron-sulfur (Fe-S) clusters are one of the most ancient yet essential cofactors mediating various essential biological processes. In prokaryotes, Fe-S clusters are generated via several distinctive biogenesis mechanisms, among which the ISC (Iron-Sulfur Cluster) mechanism plays a house-keeping role to satisfy cellular needs for Fe-S clusters. The Escherichia coli ISC mechanism is maintained by several essential protein factors, whose structural characterization has been of great interest to reveal mechanistic details of the Fe-S cluster biogenesis mechanisms. In particular, nuclear magnetic resonance (NMR) spectroscopic approaches have contributed much to elucidate dynamic features not only in the structural states of the protein components but also in the interaction between them. The present minireview discusses recent advances in elucidating structural features of IscU, the key player in the E. coli ISC mechanism. IscU accommodates exceptional structural flexibility for its versatile activities, for which NMR spectroscopy was particularly successful. We expect that understanding to the structural diversity of IscU provides critical insight to appreciate functional versatility of the Fe-S cluster biogenesis mechanism.

• Journal of Microbiology and Biotechnology
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• v.18 no.10
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• pp.1672-1677
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• 2008

IscR (iron-sulfur cluster regulator) has been reported to be a repressor of the iscRSUA operon, and in vitro transcription reactions have revealed that IscR has a repressive effect on the iscR promoter in the case of [$Fe_{2}S_{2}$] cluster loading. In the present study, the iscR gene from A. ferrooxidans ATCC 23270 was cloned and successfully expressed in Escherichia coli, and then purified by one-step affinity chromatography to homogeneity. The molecular mass of the IscR was 18 kDa by SDS-PAGE. The optical and EPR spectra results for the recombinant IscR confirmed that an iron-sulfur cluster was correctly inserted into the active site of the protein. However, no [$Fe_{2}S_{2}$] cluster was assembled in apoIscR with ferrous iron and sulfide in vitro. Therefore, the [$Fe_{2}S_{2}$] cluster assembly in IscR in vivo would appear to require scaffold proteins and follow the Isc "AUS" pathway.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1070-1075
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• 2008

IscA was proposed to be involved in the iron-sulfur cluster assembly encoded by the iscSUA operon, but the role of IscA in the iron-sulfur cluster assembly still remains controversial. In our previous study, the IscA from A. ferrooxidans was successfully expressed in Escherichia coli, and purified to be a [$Fe_4S_4$] -cluster-containing protein. Cys35, Cys99, and Cys101 were important residues in ligating with the [$Fe_4S_4$] cluster. In this study, Asp97 was found to be another ligand for the iron-sulfur cluster binding according to site-directed mutagenesis results. Molecular modeling for the IscA also showed that Asp97 was a strong ligand with the [$Fe_4S_4$] cluster, which was in good agreement with the experimental results. Thus, the [$Fe_4S_4$] cluster in IscA from A. ferrooxidans was ligated by three cysteine residues and one aspartic acid.

• Journal of Microbiology and Biotechnology
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• v.21 no.2
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• pp.124-128
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• 2011

Ferredoxin is a typical iron-sulfur protein that is ubiquitous in biological redox systems. This study investigates the in vitro assembly of a [$Fe_2S_2$] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins: IscA, IscS, and IscU. The spectra and MALDI-TOF MS results for the reconstituted ferredoxin confirm that the iron-sulfur cluster was correctly assembled in the protein. The inactivation of cysteine desulfurase by L-allylglycine completely blocked any [$Fe_2S_2$] cluster assembly in the ferredoxin in E. coli, confirming that cysteine desulfurase is an essential component for iron-sulfur cluster assembly. The present results also provide strong evidence that [$Fe_2S_2$] cluster assembly in ferredoxin follows the AUS pathway.

• Clinical Nutrition Research
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• v.11 no.4
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• pp.316-330
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• 2022

Iron plays a role in energy metabolism as a component of vital enzymes and electron transport chains (ETCs) for adenosine triphosphate (ATP) synthesis. The tricarboxylic acid (TCA) cycle and oxidative phosphorylation are crucial in generating ATP in mitochondria. At the mitochondria matrix, heme and iron-sulfur clusters are synthesized. Iron-sulfur cluster is a part of the aconitase in the TCA cycle and a functional or structural component of electron transfer proteins. Heme is the prosthetic group for cytochrome c, a principal component of the respiratory ETC. Regarding fat metabolism, iron regulates mitochondrial fat oxidation and affects the thermogenesis of brown adipose tissue (BAT). Thermogenesis is a process that increases energy expenditure, and BAT is a tissue that generates heat via mitochondrial fuel oxidation. Iron deficiency may impair mitochondrial fuel oxidation by inhibiting iron-containing molecules, leading to decreased energy expenditure. Although it is expected that impaired mitochondrial fuel oxidation may be restored by iron supplementation, its underlying mechanisms have not been clearly identified. Therefore, this review summarizes the current evidence on how iron regulates energy metabolism considering the TCA cycle, oxidative phosphorylation, and thermogenesis. Additionally, we relate iron-mediated metabolic regulation to obesity and obesity-related complications.

• Bulletin of the Korean Chemical Society
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• v.24 no.12
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• pp.1849-1852
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• 2003

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.294-300
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• 2010

The Iro protein is a member of the HiPIP family with the [$Fe_4S_4$] cluster for electron transfer. Many reports proposed that the conserved aromatic residues might be responsible for the stability of the iron-sulfur cluster in HiPIP. In this study, Tyr10 was found to be a critical residue for the stability of the [$Fe_4S_4$] cluster, according to site-directed mutagenesis results. Tyr10, Phe26, and Phe48 were essential for the stability of the [$Fe_4S_4$] cluster under acidic condition. Trp44 was not involved in the stability of the [$Fe_4S_4$] cluster. Molecular structure modeling for the mutant Tyr10 proteins revealed that the aromatic group of Tyr10 may form a hydrophobic barrier to protect the [$Fe_4S_4$] cluster from solvent.

• Journal of Life Science
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• v.28 no.3
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• pp.351-356
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• 2018

L-Serine dehydratase (LSD) is an iron-sulfur containing enzyme that catalyzes the conversion of L-serine to pyruvate and ammonia. Among the bacterial amino acid dehydratases, it appears that only the L-serine specific enzymes utilize an iron-sulfur cluster at their catalytic site. Moreover, bacterial LSDs are classified into four types based on structural characteristics and domain arrangement. To date, only the LSD enzymes from a few bacterial strains have been studied, but more detailed investigations are required to understand the catalytic mechanism of various bacterial LSDs. In this study, LSD type II from Mycobacterium tuberculosis (MtLSD) H37Rv was expressed and purified to elucidate the biochemical and catalytic properties using the enzyme kinetic method. The L-serine saturation curve of MtLSD exhibited a typically sigmoid character, indicating an allosteric cooperativity. The values of $K_m$ and $k_{cat}$ were estimated to be $59.35{\pm}1.23mM$ and $18.12{\pm}0.20s^{-1}$, respectively. Moreover, the plot of initial velocity versus D-serine concentration at fixed L-serine concentrations showed a non-linear hyperbola decay shape and exhibited a competitive inhibition for D-serine with an apparent $K_i$ value of $30.46{\pm}5.93mM$ and with no change in the $k_{cat}$ value. These results provide insightful biochemical information regarding the catalytic properties and the substrate specificity of MtLSD.

• Journal of the Korean Magnetic Resonance Society
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• v.28 no.2
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• pp.6-9
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• 2024

HscA is a Hsp70-type chaperone protein that plays an essential role to mediate the iron-sulfur (Fe-S) cluster biogenesis mechanism in Escherichia coli. Like other Hsp70 chaperones, HscA is composed of two domains: the nucleotide binding domain (NBD), which can hydrolyze ATP and use its chemical energy to facilitate the Fe-S cluster transfer process, and the substrate binding domain (SBD), which directly interacts with the substrate, IscU, the scaffold protein of an Fe-S cluster. In the present work, we prepared the isolated SBD construct of HscA (HscA(SBD)) and conducted the solution-state nuclear magnetic resonance (NMR) experiments to have its backbone chemical shift assignment information. Due to low spectral quality of HscA(SBD), we obtained all the NMR data from the sample containing the peptide LPPVKIHC, the HscA-interaction motif of IscU, from which the chemical shift assignment could be done successfully. We expect that this information provides an important basis to execute detailed structural characterization of HscA and appreciate its interaction with IscU.

• Proceedings of the Microbiological Society of Korea Conference
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• 2006.05a
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• pp.66-68
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• 2006

SUF machinery in Echerichia coli, responsible for the biosynthesis of iron-sulfur clusters, is composed of six protein components (SufABCDSE), among which SufB, SufC, and SufD associate in a complex. We have determined the structures of SufA, SufC, and SufD by X-ray crystallography. SufA is a dimer, in which C-terminal segments containing essential cysteine residues (Cys-Gly-Cys) are positioned to allow coordination of an Fe-S cluster and/or an Fe atom. SufC has the overall structure similar to that of ABC-ATPase but takes an inactive form. SufD has a ${\beta}-helix$ flanked with a-helical domains. We also studied the functional roles of the residues in SufD by mutagenesis and determined the crystal structure of SufCD complex. Molecular mechanism of Fe-S cluster biosynthesis is discussed on the basis of the structural and functional evidence.