scholarly journals Monitoring iron-sulfur cluster occupancy across the E. coli proteome using chemoproteomics

2021 ◽  
Author(s):  
Daniel W Bak ◽  
Eranthie Weerapana
Keyword(s):  
Scaffold Proteins ◽  
Iron Sulfur Cluster ◽  
Protein Targets ◽  
E Coli ◽  
Iron Incorporation ◽  
Iron Sulfur ◽  
Atpase Subunits ◽  
Radical Generation ◽  
S Proteins

Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors found across diverse protein families, where they perform myriad functions including redox chemistry, radical generation, and gene regulation. Monitoring Fe-S cluster occupancy in protein targets directly within native biological systems has been challenging. Commonly utilized spectroscopic methods to detect Fe-S clusters require purification of proteins prior to analysis. Global iron incorporation into the proteome can be monitored using radiolabeled iron, but limitations include the low resolution afforded by gel-based autoradiography. Here, we report the development of a mass spectrometry-based strategy to assess Fe-S cluster binding in a native proteome. This chemoproteomic strategy relies on monitoring changes in the reactivity of Fe-S cluster cysteine ligands upon disruption of Fe-S cluster incorporation. Application to E. coli cells cultured under iron-depleted conditions enabled monitoring of disruptions to Fe-S cluster incorporation broadly across the E. coli Fe-S proteome. Evaluation of E. coli deletion strains of three scaffold proteins within the Isc Fe-S biogenesis pathway enabled the identification of Fe-S clients that are reliant on each individual scaffold protein for proper cluster installation. Lastly, cysteine-reactivity changes characteristic of Fe-S ligands were used to identify previously unannotated Fe-S proteins, including the tRNA hydroxylase, TrhP, and a member of a family of membrane transporter ATPase subunits, DppD. In summary, the chemoproteomic strategy described herein provides a powerful tool to report on Fe-S cluster incorporation directly within a native proteome, to interrogate the role of scaffold and accessory proteins within Fe-S biogenesis pathways, and to identify previously uncharacterized Fe-S proteins.

scholarly journals The Role of CyaY in Iron Sulfur Cluster Assembly on the E. coli IscU Scaffold Protein

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e21992 ◽  
Author(s):  
Clara Iannuzzi ◽  
Salvatore Adinolfi ◽  
Barry D. Howes ◽  
Ricardo Garcia-Serres ◽  
Martin Clémancey ◽  
...  
Keyword(s):  
Scaffold Protein ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

scholarly journals Essential role of Isd11 in mitochondrial iron–sulfur cluster synthesis on Isu scaffold proteins

2005 ◽  
Vol 25 (1) ◽  
pp. 184-195 ◽  
Author(s):  
Nils Wiedemann ◽  
Eugen Urzica ◽  
Bernard Guiard ◽  
Hanne Müller ◽  
Christiane Lohaus ◽  
...  
Keyword(s):  
Essential Role ◽  
Scaffold Proteins ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Mitochondrial Iron

scholarly journals The Yeast Scaffold Proteins Isu1p and Isu2p Are Required inside Mitochondria for Maturation of Cytosolic Fe/S Proteins

2004 ◽  
Vol 24 (11) ◽  
pp. 4848-4857 ◽  
Author(s):  
Jana Gerber ◽  
Karina Neumann ◽  
Corinna Prohl ◽  
Ulrich Mühlenhoff ◽  
Roland Lill
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Mitochondrial Targeting ◽  
Protein Maturation ◽  
Iron Sulfur Cluster ◽  
Subcellular Compartment ◽  
S Protein ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
S Proteins

ABSTRACT Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

scholarly journals Understanding the role of dynamics in the iron sulfur cluster molecular machine

2017 ◽  
Vol 1861 (1) ◽  
pp. 3154-3163 ◽  
Author(s):  
Danilo di Maio ◽  
Balasubramanian Chandramouli ◽  
Robert Yan ◽  
Giuseppe Brancato ◽  
Annalisa Pastore
Keyword(s):  
Molecular Machine ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals Minor Alterations in Core Promoter Element Positioning Reveal Functional Plasticity of a Bacterial Transcription Factor

mBio ◽  
2021 ◽  
Author(s):  
Wamiah P. Chowdhury ◽  
Kenneth A. Satyshur ◽  
James L. Keck ◽  
Patricia J. Kiley
Keyword(s):  
Transcription Factor ◽  
Core Promoter ◽  
Promoter Element ◽  
Functional Plasticity ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
E Coli ◽  
Bacterial Transcription ◽  
Iron Sulfur ◽  
Living Organisms

Transcription regulation is a key process in all living organisms, involving a myriad of transcription factors. In E. coli , the regulator of the iron-sulfur cluster biogenesis pathway, IscR, acts as a global transcription factor, activating the transcription of some pathways and repressing others.

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