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

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.

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Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.03153-20 ◽

2021 ◽

Vol 87 (10) ◽

Author(s):

Xiaojun Ren ◽

Feng Liang ◽

Zhengfen He ◽

Bingqian Fan ◽

Zhirong Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Cold Stress ◽

Stress Conditions ◽

Iron Binding ◽

Cluster Assembly ◽

Iron Sulfur Cluster ◽

Content Type ◽

Sulfur Cluster ◽

E Coli ◽

Iron Sulfur

ABSTRACT Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli. Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system. IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

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Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli

10.1101/2021.10.06.463429 ◽

2021 ◽

Author(s):

Katherine Picott ◽

Robert Flick ◽

Elizabeth Anne Edwards

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Specific Activity ◽

Expression System ◽

Small Scale ◽

Anaerobic Conditions ◽

Iron Sulfur Cluster ◽

E Coli ◽

Nickel Affinity Chromatography ◽

Iron Sulfur

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, primarily because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. In this work we outline a novel respiratory RDase expression system using Escherichia coli as the host. The overexpression of E. coli's cobamide transport system, btu, and RDase expression under anaerobic conditions were established to be essential for the expression of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six RDase enzymes with amino acid sequence identities ranging from >30-95%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of both the cobamide and iron-sulfur cluster cofactors was verified, and the specific activity of TmrA was found to be consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases.

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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 ◽

10.1099/mic.0.27476-0 ◽

2005 ◽

Vol 151 (5) ◽

pp. 1421-1431 ◽

Cited By ~ 34

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.

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Evidence for an oxygen-sensitive iron–sulfur cluster in an immature large subunit species of Escherichia coli [NiFe]-hydrogenase 2

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2012.06.096 ◽

2012 ◽

Vol 424 (1) ◽

pp. 158-163 ◽

Cited By ~ 10

Author(s):

Basem Soboh ◽

Martin Kuhns ◽

Mario Braussemann ◽

Mandy Waclawek ◽

Enrico Muhr ◽

...

Keyword(s):

Escherichia Coli ◽

Large Subunit ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Oxygen Sensitive ◽

Hydrogenase 2

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Positively Charged Residues within the Iron–Sulfur Cluster Loop of E. coli MutY Participate in Damage Recognition and Removal

Archives of Biochemistry and Biophysics ◽

10.1006/abbi.2000.1890 ◽

2000 ◽

Vol 380 (1) ◽

pp. 11-19 ◽

Cited By ~ 19

Author(s):

Cindy Lou Chepanoske ◽

Marie-Pierre Golinelli ◽

Scott D. Williams ◽

Sheila S. David

Keyword(s):

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Damage Recognition ◽

Iron Sulfur ◽

Charged Residues ◽

Positively Charged

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Engineering a Novel Iron-Sulfur Cluster into the Catalytic Subunit of Escherichia coli Dimethyl-sulfoxide Reductase

Journal of Biological Chemistry ◽

10.1074/jbc.271.9.4620 ◽

1996 ◽

Vol 271 (9) ◽

pp. 4620-4626 ◽

Cited By ~ 13

Author(s):

Catharine A. Trieber ◽

Richard A. Rothery ◽

Joel H. Weiner

Keyword(s):

Escherichia Coli ◽

Dimethyl Sulfoxide ◽

Catalytic Subunit ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

Iron Sulfur ◽

Dimethyl Sulfoxide Reductase

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Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

Genes ◽

10.3390/genes10010034 ◽

2019 ◽

Vol 10 (1) ◽

pp. 34 ◽

Cited By ~ 4

Author(s):

Natalie Gugala ◽

Kate Chatfield-Reed ◽

Raymond J. Turner ◽

Gordon Chua

Keyword(s):

Escherichia Coli ◽

Cell Envelope ◽

Antimicrobial Properties ◽

Iron Sulfur Cluster ◽

Nucleotide Biosynthesis ◽

E Coli ◽

Chemical Genetic ◽

Iron Sulfur ◽

Insight Into ◽

Mutant Collection

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

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Crystal Structure of RumA, an Iron-Sulfur Cluster Containing E. coli Ribosomal RNA 5-Methyluridine Methyltransferase

Structure ◽

10.1016/j.str.2004.02.009 ◽

2004 ◽

Vol 12 (3) ◽

pp. 397-407 ◽

Cited By ~ 29

Author(s):

Tom T Lee ◽

Sanjay Agarwalla ◽

Robert M Stroud

Keyword(s):

Crystal Structure ◽

Ribosomal Rna ◽

Iron Sulfur Cluster ◽

Sulfur Cluster ◽

E Coli ◽

Iron Sulfur

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Zinc Toxicity and Iron-Sulfur Cluster Biogenesis inEscherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01967-18 ◽

2019 ◽

Vol 85 (9) ◽

Cited By ~ 8

Author(s):

Jianghui Li ◽

Xiaojun Ren ◽

Bingqian Fan ◽

Zhaoyang Huang ◽

Wu Wang ◽

...

Keyword(s):

Binding Activity ◽

Zinc Toxicity ◽

Cluster Assembly ◽

Intracellular Zinc ◽

Iron Sulfur Cluster ◽

Content Type ◽

Sulfur Cluster ◽

E Coli ◽

Iron Sulfur ◽

Assembly Proteins

ABSTRACTWhile zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases inEscherichia coli. Here, we report that the intracellular zinc overload inE. colicells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene clusteriscSUA-hscBA-fdx-iscXinE. colicells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of theE. colimutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin inE. colicells.IMPORTANCEZinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload inEscherichia colicells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

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