Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

Funding Information: We thank L?gia S. Nobre and Joana M. Baptista for contribution at the initial stage of the work, and Cl?udia S. Freitas for technical support. We also thank Professor Miguel Teixeira of ITQB-NOVA for critical reading of the manuscript. We thank the XALOC staff and floor coordinators at the synchrotron ALBA for the YtfEM data collection. We acknowledge the ESRF for provision of synchrotron radiation facilities and we would like to thank Gianluca Santoni for assistance using the beamline ID30A-3 for the YtfEM-E159L data collection. We also thank Diamond Light Source for beamtime and the staff of beamline I04 for assistance with crystal testing and data collection of YtfEM-E125L. Funding. This work was financially supported by Funda??o para a Ci?ncia e Tecnologia (Portugal) through fellowship SFRH/BD/118545/2016 (LOS) and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) co-funded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. This work was partially supported by PPBI ? Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE ? ?Or?amento de Estado? and by European funds from FEDER ? ?Fundo Europeu de Desenvolvimento Regional.? We also acknowledge funding from the European Union?s Horizon 2020 Research and Innovation Program under grant agreement no. 810856. Publisher Copyright: © Copyright © 2021 Silva, Matias, Romão and Saraiva. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.publishersversionpublishe

Superoxide reductase from Giardia intestinalis: structural characterization of the first sor from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage

Insights into the role of three Endonuclease III enzymes for oxidative stress resistance in the extremely radiation resistant bacterium Deinococcus radiodurans

The extremely radiation and desiccation resistant bacterium Deinococcus radiodurans possesses three genes encoding Endonuclease III-like enzymes (DrEndoIII1, DrEndoIII2, DrEndoIII3). In vitro enzymatic activity measurements revealed that DrEndoIII2 is the main Endonuclease III in this organism, while DrEndoIII1 and 3 possess unusual and, so far, no detectable EndoIII activity, respectively. In order to understand the role of these enzymes at a cellular level, DrEndoIII knockout mutants were constructed and subjected to various oxidative stress related conditions. The results showed that the mutants are as resistant to ionizing and UV-C radiation as well as H2O2 exposure as the wild type. However, upon exposure to oxidative stress induced by methyl viologen, the knockout strains were more resistant than the wild type. The difference in resistance may be attributed to the observed upregulation of the EndoIII homologs gene expression upon addition of methyl viologen. In conclusion, our data suggest that all three EndoIII homologs are crucial for cell survival in stress conditions, since the knockout of one of the genes tend to be compensated for by overexpression of the genes encoding the other two

An internal promoter drives the expression of a truncated form of ccc1 capable of protecting yeast from iron toxicity

Funding Information: This work was supported by Project LISBOA-01-0145-FEDER-007660 (?Microbiologia Molecular, Estrutural e Celular?) funded by FEDER funds through COMPETE2020??Programa Operacional Competitividade e Internacionaliza??o? (POCI); ?Funda??o para a Ci?ncia e a Tecnologia? (FCT) grants EXPL/BIA-MIC/2525/2013 and programme IF (IF/00124/2015) to C.P.; grant PTDC/BIA-BQM/31317/2017 to C.V.R.; the European Union?s Horizon 2020 research and innovation programme under grant agreement No. 810856 and 857203; COST Action CA15133, supported by COST (European Cooperation in Science and Technology) and PPBI?Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE??Or?amento de Estado? and by FEDER. C.A. (SFRH/BPD/74294/2010), S.M.C. (SFRH/BD/91077/2012), C.V.R. (SFRH/BPD/94050/2013) and A.G.-C. (SFRH/BD/118866/2016) were supported by FCT contracts or fellowships. Funding Information: Funding: This work was supported by Project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, ?strutural e Celular”) funded by F?D?R funds through COMP?T?2020—“Programa Operacional Competitividade e Internacionalização” (POCI); “Fundação para a Ciência e a Tecno-logia” (FCT) grants ?XPL/BIA-MIC/2525/2013 and programme IF (IF/00124/2015) to C.P.; grant PTDC/BIA-BQM/31317/2017 to C.V.R.; the ?uropean Union’s Horizon 2020 research and innovation programme under grant agreement No. 810856 and 857203; COST Action CA15133, supported by COST (European Cooperation in Science and Technology) and PPBI—Portuguese Platform of Bi-oImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE—“Orçamento de Estado” and by FEDER. C.A. (SFRH/BPD/74294/2010), S.M.C. (SFRH/BD/91077/2012), C.V.R. (SFRH/BPD/94050/2013) and A.G.-C. (SFRH/BD/118866/2016) were supported by FCT contracts or fellowships. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.In yeast, iron storage and detoxification depend on the Ccc1 transporter that mediates iron accumulation in vacuoles. While deletion of the CCC1 gene renders cells unable to survive under iron overload conditions, the deletion of its previously identified regulators only partially affects survival, indicating that the mechanisms controlling iron storage and detoxification in yeast are still far from well understood. This work reveals that CCC1 is equipped with a complex transcriptional structure comprising several regulatory regions. One of these is located inside the coding sequence of the gene and drives the expression of a short transcript encoding an N-terminally truncated protein, designated as s-Ccc1. s-Ccc1, though less efficiently than Ccc1, is able to promote metal accumulation in the vacuole, protecting cells against iron toxicity. While the expression of the s-Ccc1 appears to be repressed in the normal genomic context, our current data clearly demonstrates that it is functional and has the capacity to play a role under iron overload conditions.publishersversionpublishe

The amino acids motif-32GSSYN36-in the catalytic domain of E. coli flavorubredoxin NO reductase is essential for its activity

Funding Information: Funding: This study was financially supported by the Portuguese Fundação para a Ciência e Tec-nologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Funding Information: This study was financially supported by the Portuguese Funda??o para a Ci?ncia e Tecnologia (FCT), grants PTDC/BIA-BQM/27959/2017 and PTDC/BIA-BQM/0562/2020, and Project MOSTMICRO-ITQB with references UIDB/04612/2020 and UIDP/04612/2020. This project has also received funding from the European Union?s Horizon 2020 research and innovation program under grant agreement 810856. MCM is the recipient of FCT grant SFRH/BD/143651/2019. BAS is the recipient of FCT grant DFA/BD/8066/2020. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Flavodiiron proteins (FDPs) are a family of modular and soluble enzymes endowed with nitric oxide and/or oxygen reductase activities, producing N2O or H2O, respectively. The FDP from Escherichia coli, which, apart from the two core domains, possesses a rubredoxin-like domain at the C-terminus (therefore named flavorubredoxin (FlRd)), is a bona fide NO reductase, exhibiting O2 reducing activity that is approximately ten times lower than that for NO. Among the flavorubredoxins, there is a strictly conserved amino acids motif,-G[S,T]SYN-, close to the catalytic diiron center. To assess its role in FlRd’s activity, we designed several site-directed mutants, replacing the conserved residues with hydrophobic or anionic ones. The mutants, which maintained the general characteristics of the wild type enzyme, including cofactor content and integrity of the diiron center, revealed a decrease of their oxygen reductase activity, while the NO reductase activity—specifically, its physiological function—was almost completely abolished in some of the mutants. Molecular modeling of the mutant proteins pointed to subtle changes in the predicted structures that resulted in the reduction of the hydration of the regions around the conserved residues, as well as in the elimination of hydrogen bonds, which may affect proton transfer and/or product release.publishe

Desulfovibrio vulgarisCbiKPcobaltochelatase: evolution of a haem binding protein orchestrated by the incorporation of two histidine residues

The sulfate-reducing bacteria of the Desulfovibrio genus make three distinct modified tetrapyrroles, haem, sirohaem and adenosylcobamide, where sirohydrochlorin acts as the last common biosynthetic intermediate along the branched tetrapyrrole pathway. Intriguingly, D. vulgaris encodes two sirohydrochlorin chelatases, CbiKP and CbiKC, that insert cobalt/iron into the tetrapyrrole macrocycle but are thought to be distinctly located in the periplasm and cytoplasm respectively. Fusing GFP onto the C-terminus of CbiKP confirmed that the protein is transported to the periplasm. The structure-function relationship of CbiKP was studied by constructing eleven site-directed mutants and determining their chelatase activities, oligomeric status and haem binding abilities. Residues His154 and His216 were identified as essential for metal-chelation of sirohydrochlorin. The tetrameric form of the protein is stabilized by Arg54 and Glu76, which form hydrogen bonds between two subunits. His96 is responsible for the binding of two haem groups within the main central cavity of the tetramer. Unexpectedly, CbiKP is shown to bind two additional haem groups through interaction with His103. Thus, although still retaining cobaltochelatase activity, the presence of His96 and His103 in CbiKP, which are absent from all other known bacterial cobaltochelatases, has evolved CbiKP a new function as a haem binding protein permitting it to act as a potential haem chaperone or transporter

Characterization of the [NiFe] Hydrogenase from the sulfate reducer Desulfovibrio vulgaris Hildenborough

The [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough was isolated from the cytoplasmic membranes and characterized by EPR spectroscopy. It has a total molecular mass of 98.7 kDa (subunits of 66.4 and 32.3 kDa), and contains 1 nickel and 12 Fe atoms per heterodimer. The catalytic activities for hydrogen consumption and production were determined to be 174 and 89 umol H2 min-1 mg -1, respectively. As isolated, under aerobic conditions, this hydrogenase exhibits EPR signals characteristic of the nickel centers in [NiFe] hydrogenases (Ni-A signal at gx,y,z=2.32, 2.23 and ~2.0 and Ni-B signal at gx,y,z=2.33, 2.16 and ~2.0) as well as an intense quasi-isotropic signal centered at g=2.02 due to the oxidized [3Fe-4S] center. The redox profile under hydrogen atmosphere is remarkably similar to that of other [NiFe] hydrogenases. The signals observed for the oxidized state disappear, first being substituted by the Ni-C type signal (gx,y,z=2.19, 2.14, ~2.01), which upon long incubation under hydrogen yields the split Ni-C signal due to interaction with the reduced [4Fe-4S] centers

Repair of Iron Center Proteins—A Different Class of Hemerythrin-like Proteins

Repair of Iron Center proteins (RIC) form a family of di-iron proteins that are widely spread in the microbial world. RICs contain a binuclear nonheme iron site in a four-helix bundle fold, two basic features of hemerythrin-like proteins. In this work, we review the data on microbial RICs including how their genes are regulated and contribute to the survival of pathogenic bacteria. We gathered the currently available biochemical, spectroscopic and structural data on RICs with a particular focus on Escherichia coli RIC (also known as YtfE), which remains the best-studied protein with extensive biochemical characterization. Additionally, we present novel structural data for Escherichia coli YtfE harboring a di-manganese site and the protein’s affinity for this metal. The networking of protein interactions involving YtfE is also described and integrated into the proposed physiological role as an iron donor for reassembling of stress-damaged iron-sulfur centers

How superoxide reductases and flavodiiron proteins combat oxidative stress in anaerobes

Project TIMB3 - Twin to Illuminate Metals in Biology and Biocatalysis Through BiospectroscopyMicrobial anaerobes are exposed in the natural environment and in their hosts, even if transiently, to fluctuating concentrations of oxygen and its derived reactive species, which pose a considerable threat to their anoxygenic lifestyle. To counteract these stressful conditions, they contain a multifaceted array of detoxifying systems that, in conjugation with cellular repairing mechanisms and in close crosstalk with metal homeostasis, allow them to survive in the presence of O 2 and reactive oxygen species. Some of these systems are shared with aerobes, but two families of enzymes emerged more recently that, although not restricted to anaerobes, are predominant in anaerobic microbes. These are the iron-containing superoxide reductases, and the flavodiiron proteins, endowed with O 2 and/or NO reductase activities, which are the subject of this Review. A detailed account of their physicochemical, physiological and molecular mechanisms will be presented, highlighting their unique properties in allowing survival of anaerobes in oxidative stress conditions, and comparing their properties with the most well-known detoxifying systems.preprintpublishe