Cysteine mutational studies provide insight into a thiol-based redox switch mechanism of metal and DNA binding in FurA from Anabaena sp. PCC 7120

Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. Results: Residue C101 that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C101 by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C101 is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C101 varies with the presence or absence of C104 or C133, suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C101 is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria

Exploring the ability of cyanobacterial ferric uptake regulator (FUR) proteins to increase yeast tolerance to abiotic stresses

Chapter 7. -- 18 pages.Ferric uptake regulator (FUR) proteins are involved in the regulation of genes related to metal homeostasis and the oxidative stress response in most prokaryotes. The cyanobacterium Anabaena sp. PCC 7120 contains three FUR paralogs: FurA, FurB, and FurC. Previous studies demonstrated that overexpression of FurA and FurB increased oxidative stress tolerance in Escherichia coli. Hereby, we analyzed the effects of FurA and FurB expression in Saccharomyces cerevisiae. We tested the tolerance of the yeast strains expressing these cyanobacterial genes against common abiotic stresses: divalent metals, oxidative stress inducers, membrane damaging reagents, and salt. Both strains exhibited either the same or lower growth than the control under most of the conditions tested. However, the FurA-derivative strain showed better growth under oxidative stress induced with low concentrations of hydrogen peroxide and t-butyl hydroperoxide. The FurB-derivative grew better than the control under a broader range of oxidative conditions. To our knowledge, this is the first report demonstrating that FUR proteins may increase oxidative stress tolerance when heterologously expressed in a eukaryotic organism.Peer reviewe

Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120

44 Pags.- 15 Figs.- 2 Tabls. The definitive version is available at: http://www.biochemj.org/FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB–DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB–DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.This work has been supported by grants B18 from Gobierno de Aragón, BFU2012- 31458/FEDER & BFU2016-77671-P/FEDER from MINECO and UZ2016-BIO-02 from University of Zaragoza. VCS was recipient of a fellowship from Gobierno de Aragón.Peer reviewe

Cysteine Mutational Studies Provide Insight into a Thiol-Based Redox Switch Mechanism of Metal and DNA Binding in FurA from Anabaena

Aims: The ferric uptake regulator (Fur) is the main transcriptional regulator of genes involved in iron homeostasis in most prokaryotes. FurA from Anabaena sp. PCC 7120 contains five cysteine residues, four of them arranged in two redox-active CXXC motifs. The protein needs not only metal but also reducing conditions to remain fully active in vitro. Through a mutational study of the cysteine residues present in FurA, we have investigated their involvement in metal and DNA binding. Results: Residue C(101) that belongs to a conserved CXXC motif plays an essential role in both metal and DNA binding activities in vitro. Substitution of C(101) by serine impairs DNA and metal binding abilities of FurA. Isothermal titration calorimetry measurements show that the redox state of C(101) is responsible for the protein ability to coordinate the metal corepressor. Moreover, the redox state of C(101) varies with the presence or absence of C(104) or C(133), suggesting that the environments of these cysteines are mutually interdependent. Innovation: We propose that C(101) is part of a thiol/disulfide redox switch that determines FurA ability to bind the metal corepressor. Conclusion: This mechanism supports a novel feature of a Fur protein that emerges as a regulator, which connects the response to changes in the intracellular redox state and iron management in cyanobacteria. Antioxid. Redox Signal. 24, 173–185

Phytoglobins in the nuclei, cytoplasm, and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Rubio MC, Calvo-Begueria L, Diaz-Mendoza M, et al. Phytoglobins in the nuclei, cytoplasm, and chloroplasts modulate nitric oxide signaling and interact with abscisic acid. The Plant journal : for cell and molecular biology. 2019;100(1):38-54.Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of nonsymbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine NO scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and ABA responses. We found that Glbs occur in the nuclei, chloroplasts, and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A), and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly up-regulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved

Subcellular localization and nitric oxide-scavenging activity of plant hemoglobins

1 Pag.Symbioc hemoglobins provide O2 to N2-fixing bacteria within legume nodules. However, the funcons of nonsymbioc hemoglobins or phytoglobins (Glbs) are less defined. Three Glb classes can be disnguished based on phylogenec and biochemical analyses and may coexist in plant ssues: class 1 Glbs have extreme O2 affinity and are induced by hypoxia; class 2 Glbs have moderate O2 affinity and are precursors of leghemoglobins; and class 3 have low O2 affinity and high sequence homology with bacterial truncated hemoglobins. Immunogold labeling combined with confocal microscopy of Glbs tagged with GFP at the C-terminus was used to determine the subcellular localizaons of Glbs in the model plants Arabidopsis and Lotus japonicus. To this end, we used overexpressing and knockout or silenced lines of Arabidopsis, performed quantave immunolabeling, and monitored the GFP-tagged proteins in leaf cells and protoplasts.This work was funded by grant AGL2017-85775-R from the Spanish Ministry of Economy, Industry and Competitiveness/European Regional Development Fund.Peer reviewe