Ferric Reductase Research Articles

Carbonate-Induced Chemical Reductants Are Responsible for Iron Acquisition in Strategy I Wild Herbaceous Plants Native to Calcareous Grasslands.

Significant progress has been made in understanding Strategy I iron (Fe) acquisition using crop/model plants under controlled conditions in laboratories. However, plant species native to calcareous soils may have evolved unique strategies for adaptation to high carbonate/pH-induced Fe deficiency. Until now, little information is available on the Fe acquisition mechanisms in these plants. Here, we explored the Fe acquisition mechanisms in wild dicot species native to calcareous grasslands, by monitoring the Fe nutrition-related rhizosphere processes in field and greenhouse conditions. Most of these wild species displayed comparable shoot Fe concentration to those of crops, and some dicots actually accumulated very high shoot Fe. However, these species did not exhibit ferric reductase oxidase (FRO)-dependent Strategy I responses to Fe deficiency, including visual rhizosphere acidification and increased Fe3+ reduction. In contrast, chemical reductants exuded by roots of dicots were responsible for Fe3+ reduction in these wild plants. These features were not observed in the FRO-dependent Strategy I crop plant cucumber. Neither leaf chlorophyll nor shoot/root Fe was depressed by 10% CaCO3 application in all the examined wild species. Furthermore, their root exudation was significantly activated by CaCO3, leading to an increased Fe3+ reduction. We show that chemical reductant-mediated Fe3+ reduction occurs preferentially in these wild dicots and that these mechanisms are not sensitive to high soil carbonate/pH. Our findings support that Fe acquisition in Strategy I wild plants native to calcareous soils is substantially different from the enzyme-dependent system of Strategy I plants.

MIR164b represses iron uptake by regulating the NAC domain transcription factor5-Nuclear Factor Y, Subunit A8 module in Arabidopsis.

Recent findings have revealed the important roles of microRNAs (miRNAs) in the secondary responses to oxidative damage caused by iron (Fe) excess. However, the functional importance of miRNAs in plant responses to Fe deficiency remains to be explored. Here, we show that the expression level of miR164 in Arabidopsis (Arabidopsis thaliana) roots was repressed by Fe deficiency. Primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2) were higher in the mir164b mutant than in the wild-type (WT) under Fe-deficient conditions. Analysis of the Fe concentrations and ferric reductase activities in the roots of miR164 knockdown transgenic plants showed that members of the miR164 family had different functions in Fe-deficiency responses. Promoter::GUS analysis showed that NAM/ATAF/CUC (NAC) domain transcription factor5 (NAC5) is regulated at both transcriptional and posttranscriptional levels under Fe-deficient conditions. Transgenic Arabidopsis plants overexpressing NAC5 were more tolerant of Fe deficiency than the WT. NAC5 has transactivation activity and directly transactivates the expression of Nuclear Factor Y, Subunit A8 (NFYA8), as demonstrated by chromatin immunoprecipitation followed by quantitative polymerase chain reaction, electrophoretic mobility shift assay (EMSA), and dual-luciferase reporter assay. Like overexpression of NAC5, overexpression of NFYA8 increases primary root length, lateral root number, ferric reductase activity, and mRNA abundance of IRT1 and FRO2 under Fe-deficient conditions. Thus, MIR164b is important for Fe-deficiency responses by its regulation of the NAC5-NFYA8 module.

Identification and analysis of iron transporters from the fission yeast Schizosaccharomyces pombe.

Iron is an essential trace metal ion required for all living organisms, and is taken up by iron transporters. Here, we identified and characterized three-candidate high-affinity (Fio1, Frp1 and Frp2) and two-candidate low-affinity iron transporters (Fet4 and Pdt1) from the fission yeast Schizosaccharomyces pombe. Protein sequence analyses revealed that Fio1 is a multicopper oxidase that contains three cupredoxin domains with eleven candidate iron-binding ligands, whereas Frp1 harbors a ferric reductase domain with three-candidate heme-binding ligands. Protein sequence analyses also revealed that Fet4 and Pdt1 are integral membrane proteins with 10 and 11 transmembrane regions, respectively. Deletion of fio1 and, to a lesser extent, frp1 impaired growth under iron-depleted conditions, whereas deletion of frp1 and, to a lesser extent, frp2 inhibited growth under iron-replete conditions. Deletion of fet4 and pdt1 did not affect the growth of cells under iron-depleted and iron-replete conditions. Deletion of fio1 or frp1 also increased the sensitivity of cells to other transition metals. The copper sensitivity of Δfio1 cells could be rescued by iron, suggesting that the addition of iron mightdecrease the uptake of potentially toxic copper in Δfio1 cells. The copper sensitivity of Δfio1 cells could also be rescued by deletion of frp1, suggesting that Fio1 and Frp1 may function together in iron and copper uptakes in S. pombe. Our results revealed that iron and copper uptake systems may be partially overlapped in S. pombe.

Screening for genes involved in cellulase regulation by expression under the control of a novel constitutive promoter in Trichoderma reesei

• We characterized the upstream region of cfe1 as promising promoter without upstream open reading frames (uORFs) or introns. • The cfe1 promoter enabled up to 400 fold overexpression. • We analyzed transcriptomic data with support from machine learning to select target genes. • We report three genes affecting cellulase regulation. The filamentous ascomycete Trichoderma reesei is a biotechnological workhorse used in the production of homologous and heterologous proteins for diverse applications including biofuel production, textile finishing and food additives. This fungus uses a complex adaptation machinery to regulate its cellulases in response to environmental conditions. Detailed understanding of this regulation allows for improvement of enzyme production using the strong enzyme gene promotors. Here, we selected six genes with characteristic transcript levels associated with cellulase production to be tested for their enzyme regulatory function. Machine learning for inference of a gene regulatory network (GRN) was applied to support the association of these genes with cellulase induction. Additionally, we screened available transcriptomic data for genes with strong constitutive transcript levels and selected the promoter of the gene cfe1 , whose transcript levels were above those of tef1 and cDNA1 and near those of cbh1 , for gene overexpression testing. Using this promoter, we explored the relevance to cellulose degradation efficiency of three transporters, two ferric reductases and one gene of unknown function, which were overexpressed in T. reesei grown on cellulose. This promoter enabled up to 400 fold overexpression and yielded transcript levels above those of tef1 or cDNA1. We provide evidence of effects of a ferric reductase, an ammonium permease and a gene of unknown function on the regulatory machinery of cellulase expression in T. reesei . In summary we identified the cfe1 promoter, a novel constitutive promoter with promising efficiency, as well as three genes relevant to cellulase regulation in T. reesei .

Several Yeast Species Induce Iron Deficiency Responses in Cucumber Plants (Cucumis sativus L.).

Iron (Fe) deficiency is a first-order agronomic problem that causes a significant decrease in crop yield and quality. Paradoxically, Fe is very abundant in most soils, mainly in its oxidized form, but is poorly soluble and with low availability for plants. In order to alleviate this situation, plants develop different morphological and physiological Fe-deficiency responses, mainly in their roots, to facilitate Fe mobilization and acquisition. Even so, Fe fertilizers, mainly Fe chelates, are widely used in modern agriculture, causing environmental problems and increasing the costs of production, due to the high prices of these products. One of the most sustainable and promising alternatives to the use of agrochemicals is the better management of the rhizosphere and the beneficial microbial communities presented there. The main objective of this research has been to evaluate the ability of several yeast species, such as Debaryomyces hansenii, Saccharomyces cerevisiae and Hansenula polymorpha, to induce Fe-deficiency responses in cucumber plants. To date, there are no studies on the roles played by yeasts on the Fe nutrition of plants. Experiments were carried out with cucumber plants grown in a hydroponic growth system. The effects of the three yeast species on some of the most important Fe-deficiency responses developed by dicot (Strategy I) plants, such as enhanced ferric reductase activity and Fe2+ transport, acidification of the rhizosphere, and proliferation of subapical root hairs, were evaluated. The results obtained show the inductive character of the three yeast species, mainly of Debaryomyces hansenii and Hansenula polymorpha, on the Fe-deficiency responses evaluated in this study. This opens a promising line of study on the use of these microorganisms as Fe biofertilizers in a more sustainable and environmentally friendly agriculture.

Membrane-Bound Flavocytochrome MsrQ Is a Substrate of the Flavin Reductase Fre in Escherichia coli.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

Insilico analysis of Arabidopsis ferric reductase oxidases (FRO) proteins associated with iron homeostasis

The ferric reduction oxidase (FRO) gene family is involved in various biological processes of plants and plays an essential role in metal homeostasis, tolerance, and signaling networks in response to several abiotic stresses. Our study describes the structural, functional characterization, and evolutionary relationships of eight Arabidopsis FRO proteins. The studies predicted the subcellular localization of FRO proteins to the plasma membrane, mitochondria, and chloroplast organelles. The structural analysis revealed localization of proteins onto the first and fifth chromosomes having 8-9exons and 8-10 transmembrane helices. The protein features of FRO proteins revealed 699-747 amino acids having 79600.02-84126.3 (Da) molecular weight. The six highly conserved protein motifs were predicted with 45-50 amino acids long representing ferric chelate reductase family domains. The phylogeny tree constructed using Clustal W divided the FRO proteins into two clusters and the interactome network revealed the co-expression of COPT1, NRAMP1, NRAMP3, NRAMP4, FRD3, OPT3, IRT1, IRT2, ZIF1, PYE proteins along with the seven FRO proteins.

Ferric reductase activity and production of phytosiderophore in fresh roots of rice varieties grown under iron deficiency conditions and chlorosis degrees in leaves

Bu çalışmanın amacı, demir noksanlığı şartlarında yetiştirilen çeltik çeşitlerinin taze köklerinde fitosiderofor üretimi ve ferrikredüktaz aktivitesi ile yapraklarında kloroz derecelerini belirlemektir. Fitosiderofor salgısı üretimi ve ferrik redüktaz aktivitesibelirlemek amacıyla çeltik çeşitleri 10 gün süreyle demirsiz besin çözeltisi uygulayarak kum kültüründe yetiştirilmiştir. Ayrıca,kloroz derecesinin ve kloroza tolerans indeks değerinin belirlenmesi amacıyla 5×2 faktöriyel deneme deseninde çalışmayürütülmüş olup, bu çalışmada 5 farklı çeltik çeşidine 0 ve 45 µM Fe dozlarında Fe-EDDHA içeren besin çözeltisi uygulanmıştır. Tazekökte üretilen fitosiderofor miktarı en yüksek olan çeşidin Hamzadere çeltik çeşidi olduğu görülmüştür. Köklerde fitosideroforüretimleri bakımından geri kalan diğer çeşitler arası fark istatistiksel olarak önemsiz olup, birbirlerine yakın bulunmuştur. Demirnoksanlığı şartlarında yetiştirilen çeltik çeşitleri arasında taze köklerde ferrik redüktaz aktivitesi en yüksek olan çeşidin Ronaldoçeltik çeşidi olduğu; buna karşın, en düşük çeşidin ise Biga incisi çeltik çeşidi olduğu görülmüştür. Hamzadere ve Ronaldo çeltikçeşitlerinin yapraklarında gözlenen kloroz derecesi şiddetli olup, bu çeşitlerin demir noksanlığına tolerans indeks değerlerisırasıyla %41.24 ve %41.52 olarak hesaplanmıştır. Bu sonuçlara göre, araştırmada incelenen çeltik çeşitleri arasında demirnoksanlığına en hassas çeşidin Hamzadere çeltik çeşidi olduğu belirlenmiştir.

Supraoptimal Iron Nutrition of Brassica napus Plants Suppresses the Iron Uptake of Chloroplasts by Down-Regulating Chloroplast Ferric Chelate Reductase.

Iron (Fe) is an essential micronutrient for plants. Due to the requirement for Fe of the photosynthetic apparatus, the majority of shoot Fe content is localised in the chloroplasts of mesophyll cells. The reduction-based mechanism has prime importance in the Fe uptake of chloroplasts operated by Ferric Reductase Oxidase 7 (FRO7) in the inner chloroplast envelope membrane. Orthologue of Arabidopsis thaliana FRO7 was identified in the Brassica napus genome. GFP-tagged construct of BnFRO7 showed integration to the chloroplast. The time-scale expression pattern of BnFRO7 was studied under three different conditions: deficient, optimal, and supraoptimal Fe nutrition in both leaves developed before and during the treatments. Although Fe deficiency has not increased BnFRO7 expression, the slight overload in the Fe nutrition of the plants induced significant alterations in both the pattern and extent of its expression leading to the transcript level suppression. The Fe uptake of isolated chloroplasts decreased under both Fe deficiency and supraoptimal Fe nutrition. Since the enzymatic characteristics of the ferric chelate reductase (FCR) activity of purified chloroplast inner envelope membranes showed a significant loss for the substrate affinity with an unchanged saturation rate, protein level regulation mechanisms are suggested to be also involved in the suppression of the reduction-based Fe uptake of chloroplasts together with the saturation of the requirement for Fe.

Iron Uptake in Drosophila Sg4 Cells Does Not Require Endocytosis

Iron is essential for all organisms including insects; however, at high levels it is toxic. Therefore, the transport and uptake of iron must be well-regulated, and in insects this process is poorly understood. In mammals, the most widely known mechanism of iron uptake is receptor-mediated endocytic uptake of Fe3+-transferrin. In the acidified endosome, iron is released from transferrin, reduced by a ferric reductase to Fe2+, and transported across the endosome membrane through divalent metal transporter 1 (DMT1). The goal of this project was to determine whether iron uptake by cultured Drosophila Sg4 cells (an isolate of embryonically derived S2 cells) requires endocytosis. To test this hypothesis, we blocked endocytosis and then measured cellular iron content. We performed RNAi-mediated knockdown of rab5, which is expected to block both clathrin-mediated and caveolar endocytic pathways, and we also treated cells with one of three chemical inhibitors: 200 nM bafilomycin-A1, which inhibits clathrin-mediated endocytosis in S2 cells, 5 mM methyl-beta-cyclodextrin, which blocks caveolae-mediated endocytosis, and 1 µM cytochalasin D, which blocks phagocytosis and macropinocytosis. The rab5 RNAi and chemical treatments caused a slight decrease in cell growth and viability, but they did not decrease iron content. The results of these experiments suggest that endocytosis is not required for iron uptake by Sg4 cells. To test our hypothesis using a different strategy, we are evaluating the role in iron uptake of an insect homolog of DMT1, Malvolio (Mvl). Based on previous studies, Mvl appears to be a divalent metal transporter that is present mainly in endosome membranes. We used RT-PCR to verify that Sg4 cells express Mvl, and we are currently performing RNAi to assess the effect of Mvl knockdown on cellular iron content. Given that blocking endocytosis had no effect on cellular iron content, if Mvl participates in endocytic uptake of protein-bound iron, we expect that knockdown of Mvl will also have no effect on cellular iron content.

Expression Patterns in Reductive Iron Assimilation and Functional Consequences during Phagocytosis of Lichtheimia corymbifera, an Emerging Cause of Mucormycosis.

Iron is an essential micronutrient for most organisms and fungi are no exception. Iron uptake by fungi is facilitated by receptor-mediated internalization of siderophores, heme and reductive iron assimilation (RIA). The RIA employs three protein groups: (i) the ferric reductases (Fre5 proteins), (ii) the multicopper ferroxidases (Fet3) and (iii) the high-affinity iron permeases (Ftr1). Phenotyping under different iron concentrations revealed detrimental effects on spore swelling and hyphal formation under iron depletion, but yeast-like morphology under iron excess. Since access to iron is limited during pathogenesis, pathogens are placed under stress due to nutrient limitations. To combat this, gene duplication and differential gene expression of key iron uptake genes are utilized to acquire iron against the deleterious effects of iron depletion. In the genome of the human pathogenic fungus L. corymbifera, three, four and three copies were identified for FRE5, FTR1 and FET3 genes, respectively. As in other fungi, FET3 and FTR1 are syntenic and co-expressed in L. corymbifera. Expression of FRE5, FTR1 and FET3 genes is highly up-regulated during iron limitation (Fe-), but lower during iron excess (Fe+). Fe- dependent upregulation of gene expression takes place in LcFRE5 II and III, LcFTR1 I and II, as well as LcFET3 I and II suggesting a functional role in pathogenesis. The syntenic LcFTR1 I–LcFET3 I gene pair is co-expressed during germination, whereas LcFTR1 II- LcFET3 II is co-expressed during hyphal proliferation. LcFTR1 I, II and IV were overexpressed in Saccharomyces cerevisiae to represent high and moderate expression of intracellular transport of Fe3+, respectively. Challenge of macrophages with the yeast mutants revealed no obvious role for LcFTR1 I, but possible functions of LcFTR1 II and IVs in recognition by macrophages. RIA expression pattern was used for a new model of interaction between L. corymbifera and macrophages.

Influence of Ethylene Signaling in the Crosstalk Between Fe, S, and P Deficiency Responses in Arabidopsis thaliana.

To cope with P, S, or Fe deficiency, dicot plants, like Arabidopsis, develop several responses (mainly in their roots) aimed to facilitate the mobilization and uptake of the deficient nutrient. Within these responses are the modification of root morphology, an increased number of transporters, augmented synthesis-release of nutrient solubilizing compounds and the enhancement of some enzymatic activities, like ferric reductase activity (FRA) or phosphatase activity (PA). Once a nutrient has been acquired in enough quantity, these responses should be switched off to minimize energy costs and toxicity. This implies that they are tightly regulated. Although the responses to each deficiency are induced in a rather specific manner, crosstalk between them is frequent and in such a way that P, S, or Fe deficiency can induce responses related to the other two nutrients. The regulation of the responses is not totally known but some hormones and signaling substances have been involved, either as activators [ethylene (ET), auxin, nitric oxide (NO)], or repressors [cytokinins (CKs)]. The plant hormone ET is involved in the regulation of responses to P, S, or Fe deficiency, and this could partly explain the crosstalk between them. In spite of these crosslinks, it can be hypothesized that, to confer the maximum specificity to the responses of each deficiency, ET should act in conjunction with other signals and/or through different transduction pathways. To study this latter possibility, several responses to P, S, or Fe deficiency have been studied in the Arabidopis wild-type cultivar (WT) Columbia and in some of its ethylene signaling mutants (ctr1, ein2-1, ein3eil1) subjected to the three deficiencies. Results show that key elements of the ET transduction pathway, like CTR1, EIN2, and EIN3/EIL1, can play a role in the crosstalk among nutrient deficiency responses.

Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

Iron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins.Graphic abstract

Fungal cross-talk: an integrated approach to study distance communication

Despite the interest on fungi as eukaryotic model systems, the molecular mechanisms regulating the fungal non-self-recognition at a distance have not been studied so far. This paper investigates the molecular mechanisms regulating the cross-talk at a distance between two filamentous fungi, Trichoderma gamsii and Fusarium graminearum which establish a mycoparasitic interaction where T. gamsii and F. graminearum play the roles of mycoparasite and prey, respectively. In the present work, we use an integrated approach involving dual culture tests, comparative genomics and transcriptomics to investigate the fungal interaction before contact (‘sensing phase’).Dual culture tests demonstrate that growth rate of F. graminearum accelerates in presence of T. gamsii at the sensing phase. T. gamsii up-regulates the expression of a ferric reductase involved in iron acquisition, while F. graminearum up-regulates the expression of genes coding for transmembrane transporters and killer toxins. At the same time, T. gamsii decreases the level of extracellular interaction by down-regulating genes coding for hydrolytic enzymes acting on fungal cell wall (chitinases).Given the importance of fungi as eukaryotic model systems and the ever-increasing genomic resources available, the integrated approach hereby presented can be applied to other interactions to deepen the knowledge on fungal communication at a distance.

Functional Assembly of Caenorhabditis elegans Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity.

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O2 in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a F420H2:NADP+-like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome b subunits of the mitochondrial complex III and photosynthetic cytochrome b6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes b act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome b domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome b enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.

A ferric reductase of Trypanosoma cruzi (TcFR) is involved in iron metabolism in the parasite

Trypanosoma cruzi is a parasitic protozoan that infects various species of domestic and wild animals, triatomine bugs and humans. It is the etiological agent of American trypanosomiasis, also known as Chagas Disease, which affects about 17 million people in Latin America and is emerging elsewhere in the world. Iron (Fe) is a crucial micronutrient for almost all cells, acting as a cofactor for several metabolic enzymes. T. cruzi has a high requirement for Fe, using heminic and non-heminic Fe for growth and differentiation. Fe occurs in the oxidized (Fe3+) form in aerobic environments and needs to be reduced to Fe2+ before it enters cells. Fe-reductase, located in the plasma membranes of some organisms, catalyzes the Fe3+⇒ Fe2+ conversion. In the present study we found an amino acid sequence in silico that allowed us to identify a novel 35 kDa protein in T. cruzi with two transmembrane domains in the C-terminal region containing His residues that are conserved in the Ferric Reductase Domain Superfamily and are required for catalyzing Fe3+ reduction. Accordingly, we named this protein TcFR. Intact epimastigotes from the T. cruzi DM28c strain reduced the artificial Fe3+-containing substrate potassium ferricyanide in a cell density-dependent manner, following Michaelis-Menten kinetics. The TcFR activity was more than eightfold higher in a plasma membrane-enriched fraction than in whole homogenates, and this increase was consistent with the intensity of the 35 kDa band on Western blotting images obtained using anti-NOX5 raised against the human antigen. Immunofluorescence experiments demonstrated TcFR on the parasite surface. That TcFR is part of a catalytic complex allowing T. cruzi to take up Fe from the medium was confirmed by experiments in which DM28c was assayed after culturing in Fe-depleted medium: (i) proliferation during the stationary growth phase was five times slower; (ii) the relative expression of TcFR (qPCR) was 50% greater; (iii) intact cells had 120% higher Fe-reductase activity. This ensemble of results indicates that TcFR is a conserved enzyme in T. cruzi, and its catalytic properties are modulated in order to respond to external Fe fluctuations.

Regulation of Zn uptake and redox status confers Zn deficiency tolerance in tomato

Zinc (Zn) is a well-known nutritional problem affecting plant growth and development. Due to Zn deficiency in the soil, tomato plants exhibit yield loss. This study was conducted on two contrasting tomato genotypes (Marglobe and Ratan) subjected to Zn deficiency to unveil the mechanistic foundation of Zn-deficiency tolerance in this vegetable. We found that Zn deficiency adversely affected plant biomass and photosynthesis parameters (chlorophyll score, quantum efficiency of photosystem, photosynthesis performance index) in Zn-deficiency sensitive Ratan. Furthermore, Zn-shortage showed increased electrolyte leakage, cell death %, and hydrogen peroxide concentration in Ratan (Zn-deficiency sensitive), which was not evident in Marglobe (Zn-deficiency tolerant). The metal analysis showed no significant decrease of Zn and Fe in Marglobe; however, Ratan exhibited a significant decline in Zn and Fe in both root and shoot subjected to Zn shortage. It suggests that maintaining Zn status is one of the adaptive mechanisms that Marglobe exhibits subjected to Zn shortage. This was further supported by the constitutive expression of SlIRT1 (Fe-regulated transporter) along with increased ferric reductase activity in addition to the upregulation of ZIP (Irt-like proteins) transporters (SlZIPL and SlZIP2) in roots of Marglobe. It also implies that Zn acquisition was tightly regulated and may also be dependent on Fe homeostasis in Marglobe. Together with Zn status, the elevated CAT (catalase) and SOD (superoxide dismutase) activities were actively associated with Zn-deficiency induced ROS (reactive oxygen species) regulation in Marglobe. Finally, auxin inhibitor resulted in a marked interruption in photosynthesis, growth, and ROS scavenging, indicating its crucial involvement in the adaptive mechanisms that Marglobe showed under Zn deficiency. This study uncovers the mechanistic strategies that confer tolerance to Zn shortage in tomato plants.

Physiological Responses to Fe Deficiency in Split-Root Tomato Plants: Possible Roles of Auxin and Ethylene?

Iron (Fe) bioavailability in soils is often limited and can be further exacerbated by a non- homogeneous distribution in the soil profile, which has been demonstrated to vary both in space and time. Consequently, plants respond with morphological and physiological modifications at the root level involving a complex local and systemic signaling machinery. The present work unravels the role of two phytohormones (i.e., ethylene and auxin) and their integrated signaling in plant response to Fe deficiency. Inhibitors of auxin polar transport and of ethylene biosynthesis (N-1-naphthylphthalamic acid - NPA and aminoethoxyvinylglycine - AVG, respectively) were applied on tomato (Solanum lycopersicum L.) plants grown by the split-root technique, which allows to simulate condition of Fe heterogeneous distribution. Results showed that plants, exposed to an uneven Fe supply, triggered a complex auxin-ethylene signaling. A systemic action of auxin on FERRIC REDUCTASE OXIDASE 1 (SlFRO1) expression was revealed, while ethylene signaling was effective both locally and systemically. In addition, the investigation of Fe concentration in tissues showed that when leaves overcame Fe deficiency a Fe “steady state” was maintained. Therefore, physiological adaptation to this heterogeneous Fe supply could be mediated by the integration of the complex signaling pathways prompted by both auxin and ethylene activities.

Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1)

Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2–STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer.