Genetic basis of the historical iron-accumulating dgl and brz mutants in pea.

The Pisum sativum (pea) mutants degenerate leaves (dgl) and bronze (brz) accumulate large amounts of iron in leaves. First described several decades ago, the two mutants have provided important insights into iron homeostasis in plants but the underlying mutations have remained unknown. Using exome sequencing we identified an in-frame deletion associated with dgl in a BRUTUS homolog. The deletion is absent from wild type and the original parent line. BRUTUS belongs to a small family of E3 ubiquitin ligases acting as negative regulators of iron uptake in plants. The brz mutation was previously mapped to chromosome 4, and superimposing this region to the pea genome sequence uncovered a mutation in OPT3, encoding an oligopeptide transporter with a plant-specific role in metal transport. The causal nature of the mutations was confirmed by additional genetic analyses. Identification of the mutated genes rationalizes many of the previously described phenotypes and provides new insights into shoot-to-root signaling of iron deficiency. Furthermore, the non-lethal mutations in these essential genes suggest new strategies for biofortification of crops with iron.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

Bacterial hemerythrin domain-containing oxygen and redox sensors: Versatile roles for oxygen and redox signaling.

Hemerythrin is an oxygen-binding protein originally found in certain marine invertebrates. Oxygen reversibly binds at its non-heme diiron center, which consists of two oxo-bridged iron atoms bound to a characteristic conserved set of five His residues, one Glu residue, and one Asp residue. It was recently discovered that several bacteria utilize hemerythrin as an oxygen- and redox-sensing domain in responding to changes in cellular oxygen concentration or redox status, and immediately adapt to these environmental changes in order to maintain important physiological processes, including chemotaxis and c-di-GMP synthesis and degradation. This Mini Review focuses on the recent progress made on structural and functional aspects of these emerging bacterial hemerythrin domain-containing oxygen and redox sensors, revealing characteristic features of this family of proteins.

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.

Iron in leaves: chemical forms, signalling, and in-cell distribution.

Iron (Fe) is an essential transition metal. Based on its redox-active nature under biological conditions, various Fe compounds serve as cofactors in redox enzymes. In plants, the photosynthetic machinery has the highest demand for Fe. In consequence, the delivery and incorporation of Fe into cofactors of the photosynthetic apparatus is the focus of Fe metabolism in leaves. Disturbance of foliar Fe homeostasis leads to impaired biosynthesis of chlorophylls and composition of the photosynthetic machinery. Nevertheless, mitochondrial function also has a significant demand for Fe. The proper incorporation of Fe into proteins and cofactors as well as a balanced intracellular Fe status in leaf cells require the ability to sense Fe, but may also rely on indirect signals that report on the physiological processes connected to Fe homeostasis. Although multiple pieces of information have been gained on Fe signalling in roots, the regulation of Fe status in leaves has not yet been clarified in detail. In this review, we give an overview on current knowledge of foliar Fe homeostasis, from the chemical forms to the allocation and sensing of Fe in leaves.

The response of Naegleria gruberi to oxidative stress.

Aerobic organisms require oxygen for respiration but must simultaneously cope with oxidative damages inherently linked with this molecule. Unicellular amoeboflagellates of the genus Naegleria, containing both free-living species and opportunistic parasites, thrive in aerobic environments. However, they are also known to maintain typical features of anaerobic organisms. Here, we describe the mechanisms of oxidative damage mitigation in Naegleria gruberi and focus on the molecular characteristics of three noncanonical proteins interacting with oxygen and its derived reactive forms. We show that this protist expresses hemerythrin, protoglobin, and an aerobic-type rubrerythrin, with spectral properties characteristic of the cofactors they bind. We provide evidence that protoglobin and hemerythrin interact with oxygen in vitro and confirm the mitochondrial localization of rubrerythrin by immunolabeling. Our proteomic analysis and immunoblotting following heavy metal treatment revealed upregulation of hemerythrin, while rotenone treatment resulted in an increase in rubrerythrin protein levels together with a vast upregulation of alternative oxidase. Our study provided new insights into the mechanisms employed by N. gruberi to cope with different types of oxidative stress and allowed us to propose specific roles for three unique and understudied proteins: hemerythrin, protoglobin, and rubrerythrin.

Diversity of structures and functions of oxo-bridged non-heme diiron proteins.

Oxo-bridged diiron proteins are a distinct class of non-heme iron proteins. Their active sites are composed of two irons that are coordinated by amino acid side chains, and a bridging oxygen that interacts with each iron. These proteins are members of the ferritin superfamily and share the structural feature of a four α-helix bundle that provides the residues that coordinate the irons. The different proteins also display a wide range of structures and functions. A prototype of this family is hemerythrin, which functions as an oxygen transporter. Several other hemerythrin-like proteins have been described with a diversity of functions including oxygen and iron sensing, and catalytic activities. Rubrerythrins react with hydrogen peroxide and rubrerythrin-like proteins possess a rubredoxin domain, in addition to the oxo-bridged diiron center. Other redox enzymes with oxo-bridged irons include flavodiiron proteins that act as O2 or NO reductases, ribonucleotide reductase and methane monooxygenase. Ferritins have an oxo-bridged diiron in the ferroxidase center of the protein, which plays a role in the iron storage function of these proteins. There are also bacterial ferritins that exhibit catalytic activities. The structures and functions of this broad class of oxo-bridged diiron proteins are described and compared in this review.

Screening a Spliced Leader-Based Symbiodinium microadriaticum cDNA Library Using the Yeast-Two Hybrid System Reveals a Hemerythrin-Like Protein as a Putative SmicRACK1 Ligand.

The dinoflagellate Symbiodiniaceae family plays a central role in the health of the coral reef ecosystem via the symbiosis that establishes with its inhabiting cnidarians and supports the host metabolism. In the last few decades, coral reefs have been threatened by pollution and rising temperatures which have led to coral loss. These events have raised interest in studying Symbiodiniaceae and their hosts; however, progress in understanding their metabolism, signal transduction pathways, and physiology in general, has been slow because dinoflagellates present peculiar characteristics. We took advantage of one of these peculiarities; namely, the post-transcriptional addition of a Dino Spliced Leader (Dino-SL) to the 5' end of the nuclear mRNAs, and used it to generate cDNA libraries from Symbiodinium microadriaticum. We compared sequences from two Yeast-Two Hybrid System cDNA Libraries, one based on the Dino-SL sequence, and the other based on the SMART technology (Switching Mechanism at 5' end of RNA Transcript) which exploits the template switching function of the reverse transcriptase. Upon comparison of the performance of both libraries, we obtained a significantly higher yield, number and length of sequences, number of transcripts, and better 5' representation from the Dino-SL based library than from the SMART library. In addition, we confirmed that the cDNAs from the Dino-SL library were adequately expressed in the yeast cells used for the Yeast-Two Hybrid System which resulted in successful screening for putative SmicRACK1 ligands, which yielded a putative hemerythrin-like protein.

Copper detoxification machinery of the brain-eating amoeba Naegleria fowleri involves copper-translocating ATPase and the antioxidant system.

Copper is a trace metal that is necessary for all organisms but toxic when present in excess. Different mechanisms to avoid copper toxicity have been reported to date in pathogenic organisms such as Cryptococcus neoformans and Candida albicans. However, little if anything is known about pathogenic protozoans despite their importance in human and veterinary medicine. Naegleria fowleri is a free-living amoeba that occurs naturally in warm fresh water and can cause a rapid and deadly brain infection called primary amoebic meningoencephalitis (PAM). Here, we describe the mechanisms employed by N. fowleri to tolerate high copper concentrations, which include various strategies such as copper efflux mediated by a copper-translocating ATPase and upregulation of the expression of antioxidant enzymes and obscure hemerythrin-like and protoglobin-like proteins. The combination of different mechanisms efficiently protects the cell and ensures its high copper tolerance, which can be advantageous both in the natural environment and in the host. Nevertheless, we demonstrate that copper ionophores are potent antiamoebic agents; thus, copper metabolism may be considered a therapeutic target.

Recent Insights into the Diversity and Evolution of Invertebrate Hemerythrins and Extracellular Globins.

There are three broad groups of oxygen-transport proteins found in the haemolymph (blood) of invertebrates, namely the hemocyanins, the hemerythrins and the globins. Both hemerythrins and extracellular globins are iron-based proteins that are understudied when compared to the copper-containing hemocyanins. Recent evidence suggests that hemerythrins and (giant) extracellular globins (and their linker chains) are more widely distributed than previously thought and may have biological functions beyond oxygen transport and storage. Herein, we review contemporary literature of these often-neglected proteins with respect to their structural configurations on formation and ancestral states.

Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Iron economy in Naegleria gruberi reflects its metabolic flexibility.

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.

Harnessing the evolutionary information on oxygen binding proteins through Support Vector Machines based modules.

OBJECTIVES: The arrival of free oxygen on the globe, aerobic life is becoming possible. However, it has become very clear that the oxygen binding proteins are widespread in the biosphere and are found in all groups of organisms, including prokaryotes, eukaryotes as well as in fungi, plants, and animals. The exponential growth and availability of fresh annotated protein sequences in the databases motivated us to develop an improved version of "Oxypred" for identifying oxygen-binding proteins. RESULTS: In this study, we have proposed a method for identifying oxy-proteins with two different sequence similarity cutoffs 50 and 90%. A different amino acid composition based Support Vector Machines models was developed, including the evolutionary profiles in the form position-specific scoring matrix (PSSM). The fivefold cross-validation techniques were applied to evaluate the prediction performance. Also, we compared with existing methods, which shows nearly 97% recognition, but, our newly developed models were able to recognize almost 99.99 and 100% in both oxy-50 and 90% similarity models respectively. Our result shows that our approaches are faster and achieve a better prediction performance over the existing methods. The web-server Oxypred2 was developed for an alternative method for identifying oxy-proteins with more additional modules including PSSM, available at http://bioinfo.imtech.res.in/servers/muthu/oxypred2/home.html .

Structure, function and evolution of the hemerythrin-like domain superfamily.

Hemerythrin-like proteins have generally been studied for their ability to reversibly bind oxygen through their binuclear nonheme iron centers. However, in recent years, it has become increasingly evident that some members of the hemerythrin-like superfamily also participate in many other biological processes. For instance, the binuclear nonheme iron site of YtfE, a hemerythrin-like protein involved in the repair of iron centers in Escherichia coli, catalyzes the reduction of nitric oxide to nitrous oxide, and the human F-box/LRR-repeat protein 5, which contains a hemerythrin-like domain, is involved in intracellular iron homeostasis. Furthermore, structural data on hemerythrin-like domains from two proteins of unknown function, PF0695 from Pyrococcus furiosus and NMB1532 from Neisseria meningitidis, show that the cation-binding sites, typical of hemerythrin, can be absent or be occupied by metal ions other than iron. To systematically investigate this functional and structural diversity of the hemerythrin-like superfamily, we have collected hemerythrin-like sequences from a database comprising fully sequenced proteomes and generated a cluster map based on their all-against-all pairwise sequence similarity. Our results show that the hemerythrin-like superfamily comprises a large number of protein families which can be classified into three broad groups on the basis of their cation-coordinating residues: (a) signal-transduction and oxygen-carrier hemerythrins (H-HxxxE-HxxxH-HxxxxD); (b) hemerythrin-like (H-HxxxE-H-HxxxE); and, (c) metazoan F-box proteins (H-HExxE-H-HxxxE). Interestingly, all but two hemerythrin-like families exhibit internal sequence and structural symmetry, suggesting that a duplication event may have led to the origin of the hemerythrin domain.

The Rv2633c protein of Mycobacterium tuberculosis is a non-heme di-iron catalase with a possible role in defenses against oxidative stress.

The Rv2633c gene in Mycobacterium tuberculosis is rapidly up-regulated after macrophage infection, suggesting that Rv2633c is involved in M. tuberculosis pathogenesis. However, the activity and role of the Rv2633c protein in host colonization is unknown. Here, we analyzed the Rv2633c protein sequence, which revealed the presence of an HHE cation-binding domain common in hemerythrin-like proteins. Phylogenetic analysis indicated that Rv2633c is a member of a distinct subset of hemerythrin-like proteins exclusive to mycobacteria. The Rv2633c sequence was significantly similar to protein sequences from other pathogenic strains within that subset, suggesting that these proteins are involved in mycobacteria virulence. We expressed and purified the Rv2633c protein in Escherichia coli and found that it contains two iron atoms, but does not behave like a hemerythrin. It migrated as a dimeric protein during size-exclusion chromatography. It was not possible to reduce the protein or observe any evidence for its interaction with O2 However, Rv2633c did exhibit catalase activity with a kcat of 1475 s-1 and Km of 10.1 ± 1.7 mm Cyanide and azide inhibited the catalase activity with Ki values of 3.8 µm and 37.7 µm, respectively. Rv2633c's activity was consistent with a role in defenses against oxidative stress generated during host immune responses after M. tuberculosis infection of macrophages. We note that Rv2633c is the first example of a non-heme di-iron catalase, and conclude that it is a member of a subset of hemerythrin-like proteins exclusive to mycobacteria, with likely roles in protection against host defenses.

In vivo evaluation of hemerythrin-based oxygen carriers: Similarities with hemoglobin-based counterparts.

We have previously proposed the annelid-derived protein, hemerythrin, as a viable replacement for hemoglobin in the synthesis of semi-synthetic oxygen carriers ("blood substitutes"). Here, we report the first in vivo tests for potential hemerythrin-based oxygen carriers (HrBOC), using a battery of experiments involving Wistar rats and previously tested on a series of hemoglobin-based oxygen carrier candidates (HBOC). At the concentrations tested, hemerythrin appears to behave similarly to hemoglobin - including, importantly, immunological effects. The antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages.

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all-Atom Molecular Dynamics Investigation in Water Solution.

In a preliminary exploration of the dummy model for diiron proteins, random-acceleration molecular dynamics (RAMD) revealed that a pure four-helix bundle structure, like hemerythrin, constitutes an efficient cage for dioxygen (O2 ), which can only leave from defined, albeit very broad, gates. However, this well ordered structure does not constitute an archetype on which to compare O2 permeation of other diiron proteins, like the complex of soluble methane monooxygenase hydroxylase with the regulatory protein (sMMOH-MMOB). The reason is that with this complex, unlike hemerythrin, the four helices of the four-helix bundle are heavily bent, and RAMD showed that most traps for O2 lie outside them. It was also observed that, in spite of a nearly identical van der Waals radius for O2 and the natural substrate CH4 , the latter behaves under RAMD as a bulkier molecule than O2 , requiring a higher external force to be brought out of sMMOH-MMOB along trajectories of viable length. All that determined with sMMOH-MMOB multiple gates and multiple pathways to each of them through several binding pockets, for both O2 and CH4 . Of the two equally preferred pathways for O2 , at right angle with one another, one proved to be in accordance with the Xe-atom mapping for sMMOH. In contrast, none of the pathways identified for CH4 proved to be in accordance with such mapping, CH4 looking for more open avenues instead.

Copolymerization of recombinant Phascolopsis gouldii hemerythrin with human serum albumin for use in blood substitutes.

Hemerythrin is an oxygen-carrying protein found in marine invertebrates and may be a promising alternative to hemoglobin for use in blood substitutes, primarily due to its negligible peroxidative toxicity. Previous studies have shown that glutaraldehyde-induced copolymerization of hemoglobin with bovine serum albumin increases the half-life of the active oxy form of hemoglobin (i.e. decreases the auto-oxidation rate). Here, we describe a protocol for glutaraldehyde copolymerization of Hr with human serum albumin and the dioxygen-binding properties of the co-polymerized products. The copolymerization with HSA results in alteration of hemerythrin's dioxygen-binding properties in directions that may be favorable for use in blood substitutes.

A mononuclear non-heme-iron dioxygen-carrying protein?

The ability of mononuclear non-heme iron complexes to function as molecular oxygen transporters is investigated by density functional theory. The factors governing the efficiency of the reversible binding of dioxygen at the active site of the dinuclear non-heme iron enzyme hemerythrin, including antiferromagnetic coupling and the conversion of dioxygen to hydroperoxo by a proton coupled 2-electron transfer mechanism, are revisited and considered as possible tools in mononuclear non-heme complexes. Several mononuclear non-heme model complexes, including active sites of enzymes already known to interact with dioxgenic ligands, are constructed and the molecular oxygen transportation capabilities of these complexes are examined computationally. The high-spin nature of the ground state of these complexes implies an intrinsic kinetic lability of the oxy structures, as also evident from potential energy surface calculations towards iron-dioxygen cleavage. Proton affinities as calibrated with reference compounds showed that these complexes are highly unlikely to undergo protonation to form hydroperoxo-like adducts. Mixed superoxo descriptions of the dissociated dioxygenic ligands in all complexes add to the overall conclusion that these model structures are significantly disadvantaged in any attempt to be employed for molecular oxygen transportation.

Hemerythrin-related antimicrobial peptide, msHemerycin, purified from the body of the Lugworm, Marphysa sanguinea.

A ∼1.7 kDa antimicrobial peptide was purified from the acidified body extract of the Lugworm, Marphysa sanguinea, by preparative acid-urea-polyacrylamide gel electrophoresis and C18 reversed-phase high performance liquid chromatography (HPLC). The identified peptide is composed of 14 amino acids with the N-terminal acetylation. Comparison of the identified amino acid sequences and molecular weight of this peptide with those of other known proteins or peptides revealed that this peptide had high identity to the N-terminus of hemerythrin of marine invertebrates and named the msHemerycin. The full-length hemerythrin cDNA of Lugworm was contained 1027-bp, including a 5'-untranslated region (UTR) of 60-bp, a 3'-UTR of 595-bp, and an open reading frame of 372-bp encoding 123 amino acids including the msHemerycin at the N-terminus. Tissue distribution of the msHemerycin mRNA suggests that it is constitutively expressed as a non-tissue-specific manner, however, a relatively higher expression level was observed in muscle (6.8-fold) and brain (6.3-fold), and the lowest level in digestive gland. The secondary structural prediction and homology modeling studies indicate that the msHemerycin might form an unordered structure and might act via unconventional mechanism. Our results suggest that the msHemerycin might be an innate immune component related to the host defenses in the Lugworm. This is the first report on the antimicrobial function of the peptide derived from the N-terminus of hemerythrin in the Lugworm, Marphysa sanguinea.