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Many bacteria have hemerythrin (Hr) proteins that bind O 2 , including Pseudomonas aeruginosa , in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C -terminus relative to a well-characterized Hr from Methylococcus capsulatus , and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C -terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a Mhr W143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. Mhr L112Y , a variant that has a much longer-lived O 2 -bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both Mhr W143* and Mhr L112Y had secondary structures, stabilities, and O 2 -binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C -terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O 2 -binding.

Due to its ability to reversibly bind O 2 , 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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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., (© The Author(s) 2022. Published by Oxford University Press.)

The hemerythrin-like protein from Mycobacterium kansasii (Mka HLP) is a member of a distinct class of oxo-bridged diiron proteins that are found only in mycobacterial species that cause respiratory disorders in humans. Because it had been shown to exhibit weak catalase activity and a change in absorbance on exposure to nitric oxide (NO), the reactivity of Mka HLP toward NO was examined under a variety of conditions. Under anaerobic conditions, we found that NO was converted to nitrite (NO 2 - ) via an intermediate, which absorbed light at 520 nm. Under aerobic conditions NO was converted to nitrate (NO 3 - ). In each of these two cases, the maximum amount of nitrite or nitrate formed was at best stoichiometric with the concentration of Mka HLP. When incubated with NO and H 2 O 2 , we observed NO peroxidase activity yielding nitrite and water as reaction products. Steady-state kinetic analysis of NO consumption during this reaction yielded a K m for NO of 0.44 μM and a k cat /K m of 2.3 × 10 5  M -1 s -1 . This high affinity for NO is consistent with a physiological role for Mka HLP in deterring nitrosative stress. This is the first example of a peroxidase that uses an oxo-bridged diiron center and a rare example of a peroxidase utilizing NO as an electron donor and cosubstrate. This activity provides a mechanism by which the infectious Mycobacterium may combat against the cocktail of NO and superoxide (O 2 •- ) generated by macrophages to defend against bacteria, as well as to produce NO 2 - to adapt to hypoxic conditions., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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 O 2 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O 2 ) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O 2 ) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O 2 detoxification and/or protection. Tolerance of C. difficile to low O 2 tensions requires the presence of the alternative sigma factor, σ B , involved in the general stress response. Among the genes positively controlled by σ B , four encode proteins likely involved in O 2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O 2 - and H 2 O 2 -reductase activities in vitro , while purified FdpA mainly acts as an O 2 -reductase. The growth of a fdpA mutant is affected at 0.4% O 2 , while inactivation of both revRbrs leads to a growth defect above 0.1% O 2 O 2 -reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O 2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O 2 tensions such as those encountered in the colon. IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O 2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O 2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O 2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O 2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O 2 tolerance in C. difficile These enzymes are responsible for the reduction of O 2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O 2 -reductases are crucial in the ability of C. difficile to tolerate O 2 and survive to O 2 concentrations encountered in the gastrointestinal tract., (Copyright © 2020 Kint et al.)

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 K d 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., Competing Interests: The authors declare no competing interest.

Numerous hemerythrins, di-iron proteins, have been identified in prokaryote genomes, but in most cases their function remains elusive. Bacterial hemerythrin homologs (bacteriohemerythrins, Bhrs) may contribute to various cellular functions, including oxygen sensing, metal binding and antibiotic resistance. It has been proposed that methanotrophic Bhrs support methane oxidation by supplying oxygen to a core enzyme, particulate methane monooxygenase. In this study, the consequences of the overexpression or deletion of the Bhr gene (bhr) in Methylomicrobiam alcaliphillum 20ZR were investigated. We found that the bhrknockout (20ZRΔbhr) displays growth kinetics and methane consumption rates similar to wild type. However, the 20ZRΔbhr accumulates elevated concentrations of acetate at aerobic conditions, indicating slowed respiration. The methanotrophic strain overproducing Bhr shows increased oxygen consumption and reduced carbon-conversion efficiency, while its methane consumption rates remain unchanged. These results suggest that the methanotrophic Bhr proteins specifically contribute to oxygen-dependent respiration, while they have minimal, if any, input of oxygen for the methane oxidation machinery., (© FEMS 2020.)

Variable-temperature electrospray ionization combined with ion mobility spectrometry (IMS) and mass spectrometry (MS) techniques are used to monitor structural transitions of the protein myohemerythrin from peanut worm in aqueous ammonium acetate solutions from ∼15 to 92 °C. At physiological temperatures, myohemerythrin favors a four-helix bundle motif and has a diiron oxo cofactor that binds oxygen. As the solution temperature is increased from ∼15 to 35 °C, some bound oxygen dissociates; at ∼66 °C, the cofactor dissociates to produce populations of both folded and unfolded apoprotein. At higher temperatures (∼85 °C and above), the IMS-MS spectrum indicates that the folded apoprotein dominates, and provides evidence for stabilization of the structure by formation of a non-native disulfide bond. In total, we find evidence for 18 unique forms of myohemerythrin as well as information about the structures and stabilities of these states. The high-fidelity of IMS-MS techniques provides a means of examining the stabilities of individual components of complex mixtures that are inaccessible by traditional calorimetric and spectroscopic methods.

Thermococcus onnurineus NA1, an obligate anaerobic hyperthermophilic archaeon, showed variable oxygen (O 2 ) sensitivity depending on the types of substrate employed as an energy source. Unexpectedly, the culture with yeast extract as a sole energy source showed enhanced growth by 2-fold in the presence of O 2 . Genome-wide transcriptome analysis revealed the upregulation of several antioxidant-related genes encoding thioredoxin peroxidase (TON_0862), rubrerythrin (TON_0864), rubrerythrin-related protein (TON_0873), NAD(P)H rubredoxin oxidoreductase (TON_0865), or thioredoxin reductase (TON_1603), which can couple the detoxification of reactive oxygen species with the regeneration of NAD(P) + from NAD(P)H. We present a plausible mechanism by which O 2 serves to maintain the intracellular redox balance. This study demonstrates an unusual strategy of an obligate anaerobe underlying O 2 -mediated growth enhancement despite not having heme-based or cytochrome-type proteins.

" Candidatus Methanoperedens nitroreducens" is an archaeon that couples the anaerobic oxidation of methane to nitrate reduction. In natural and man-made ecosystems, this archaeon is often found at oxic-anoxic interfaces where nitrate, the product of aerobic nitrification, cooccurs with methane produced by methanogens. As such, populations of " Ca Methanoperedens nitroreducens" could be prone to regular oxygen exposure. Here, we investigated the effect of 5% (vol/vol) oxygen exposure in batch activity assays on a " Ca Methanoperedens nitroreducens" culture, enriched from an Italian paddy field. Metagenome sequencing of the DNA extracted from the enrichment culture revealed that 83% of 16S rRNA gene reads were assigned to a novel strain, " Candidatus Methanoperedens nitroreducens Verserenetto." RNA was extracted, and metatranscriptome sequencing upon oxygen exposure revealed that the active community changed, most notably in the appearance of aerobic methanotrophs. The gene expression of " Ca Methanoperedens nitroreducens" revealed that the key genes encoding enzymes of the methane oxidation and nitrate reduction pathways were downregulated. In contrast to this, we identified upregulation of glutaredoxin, thioredoxin family/like proteins, rubrerythrins, peroxiredoxins, peroxidase, alkyl hydroperoxidase, type A flavoproteins, FeS cluster assembly protein, and cysteine desulfurases, indicating the genomic potential of " Ca Methanoperedens nitroreducens Verserenetto" to counteract the oxidative damage and adapt in environments where they might be exposed to regular oxygen intrusion. IMPORTANCE " Candidatus Methanoperedens nitroreducens" is an anaerobic archaeon which couples the reduction of nitrate to the oxidation of methane. This microorganism is present in a wide range of aquatic environments and man-made ecosystems, such as paddy fields and wastewater treatment systems. In such environments, these archaea may experience regular oxygen exposure. However, " Ca Methanoperedens nitroreducens" is able to thrive under such conditions and could be applied for the simultaneous removal of dissolved methane and nitrogenous pollutants in oxygen-limited systems. To understand what machinery " Ca Methanoperedens nitroreducens" possesses to counteract the oxidative stress and survive, we characterized the response to oxygen exposure using a multi-omics approach., (Copyright © 2018 American Society for Microbiology.)

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., (Copyright © 2018 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

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 .

Early-diverging land plants such as mosses are known for their outstanding abilities to grow in various terrestrial habitats, incorporating tremendous structural and physiological innovations, as well as many lineage-specific genes. How these genes and functional innovations evolved remains unclear. In this study, we show that a dual-coding gene YAN/AltYAN in the moss Physcomitrella patens evolved from a pre-existing hemerythrin gene. Experimental evidence indicates that YAN/AltYAN is involved in fatty acid and lipid metabolism, as well as oil body and wax formation. Strikingly, both the recently evolved dual-coding YAN/AltYAN and the pre-existing hemerythrin gene might have similar physiological effects on oil body biogenesis and dehydration resistance. These findings bear important implications in understanding the mechanisms of gene origination and the strategies of plants to fine-tune their adaptation to various habitats.

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., (© 2018 The Protein Society.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

BRUTUS (BTS) is a hemerythrin (HHE) domain containing E3 ligase that facilitates the degradation of POPEYE-like (PYEL) proteins in a proteasomal-dependent manner. Deletion of BTS HHE domains enhances BTS stability in the presence of iron and also complements loss of BTS function, suggesting that the HHE domains are critical for protein stability but not for enzymatic function. The RING E3 domain plays an essential role in BTS' capacity to both interact with PYEL proteins and to act as an E3 ligase. Here we show that removal of the RING domain does not complement loss of BTS function. We conclude that enzymatic activity of BTS via the RING domain is essential for response to iron deficiency in plants. Further, we analyze possible BTS domain structure evolution and predict that the combination of domains found in BTS is specific to photosynthetic organisms, potentially indicative of a role for BTS and its orthologs in mitigating the iron-related challenges presented by photosynthesis.

Background: The evolution of oxygenic photosynthesis during Precambrian times entailed the diversification of strategies minimizing reactive oxygen species-associated damage. Four families of oxygen-carrier proteins (hemoglobin, hemerythrin and the two non-homologous families of arthropodan and molluscan hemocyanins) are known to have evolved independently the capacity to bind oxygen reversibly, providing cells with strategies to cope with the evolutionary pressure of oxygen accumulation. Oxygen-binding hemerythrin was first studied in marine invertebrates but further research has made it clear that it is present in the three domains of life, strongly suggesting that its origin predated the emergence of eukaryotes., Results: Oxygen-binding hemerythrins are a monophyletic sub-group of the hemerythrin/HHE (histidine, histidine, glutamic acid) cation-binding domain. Oxygen-binding hemerythrin homologs were unambiguously identified in 367/2236 bacterial, 21/150 archaeal and 4/135 eukaryotic genomes. Overall, oxygen-binding hemerythrin homologues were found in the same proportion as single-domain and as long protein sequences. The associated functions of protein domains in long hemerythrin sequences can be classified in three major groups: signal transduction, phosphorelay response regulation, and protein binding. This suggests that in many organisms the reversible oxygen-binding capacity was incorporated in signaling pathways. A maximum-likelihood tree of oxygen-binding hemerythrin homologues revealed a complex evolutionary history in which lateral gene transfer, duplications and gene losses appear to have played an important role., Conclusions: Hemerythrin is an ancient protein domain with a complex evolutionary history. The distinctive iron-binding coordination site of oxygen-binding hemerythrins evolved first in prokaryotes, very likely prior to the divergence of Firmicutes and Proteobacteria, and spread into many bacterial, archaeal and eukaryotic species. The later evolution of the oxygen-binding hemerythrin domain in both prokaryotes and eukaryotes led to a wide variety of functions, ranging from protection against oxidative damage in anaerobic and microaerophilic organisms, to oxygen supplying to particular enzymes and pathways in aerobic and facultative species.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

For de novo design of protein-protein interactions (PPIs), information on the shape and chemical complementarity of their interfaces is generally required. Recent advances in computational PPI design have allowed for de novo design of protein complexes, and several successful examples have been reported. In addition, a simple and easy-to-use approach has also been reported that arranges leucines on a solvent-accessible region of an α-helix and places charged residues around the leucine patch to induce interactions between the two helical peptides. For this study, we adopted this approach to de novo design a new PPI between the helical bundle proteins sulerythrin and LARFH. A non-polar patch was created on an α-helix of LARFH around which arginine residues were introduced to retain its solubility. The strongest interaction found was for the LARFH variant cysLARFH-IV-3L3R and the sulerythrin mutant 6L6D (KD=0.16 μM). This artificial protein complex is maintained by hydrophobic and ionic interactions formed by the inter-molecular helical bundle structure. Therefore, by the simple and easy-to-use approach to create de novo interfaces on the α-helices, we successfully generated an artificial PPI. We also created a second LARFH variant with the non-polar patch surrounded by positively charged residues at each end. Upon mixing this LARFH variant with 6L6D, mesh-like fibrous nanostructures were observed by atomic force microscopy. Our method may, therefore, also be applicable to the de novo design of protein nanostructures., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Amebiasis is an intestinal infection widespread throughout the world caused by the human pathogen Entamoeba histolytica. Metronidazole has been a drug of choice against amebiasis for decades despite its low efficacy against asymptomatic cyst carriers and emergence of resistance in other protozoa with similar anaerobic metabolism. Therefore, identification and characterization of specific targets is urgently needed to design new therapeutics for improved treatment against amebiasis. Toward this goal, thiol-dependent redox metabolism is of particular interest. The thiol-dependent redox metabolism in E. histolytica consists of proteins including peroxiredoxin, rubrerythrin, Fe-superoxide dismutase, flavodiiron proteins, NADPH: flavin oxidoreductase, and amino acids including l-cysteine, S-methyl-l-cysteine, and thioprolines (thiazolidine-4-carboxylic acids). E. histolytica completely lacks glutathione and its metabolism, and l-cysteine is the major intracellular low molecular mass thiol. Moreover, this parasite possesses a functional thioredoxin system consisting of thioredoxin and thioredoxin reductase, which is a ubiquitous oxidoreductase system with antioxidant and redox regulatory roles. In this review, we summarize and highlight the thiol-based redox metabolism and its control mechanisms in E. histolytica, in particular, the features of the system unique to E. histolytica, and its potential use for drug development against amebiasis., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance. Recent studies using bioinformatic analyses suggest that multiple hemerythrin-like protein coding sequences might have been acquired by lateral gene transfer and the number of hemerythrin-like proteins varies amongst different species. Mycobacterium smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility. Phylogenetic analysis indicated that these three proteins have different evolutionary origins, possibly explaining their different physiological functions. Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

In patients with a high-degree of internal carotid artery stenosis, cerebral hemodynamics and metabolism are compromised during ischemia. Revascularization improves cortical hemodynamics and oxygen metabolism during functional activity, but the process by which it occurs is still controversial. Therefore, using functional near-infrared spectroscopy (fNIRS), we investigated the process by which cerebral hemodynamics improve after revascularization surgery. Eight patients with severe carotid artery stenosis were examined using fNIRS during a motor task before and after surgery. We evaluated postoperative changes in total hemoglobin and deoxyhemoglobin (HbR), at 2 weeks after surgery, and again at 3 months after surgery. Parameters measured were the TTP (time to peak) value, defined as the time taken to reach 70% of the maximum total hemoglobin concentration, and the increase in HbR during the motor task. TTP was higher in four patients preoperatively, but this was no longer evident in two of the patients at 2 weeks after surgery. An increase in HbR during the task was observed in six patients before surgery, and was maintained at 2 weeks after surgery. However, in three of these patients, this increase was no longer evident 3 months later. These changes observed using fNIRS suggest that the increase in cerebral blood flow after revascularization surgery is followed by improvement in parenchymal vasodilation and neuronal oxygen metabolism.

Background: Entamoeba histolytica, an intestinal parasite that is the causative agent of amoebiasis, is exposed to elevated amounts of highly toxic reactive oxygen and nitrogen species during tissue invasion. A flavodiiron protein and a rubrerythrin have been characterized in this human pathogen, although their physiological reductants have not been identified., Methods: The present work deals with biochemical studies performed to reach a better understanding of the kinetic and structural properties of rubredoxin reductase and two ferredoxins from E. histolytica., Results: We complemented the characterization of two different metabolic pathways for O2 and H2O2 detoxification in E. histolytica. We characterized a novel amoebic protein with rubredoxin reductase activity that is able to catalyze the NAD(P)H-dependent reduction of heterologous rubredoxins, amoebic rubrerythrin and flavodiiron protein but not ferredoxins. In addition, the protein exhibited an NAD(P)H oxidase activity, which generates hydrogen peroxide from molecular oxygen. We describe how different ferredoxins were also efficient reducing substrates for both flavodiiron protein and rubrerythrin., Conclusions: The enzymatic systems herein characterized could contribute to the in vivo detoxification of O2 and H2O2, playing a key role for the parasite defense against reactive oxidant species., General Significance: To the best of our knowledge this is the first characterization of a eukaryotic rubredoxin reductase, including a novel kinetic study on ferredoxin-dependent reduction of flavodiiron and rubrerythrin proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objective: Reactive oxygen species are natural products of metabolism in aerobic organisms, which lead to oxidative damage, such as DNA mutation, protein inactivation and drug resistance. MSMEG_3312 was predicted as a hemerythrin-like protein, which can carry oxygen and reversibly bind to oxygen, thus it might play important roles in the process of oxygen metabolism. In this study, we explored the role of MSMEG_3312 in drug resistance., Methods: On the basis of bioinformatics, we identified the conserved sequence of HHE domain in MSMEG_3312 and it was predicted to have typical α-helix at secondary structure. To explore potential functions of MSMEG_3312, we constructed the msmeg_3312 knockout strain and compare the susceptibility to various drugs to its parent strain, mc2155. In addition, we also measured the promoter response when treatment of erythromycin., Results: Genetic results showed that MSMEG_3312 is not necessary for M. smegmatis growth at 7H9 rich medium. The msmeg_3312 knockout strain showed increased erythromycin resistance. Moreover, the drug resistance is only limited to erythromycin which its mechanism of action is by binding to the 50S subunit of the bacteria ribosomal complex and then inhibit protein synthesis. However, there were no different MICs of other antibiotics, targets for protein synthesis inhibition, but not 50S subunit, such as tetracyclines, aminoglycosides and chloramphenicol. Moreover, we also showed that the promoter of msmeg_3312 responses to erythromycin., Conclusions: Hemerythin-like protein MSMEG_3312 is involved in erythromycin resistance.

The nonheme peroxidase, rubrerythrin, shows the ability to reduce hydrogen peroxide to water without involving strongly oxidizing and free-radical-creating powerful oxidants such as compounds I and II [formally Fe(IV)] formed in peroxidases and catalases. Rubrerythrin could, therefore, be a useful ingredient in protein-based artificial oxygen carriers. Here, we report that the oxygen-carrying proteins, hemoglobin (Hb) and hemerythrin (Hr), can each be copolymerized with rubrerythrin using glutaraldehyde yielding high molecular weight species. These copolymers show additional peroxidase activity compared to Hb-only and Hr-only polymers, respectively and also generate lower levels of free radicals in reactions that involve hydrogen peroxide. Tests on human umbilical vein endothelial cells (HUVEC) reveal slightly better performance of the Rbr copolymers compared to controls, as measured at 24 h, but not at later times.

Microaerophilic bacteria are adapted to low oxygen environments, but the mechanisms by which their growth in air is inhibited are not well understood. The citric acid cycle in the microaerophilic pathogen Campylobacter jejuni is potentially vulnerable, as it employs pyruvate and 2-oxoglutarate:acceptor oxidoreductases (Por and Oor), which contain labile (4Fe-4S) centres. Here, we show that both enzymes are rapidly inactivated after exposure of cells to a fully aerobic environment. We investigated the mechanisms that might protect enzyme activity and identify a role for the hemerythrin HerA (Cj0241). A herA mutant exhibits an aerobic growth defect and reduced Por and Oor activities after exposure to 21% (v/v) oxygen. Slow anaerobic recovery of these activities after oxygen damage was observed, but at similar rates in both wild-type and herA strains, suggesting the role of HerA is to prevent Fe-S cluster damage, rather than promote repair. Another hemerythrin (HerB; Cj1224) also plays a protective role. Purified HerA and HerB exhibited optical absorption, ligand binding and resonance Raman spectra typical of μ-oxo-bridged di-iron containing hemerythrins. We conclude that oxygen lability and poor repair of Por and Oor are major contributors to microaerophily in C. jejuni; hemerythrins help prevent enzyme damage microaerobically or during oxygen transients., (© 2013 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

Background: Ruminococcus flavefaciens is an important fibre-degrading bacterium found in the mammalian gut. Cellulolytic strains from the bovine rumen have been shown to produce complex cellulosome structures that are associated with the cell surface. R. flavefaciens 007 is a highly cellulolytic strain whose ability to degrade dewaxed cotton, but not Avicel cellulose, was lost following initial isolation in the variant 007S. The ability was recovered after serial subculture to give the cotton-degrading strain 007C. This has allowed us to investigate the factors required for degradation of this particularly recalcitrant form of cellulose., Methodology/principal Findings: The major proteins associated with the bacterial cell surface and with the culture supernatant were analyzed for R. flavefaciens 007S and 007C grown with cellobiose, xylan or Avicel cellulose as energy sources. Identification of the proteins was enabled by a draft genome sequence obtained for 007C. Among supernatant proteins a cellulosomal GH48 hydrolase, a rubrerthyrin-like protein and a protein with type IV pili N-terminal domain were the most strongly up-regulated in 007C cultures grown on Avicel compared with cellobiose. Strain 007S also showed substrate-related changes, but supernatant expression of the Pil protein and rubrerythrin in particular were markedly lower in 007S than in 007C during growth on Avicel., Conclusions/significance: This study provides new information on the extracellular proteome of R. flavefaciens and its regulation in response to different growth substrates. Furthermore it suggests that the cotton cellulose non-degrading strain (007S) has altered regulation of multiple proteins that may be required for breakdown of cotton cellulose. One of these, the type IV pilus was previously shown to play a role in adhesion to cellulose in R. albus, and a related pilin protein was identified here for the first time as a major extracellular protein in R. flavefaciens.

The first demonstrated example of a regulatory function for a bacterial hemerythrin (Bhr) domain is reported. Bhrs have a characteristic sequence motif providing ligand residues for a type of non-heme diiron site that is known to bind O(2) and undergo autoxidation. The amino acid sequence encoded by the VC1216 gene from Vibrio cholerae O1 biovar El Tor str. N16961 contains an N-terminal Bhr domain connected to a C-terminal domain characteristic of bacterial diguanylate cyclases (DGCs) that catalyze formation of cyclic di-(3',5')-guanosine monophosphate (c-di-GMP) from GTP. This protein, Vc Bhr-DGC, was found to contain two tightly bound non-heme iron atoms per protein monomer. The as-isolated protein showed the spectroscopic signatures of oxo/dicarboxylato-bridged non-heme diferric sites of previously characterized Bhr domains. The diiron site was capable of cycling between diferric and diferrous forms, the latter of which was stable only under anaerobic conditions, undergoing rapid autoxidation upon being exposed to air. Vc Bhr-DGC showed approximately 10 times higher DGC activity in the diferrous than in the diferric form. The level of intracellular c-di-GMP is known to regulate biofilm formation in V. cholerae. The higher DGC activity of the diferrous Vc Bhr-DGC is consistent with induction of biofilm formation in low-dioxygen environments. The non-heme diiron cofactor in the Bhr domain thus represents an alternative to heme or flavin for redox and/or diatomic gas sensing and regulation of DGC activity.

Iron regulatory proteins play a principal role in maintaining cellular iron homeostasis by post-transcriptionally regulating factors responsible for iron uptake, utilization, and storage. An E3 ubiquitin ligase complex containing FBXL5 targets IRP2 for proteasomal degradation under iron- and oxygen-replete conditions, whereas FBXL5 itself is degraded when iron and oxygen availability decreases. FBXL5 contains a hemerythrin-like (Hr) domain at its N terminus that mediates its own differential stability. Here, we investigated the iron- and oxygen-dependent conformational changes within FBXL5-Hr that underlie its role as a cellular sensor. As predicted, FBXL5-Hr undergoes substantive structural changes when iron becomes limiting, accounting for its switch-like behavior. However, these same changes are not observed in response to oxygen depletion, indicating that this domain accommodates two distinct sensing mechanisms. Moreover, FBXL5-Hr does not behave as a dynamic sensor that continuously samples the cellular environment, assuming conformations in equilibrium with ever-changing cellular iron levels. Instead, the isolated domain appears competent to incorporate iron only at or near the time of its own synthesis. These observations have important implications for mechanisms by which these metabolites are sensed within mammalian cells.

Recently, a native bacteriohemerythrin (McHr) has been identified in Methylococcus capsulatus (Bath). Both the particulate methane monooxygenase (pMMO) and McHr are over-expressed in cells of this bacterium when this strain of methanotroph is cultured and grown under high copper to biomass conditions. It has been suggested that the role of the McHr is to provide a shuttle to transport dioxygen from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by the pMMO. Indeed, McHr enhances the activity of the pMMO when pMMO-enriched membranes are used to assay the enzyme activity. We find that McHr can dramatically improve the activity of pMMO toward the epoxidation of propylene to propylene oxide. The maximum activity is observed at a pMMO to McHr concentration ratio of 4:1, where we have obtained specific activities of 103.7nmol propylene oxide/min/mg protein and 122.8nmol propylene oxide/min/mg protein at 45°C when the turnover is driven by NADH and duroquinol, respectively. These results are consistent with the suggestion that the bacterium requires McHr to deliver dioxygen to the pMMO in the intra-cytoplasmic membranes to accomplish efficient catalysis of methane oxidation when the enzyme is over-expressed in the cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

High-resolution crystal structures of Pyrococcus furiosus rubrerythrin (PfRbr) in the resting (all-ferrous) state and at time points following exposure of the crystals to hydrogen peroxide are reported. This approach was possible because of the relativity slow turnover of PfRbr at room temperature. To this end, we were able to perform time-dependent peroxide treatment of the fully reduced enzyme, under strictly anaerobic conditions, in the crystalline state. In this work we demonstrate, for the first time, that turnover of a thermophilic rubrerythrin results in approximately 2-Å movement of one iron atom in the diiron site from a histidine to a carboxylate ligand. These results confirm that, despite the domain-swapped architecture, the hyperthermophilic rubrerythrins also utilize the classic combination of iron sites together with redox-dependent iron toggling to selectively reduce hydrogen peroxide over dioxygen. In addition, we have identified previously unobserved intermediates in the reaction cycle and observed structural changes that may explain the enzyme precipitation observed for the all-iron form of PfRbr upon oxidation to the all-ferric state.

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV-vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys(4) centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H(2)O(2) min(-1) mg(-1). Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.

The open reading frame alr3199 of the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 was cloned and overexpressed in Escherichia coli. Purified recombinant Alr3199 protein was found to be a functionally active deoxyribonuclease with novel features, such as (1) no homology to typical DNases (2) a Ca²(+)-dependent Nickase activity (3) presence of a di-hemerythrin domain, and (4) requirement of Fe²(+) conjugated to hemerythrin domains for optimal activity. Both the DNase and Nickase activities were found to be associated with the N-terminal non-hemerythrin region, but were modulated by Fe²(+) conjugated to the C-terminal hemerythrin region. This is the first report of a hemerythrin protein with DNase activity, tentatively designated as 'HE-DNase', and with a possible role in stress-induced DNA damage/repair in Anabaena., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Hemerythrin is proposed as an alternative to hemoglobin-based blood substitutes. In contrast to hemoglobin, hemerythrin exhibits negligible reactivity towards oxidative and nitrosative stress agents (peroxide, nitric oxide, nitrite). Protocols for attachment of polyethylene glycol and glutaraldehyde cross-linking of Hr are described. These derivatizations appear to have favorable effects on O(2) affinity and autoxidation rates for use in blood substitutes. Based on lessons learned from hemoglobin-based blood substitutes, these derivatizations should also help limit extravasation and antigenicity of a hemerythrin-based blood substitute.

Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100 degrees C. Addition of H(2)O(2) (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H(2)O(2) were not more resistant to the subsequent addition of H(2)O(2) (0.5-5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.

The assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S protein-mediated oxygen detoxification.

Based on the crystal structures, three possible sequence determinants have been suggested as the cause of a 285 mV increase in reduction potential of the rubredoxin domain of rubrerythrin over rubredoxin by modulating the polar environment around the redox site. Here, electrostatic calculations of crystal structures of rubredoxin and rubrerythrin and molecular dynamics simulations of rubredoxin wild-type and mutants are used to elucidate the contributions to the increased reduction potential. Asn(160) and His(179) in rubrerythrin versus valines in rubredoxins are predicted to be the major contributors, as the polar side chains contribute significantly to the electrostatic potential in the redox site region. The mutant simulations show both side chains rotating on a nanosecond timescale between two conformations with different electrostatic contributions. Reduction also causes a change in the reduction energy that is consistent with a linear response due to the interesting mechanism of shifting the relative populations of the two conformations. In addition to this, a simulation of a triple mutant indicates the side-chain rotations are approximately anticorrelated so whereas one is in the high potential conformation, the other is in the low potential conformation. However, Ala(176) in rubrerythrin versus a leucine in rubredoxin is not predicted to be a large contributor, because the solvent accessibility increases only slightly in mutant simulations and because it is buried in the interface of the rubrerythrin homodimer., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.

Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of genes responsible for iron uptake, release, use, and storage through the actions of the iron regulatory proteins IRP1 and IRP2. However, the manner in which iron levels are sensed to affect IRP2 activity is poorly understood. We found that an E3 ubiquitin ligase complex containing the FBXL5 protein targets IRP2 for proteasomal degradation. The stability of FBXL5 itself was regulated, accumulating under iron- and oxygen-replete conditions and degraded upon iron depletion. FBXL5 contains an iron- and oxygen-binding hemerythrin domain that acted as a ligand-dependent regulatory switch mediating FBXL5's differential stability. These observations suggest a mechanistic link between iron sensing via the FBXL5 hemerythrin domain, IRP2 regulation, and cellular responses to maintain mammalian iron homeostasis.

An unusual non-haem diiron protein, reverse rubrerythrin (revRbr), is known to be massively upregulated in response to oxidative stress in the strictly anaerobic bacterium Clostridium acetobutylicum. In the present study both in vivo and in vitro results demonstrate an H2O2 and O2 detoxification pathway in C. acetobutylicum involving revRbr, rubredoxin (Rd) and NADH : rubredoxin oxidoreductase (NROR). RevRbr exhibited both NADH peroxidase (NADH : H2O2 oxidoreductase) and NADH oxidase (NADH : O2 oxidoreductase) activities in in vitro assays using NROR as the electron-transfer intermediary from NADH to revRbr. Rd increased the NADH consumption rate by serving as an intermediary electron-transfer shuttle between NROR and revRbr. While H2O2 was found to be the preferred substrate for revRbr, its relative oxidase activity was found to be significantly higher than that reported for other Rbrs. A revRbr-overexpressing strain of C. acetobutylicum showed significantly increased tolerance to H2O2 and O2 exposure. RevRbr thus appears to protect C. acetobutylicum against oxidative stress by functioning as the terminal component of an NADH peroxidase and NADH oxidase.

Although psychological stress affects skin condition, the neurophysiological mechanism involved is unclear. In this study, we evaluated the relationship between skin condition and left/right asymmetry in prefrontal cortex (PFC) activity during mental stress tasks since recent studies have suggested that the right PFC dominates the regulation of the stress response system, including the hypothalamic-pituitary-adrenal (HPA) axis. Using near-infrared spectroscopy, we measured hemoglobin concentration changes in the bilateral PFC during a mental arithmetic task in normal adults and evaluated the laterality scores (i.e., [(right-left)/(right+left)]) of oxyhemoglobin concentration changes. Elicitation of stress was verified by the State-Trait Anxiety Inventory (STAI) and heart rate. The sebum levels and Propionibacterium acnes populations in the facial skin were measured before the task. The task significantly increased the STAI-II scores (p=0.00079) and heart rate (p=0.0000049). The oxyhemoglobin concentration increased in the bilateral PFC during the task, associated with a decrease in deoxyhemoglobin concentration. The laterality scores of oxyhemoglobin concentration changes were positively correlated with sebum levels (r=+0.50, p=0.026) and P. acnes populations (r=+0.49, p=0.029) in the facial skin before the task. There was a significant positive correlation between heart rate changes and the laterality scores of oxyhemoglobin concentration changes (r=+0.54, p=0.015). These results demonstrate that the subjects with higher sebum levels and higher P. acnes populations in the facial skin have a right dominant PFC activity during a mental stress task and suggest that such subjects are sensitive to mental stress associated with hyperactivity of the stress response system, including the HPA axis system.

In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Diferrous NorA binds two molecules of NO maximally. Unexpectedly, binding of NO to the diferrous NorA required an external reductant. Two different NorA-NO species could be resolved. A minor species (up to 20%) showed an S = (1/2) EPR signal with g( perpendicular) = 2.041, and g( parallel) = 2.018, typical of a paramagnetic dinitrosyl iron complex. The major species was EPR-silent, showing characteristic signals at 420 nm and 750 nm in the optical spectrum. This species is proposed to represent a novel dinitrosyl iron complex of the form Fe(2+)-[NO](2)(2-), i.e. NO is bound as NO(-). The NO binding capacity of NorA in conjunction with its high cytoplasmic concentration (20 mum) suggests that NorA regulates transcription by lowering the free cytoplasmic concentration of NO.

For a long time, the haemerythrin family of proteins was considered to be restricted to only a few phyla of marine invertebrates. When analysing differential protein expression in the methane-oxidizing bacterium, Methylococcus capsulatus (Bath), grown at a high and low copper-to-biomass ratio, respectively, we identified a putative prokaryotic haemerythrin expressed in high-copper cultures. Haemerythrins are recognized by a conserved sequence motif that provides five histidines and two carboxylate ligands which coordinate two iron atoms. The diiron site is located in a hydrophobic pocket and is capable of binding O(2). We cloned the M. capsulatus haemerythrin gene and expressed it in Escherichia coli as a fusion protein with NusA. The haemerythrin protein was purified to homogeneity cleaved from its fusion partner. Recombinant M. capsulatus haemerythrin (McHr) was found to fold into a stable protein. Sequence similarity analysis identified all the candidate residues involved in the binding of diiron (His22, His58, Glu62, His77, His81, His117, Asp122) and the amino acids forming the hydrophobic pocket in which O(2) may bind (Ile25, Phe59, Trp113, Leu114, Ile118). We were also able to model a three-dimensional structure of McHr maintaining the correct positioning of these residues. Furthermore, UV/vis spectrophotometric analysis demonstrated the presence of conjugated diiron atoms in McHr. A comprehensive genomic database search revealed 21 different prokaryotes containing the haemerythrin signature (PROSITE 00550), indicating that these putative haemerythrins may be a conserved prokaryotic subfamily.

A combination of conventional quantum chemical methods and a recently developed mixed quantum mechanical/molecular mechanical (QM/MM) method (QSite) is used to determine the different energetic components involved in reversible binding of O(2) to hemerythrin. The use of an accurate quantum chemical description of the active site and the inclusion of effects from the surrounding protein environment are both essential to achieve reversibility and thus to model accurately the binding of O(2) to the carboxylate-bridged diiron center in the protein. The major contributions from the protein environment stabilizing dioxygen binding are (1) the van der Waals interaction between the bound dioxygen and the protein atoms and (2) an increase in the hydrogen bonding energy of an imidazole group, ligated to one of the iron atoms, and a neighboring carboxylate side chain in the second coordination sphere. The protein strain energy for this system is negligible. The calculated total O(2) binding free energy is in good agreement with that derived from the experimental equilibrium constant.

Electronic mechanisms of reductive activation of O2 by oxidases coenzymes (K) are considered. Activation is induced by triplet-singlet (T-S)-transitions at the charge-transfer stage and K+...O2- radical pair (RP) formation. For flavoproteins the spin reversion is induced by spin-orbit coupling (SOC) in superoxide ion, for metal-oxidases--by exchange interaction with paramagnetic metal ion. Another type of spin activation is connected with the singlet peroxide ion O(2)2- formation in cytochromes (structures of the type Fe(3+)-O- -O-Cu2+).