Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.     

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

The clam Lucina pectinalis supports its symbiotic bacteria by H₂S transport in the open and accessible heme pocket of Lucina Hb I and by O₂ transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O₂ was removed from its active site. Structural analysis revealed that O₂ “clams up” the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O₂ affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O₂. These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins.     

Stability of the heme environment of the nitric oxide synthase from Staphylococcus aureus in the absence of pterin cofactor

We have used resonance Raman spectroscopy to probe the heme environment of a recently discovered NOS from the pathogenic bacterium Staphylococcus aureus, named SANOS. We detect two forms of the CO complex in the absence of L-arginine, with nu(Fe-CO) at 482 and 497 cm(-1) and nu(C-O) at 1949 and 1930 cm(-1), respectively. Similarly to mammalian NOS, the binding of L-arginine to SANOS caused the formation of a single CO complex with nu(Fe-CO) and nu(C-O) frequencies at 504 and 1,917 cm(-1), respectively, indicating that L-arginine induced an electrostatic/steric effect on the CO molecule. The addition of pterins to CO-bound SANOS modified the resonance Raman spectra only when they were added in combination with L-arginine. We found that (6R) 5,6,7,8 tetra-hydro-L-biopterin and tetrahydrofolate were not required for the stability of the reduced protein, which is 5-coordinate, and of the CO complex, which does not change with time to a form with a Soret band at 420 nm that is indicative of a change of the heme proximal coordination. Since SANOS is stable in the absence of added pterin, it suggests that the role of the pterin cofactor in the bacterial NOS may be limited to electron/proton transfer required for catalysis and may not involve maintaining the structural integrity of the protein as is the case for mammalian NOS.     

The adaptive value of functional and life-history traits across fertility treatments in an annual plant

Background and Aims

Plant functional traits are assumed to be adaptive. As selection acts on individuals and not on traits, interpreting the adaptive value of a trait not may be straightforward. For example, productive leaves are associated with fertile environments. However, it is not clear if productive leaves confer an advantage in these habitats, or if they are an advantage as part of a suite of coordinated traits.

Methods

Genotypes of Arabidopsis thaliana were grown in high and low nutrient treatments and low, neutral and high pH treatments. Nutrient availability is reduced in acidic or basic soils relative to neutral pH soils. pH treatments were used to alter the availability of resources rather than the amount of resources.

Key Results

Leaf function (specific leaf area, SLA) and life history (size at reproduction, age at reproduction) were variable across genotypes and were plastic. High nutrient availability induced higher SLA and larger size at reproduction. Genotypes that reproduced at large size in high nutrient conditions at neutral pH had the greatest fruit production. SLA was only indirectly related to fruit production through a causal relationship with rosette size; in high nutrient conditions, plants with high SLA were large at reproduction and had higher fruit production. In high nutrient and high pH treatments, plants were large at reproduction, but large size at reproduction was associated with low fecundity. This suggests that large size is adaptive under high nutrient availability.

Conclusions

Interpreting the adaptive value of functional traits will sometimes only be possible when these traits are considered as a suite of correlated and coordinated traits. Leaf functional traits may be important in defining adaptive strategies in A. thaliana but only through how they affect plant life history. Finally, manipulating soil pH can be a valuable tool in assessing adaptive plasticity on nutrient gradients.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Antarctic bacterial haemoglobin and its role in the protection against nitrogen reactive species

In a cold and oxygen-rich environment such as Antarctica, mechanisms for the defence against reactive oxygen and nitrogen species are needed and represent important components in the evolutionary adaptations. In the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125, the presence of multiple genes encoding 2/2 haemoglobins and a flavohaemoglobin strongly suggests that these proteins fulfil important physiological roles, perhaps associated to the peculiar features of the Antarctic habitat. In this work, the putative role of Ph-2/2HbO, encoded by the PSHAa0030 gene, was investigated by in vivo and in vitro experiments in order to highlight its involvement in NO detoxification mechanisms. The PSHAa0030 gene was cloned and then over-expressed in a flavohaemoglobin-deficient mutant of Escherichia coli, unable to metabolise NO, and the resulting strain was studied analysing its growth properties and oxygen uptake in the presence of NO. We here demonstrate that Ph-2/2HbO protects growth and cellular respiration of the heterologous host from the toxic effect of NO-donors. Unlike in Mycobacterium tuberculosis 2/2 HbN, the deletion of the N-terminal extension of Ph-2/2HbO does not seem to reduce the NO scavenging activity, showing that the N-terminal extension is not a requirement for efficient NO detoxification. Moreover, the ferric form of Ph-2/2HbO was shown to catalyse peroxynitrite isomerisation in vitro, confirming its potential role in the scavenging of reactive nitrogen species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Relationship between the expression level of SLA-DQA and Escherichia coli F18 infection in piglets

The expression of SLA-DQA was assayed by Real-time PCR to analyze the differential expression between ETEC F18-resistant and -sensitive post-weaning piglets, and then to compare the expression levels of SLA-DQA in 11 different tissues from 8-, 18-, 30- and 35-day-old ETEC F18-resistant piglets, which aimed at discussing the role of SLA-DQA in resistance to ETEC F18. The results showed that SLA-DQA is broadly expressed in 11 tissues with the highest expression level in lymph nodes, and a relatively higher expression level in lung, spleen, jejunum, and duodenum. In tissues of lymph node, lung, spleen, jejunum, and duodenum, the mRNA expression of SLA-DQA in resistant individuals was significantly higher than that in sensitive ones (P < 0.05). In most tissues, the expression of SLA-DQA increased from 8 to 18 and 30 days (weaning day), and increased persistently to 35 days of post-weaning. Expression levels of SLA-DQA on 35 days in most tissues were significant higher than that on 8, 18 and 30 days (P < 0.05). The results demonstrated that the resistance to ETEC F18 in post-weaning piglets is related to up-regulation of mRNA expression of SLA-DQA to a certain extent. The analysis suggested that SLA-DQA may be not the direct immune factor that resisted the Escherichia coli F18, but perhaps enhanced humoral immunity and cell immunity to reduce the transmembrane signal transduction of ETEC F18 bacterial LPS and then led to the resistance to ETEC F18 in piglets.     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Analysis of the formation of flower shapes in wild species and cultivars of tree peony using the MADS-box subfamily gene

Tree peony (Paeonia suffricotisa) cultivars have a unique character compared with wild species; the stamen petalody results in increased whorls of petals and generates different flower forms, which are one of the most important traits for cultivar classification. In order to investigate how petaloid stamens are formed, we obtained the coding sequence (666 bp) and genomic DNA sequence of the PsTM6 genes (belongs to B subfamily of MADS-box gene family) from 23 tree peony samples, Five introns and six exons consisted of the genomic DNA sequence. The analysis of cis-acting regulatory elements in the third and fourth intron indicated that they were highly conserved in all samples. Partial putative amino acids were analyzed and the results suggested that functional differentiation of PsTM6 paralogs apparently affected stamen petalody and flower shape formation due to due to amino acid substitution caused by differences in polarity and electronic charge. Sliding window analysis indicated that the different regions of PsTM6 were subjected to different selection forces, especially in the K domain. This is the first attempt to investigate genetic control of the stamen petalody based on the PsTM6 sequence. This will provide a basis for understanding the evolution of PsTM6 and its the function of in determining stamen morphology of tree peony.     

Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.     

青藏高原东缘高寒草原有毒植物分布与高原鼠兔、高原鼢鼠的相关性

青藏高原高寒草原生态系统是我国特有的生态系统类型,由于受到人为破坏的影响,目前该地区草原生态系统功能退化,优良牧草减少,有毒植物蔓延。高原鼠兔(Ochotona curzoniae)和高原鼢鼠(Myospalax baileyi)是青藏高原东缘高寒草原中最重要的两种小型哺乳动物,其采食行为和挖掘洞穴的生活特性必然对生态系统产生影响,但其与有毒植物之间的互作关系尚未揭示。基于此,在甘肃省玛曲县河曲马场自然生长的高寒草原生态系统中开展了有毒植物的分布与高原鼠兔、高原鼢鼠之间的相关性研究。结果表明,该高寒草原生态系统中分布有毒植物27种,分属于菊科、豆科、毛茛科等11科。在此基础上,测定了该地区有毒植物的生物多样性指数、均匀度指数和丰富度指数,并探究了单位面积条件下有毒植物的分布特征与高原鼠兔和高原鼢鼠种群密度之间的相关性,发现该地区高寒草原有毒植物的蔓延与高原鼠兔的密度之间存在密切的负相关关系(P0.05),而与高原鼢鼠的相关性不显著(P0.05)。     

A Conserved Region between the TPR and Activation Domains of p67phox Participates in Activation of the Phagocyte NADPH Oxidase

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91^phox serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91^phox requires the cytosolic proteins p67^phox, p47^phox, and Rac (a small GTPase). p67^phox, comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47^phox and there interacts with Rac; these processes are prerequisite for gp91^phox activation. Here we show that a region of p67^phox (amino acids 190–200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67^phox to support superoxide production by gp91^phox-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91^phox-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67^phox interaction with the gp91^phox NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67^phox (amino acids 190–210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91^phox.