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In our continued investigations of microbial globins, we solved the structure of a truncated hemoglobin from Shewanella benthica , an obligate psychropiezophilic bacterium. The distal side of the heme active site is lined mostly with hydrophobic residues, with the exception of a tyrosine, Tyr34 (CD1) and a histidine, His24 (B13). We found that purified SbHbN, when crystallized in the ferric form with polyethylene glycol as precipitant, turned into a green color over weeks. The electron density obtained from the green crystals accommodated a trans heme d , a chlorin-type derivative featuring a γ-spirolactone and a vicinal hydroxyl group on a pyrroline ring. In solution, exposure of the protein to one equivalent of hydrogen peroxide resulted in a similar green color change, but caused by the formation of multiple products. These were oxidation species released on protein denaturation, likely including heme d , and a species with heme covalently attached to the polypeptide. The Tyr34Phe replacement prevented the formation of both heme d and the covalent linkage. The ready modification of heme b by SbHbN expands the range of chemistries supported by the globin fold and offers a route to a novel heme cofactor. The ferric group 1 truncated hemoglobin from the extremophile Shewanella benthica KT99 reacts with peroxides. In solution, several products are obtained including a heme–protein covalent adduct. In the crystal, the product is solely trans heme d. The results add to the repertory of hemoglobin chemistry. [Display omitted] • The group 1 truncated hemoglobin of Shewanella benthica reacts with peroxides. • In the crystal (reagent polyethylene glycol), the product is heme d. • In solution (reagent H 2 O 2), products include a species with heme-protein cross-link. • Tyr34 on the distal side of the heme is implicated in the reactions. • Cyanide bound to the heme iron inhibits the reactions. [ABSTRACT FROM AUTHOR]

Abstract Background The PII proteins constitute a large superfamily, present in all domains of life. Until now, PII proteins research in Chloroplastida (green algae and land plants) has mainly focused on post-translation regulation of these signal transductors. Emerging evidence suggests that PII level is tightly controlled with regard to the nitrogen source and the physiological state of cells. Result Here we identify that a balance of positive (nitrate and nitrite) and negative (nitric oxide) signals regulates Chlamydomonas GLB1. We found that PII expression is downregulated by ammonium through a nitric oxide (NO)-dependent mechanism. We show that nitrate reductase (NR) and its partner, truncated hemoglobin 1 (THB1), participate in a signaling pathway for dual control of GLB1 expression. Moreover, NO dependent guanilate cyclase appeared to be involved in the negative control of GLB1 transcription. Conclusion This study has revealed the existence of the complex GLB1 control at transcription level, which is dependent on nitrogen source. Importantly, we found that GLB1 gene expression pattern is very similar to that observed for nitrate assimilation genes, suggesting interconnecting/coordinating PII-dependent and nitrate assimilation pathways.

Hemoglobins (Hbs) utilize heme b as a cofactor and are found in all kingdoms of life. The current knowledge reveals an enormous variability of Hb primary sequences, resulting in topological, biochemical and physiological individuality. As Hbs appear to modulate their reactivities through specific combinations of structural features, predicting the characteristics of a given Hb is still hardly possible. The unicellular green alga Chlamydomonas reinhardtii contains 12 genes encoding diverse Hbs of the truncated lineage, several of which possess extended N- or C-termini of unknown function. Studies on some of the Chlamydomonas Hbs revealed yet unpredictable structural and biochemical variations, which, along with a different expression of their genes, suggest diverse physiological roles. Chlamydomonas thus represents a promising system to analyze the diversification of Hb structure, biochemistry and physiology. Here, we report the crystal structure, resolved to 1.75 Å, of the heme-binding domain of cyanomet THB11 (Cre16.g662750), one of the pentacoordinate algal Hbs, which offer a free Fe-coordination site in the reduced state. The overall fold of THB11 is conserved, but individual features such as a kink in helix E, a tilted heme plane and a clustering of methionine residues at a putative tunnel exit appear to be unique. Both N- and C-termini promote the formation of oligomer mixtures, and the absence of the C terminus results in reduced nitrite reduction rates. This work widens the structural and biochemical knowledge on the 2/2Hb family and suggests that the N- and C-terminal extensions of the Chlamydomonas 2/2Hbs modulate their reactivity by intermolecular interactions. [ABSTRACT FROM AUTHOR]

Aims: Although the human pathogen, Mycobacterium tuberculosis (Mtb), is strictly aerobic and requires efficient supply of oxygen, it can survive long stretches of severe hypoxia. The mechanism responsible for this metabolic flexibility is unknown. We have investigated a novel mechanism by which hemoglobin O (HbO), operates and supports its host under oxygen stress. Results: We discovered that the HbO exists in a phospho-bound state in Mtb and remains associated with the cell membrane under hypoxia. Deoxy-HbO carries an autokinase activity that disrupts its dimeric assembly into monomer and facilitates its association with the cell membrane, supporting survival and adaptation of Mtb under low oxygen conditions. Consistent with these observations, deletion of the glbO gene in Mycobacterium bovis bacillus Calmette-Guerin, which is identical to the glbO gene of Mtb, attenuated its survival under hypoxia and complementation of the glbO gene of Mtb rescued this inhibition, but phosphorylation-deficient mutant did not. These results demonstrated that autokinase activity of the HbO modulates its physiological function and plays a vital role in supporting the survival of its host under hypoxia. Innovation and Conclusion: Our study demonstrates that the redox-dependent autokinase activity regulates oligomeric state and membrane association of HbO that generates a reservoir of oxygen in the proximity of respiratory membranes to sustain viability of Mtb under hypoxia. These results thus provide a novel insight into the physiological function of the HbO and demonstrate its pivotal role in supporting the survival and adaptation of Mtb under hypoxia. [ABSTRACT FROM AUTHOR]

Truncated globins are 20–40 amino acids shorter than full length globins. Till date, globins have been characterized predominantly from bacteria involved in pathogenicity, nitrogen fixation and photosynthesis, where they are implicated in bacterial virulence within the host, protection of nitrogenase from oxygen inactivation and prevention of oxidative damage to the photosynthetic machinery respectively. Myxococcus xanthus, the model myxobacterium, is an obligate aerobe with a multicellular stage in its life cycle where cells encounter oxygen limitation. This work was undertaken to investigate the potential role of the truncated globin in M. xanthus. To examine the role of globins in this unique group of bacteria, the gene coding for a putative truncated globin (HbO) was identified in the genome of M. xanthus DK 1622. The sequence analysis by bioinformatics approaches revealed that HbO from M. xanthus (Mx-HbO) likely adopts a 2-on-2 alpha helical fold of the truncated globins. The gene coding for Mx-HbO was cloned and its expression in E. coli imparted reddish tinge to the cells. The spectral analysis confirmed it to be a functional globin. The expression of Mx-HbO in the heterologous host improved its growth, resulting in the attainment of higher cell density in culture. The transcript of Mx-hbO was induced threefold in the host cells when grown under low aeration condition as compared to the cells grown under high aeration condition. In M. xanthus, an obligate aerobe, where cell growth accompanies swarming, there is a higher density of cells in the middle of the swarm. Our results suggest that Mx-HbO is a functional globin and could facilitate the growth of cells facing oxygen deprivation, the condition prevailing in the middle of the swarm. [ABSTRACT FROM AUTHOR]

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme–histidine C–N linkage similar to the C–S linkage of cytochrome c . No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (Fe III –NO) that resist reductive nitrosylation to the paramagnetic Fe II –NO forms. Dithionite reduction of Fe III –NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected Fe II –NO complex, NO binding to Fe II GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining Fe III GlbN, 15 N-labeled nitrite, and excess dithionite resulted in the formation of Fe II –H 15 NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine–heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in Fe II GlbNs: protection from NO-mediated heme loss and prevention of HNO formation. [ABSTRACT FROM AUTHOR]

Hydroxylamine (HA) is an oxidant of ferrous globins and its action has been reported to be inhibited by CO, even though this mechanism has not been clarified. Here, kinetics of the HA-mediated oxidation of ferrous carbonylated Mycobacterium tuberculosis truncated hemoglobin N and O ( Mt-trHbN(II)-CO and Mt-trHbO(II)-CO, respectively) and Campylobacter jejuni truncated hemoglobin P ( Cj-trHbP(II)-CO), at pH 7.2 and 20.0 °C, are reported. Mixing Mt-trHbN(II)-CO, Mt-trHbO(II)-CO, and Cj-trHbP(II)-CO solution with the HA solution brings about absorption spectral changes reflecting the disappearance of the ferrous carbonylated derivatives with the concomitant formation of the ferric species. HA oxidizes irreversibly Mt-trHbN(II)-CO, Mt-trHbO(II)-CO, and Cj-trHbP(II)-CO with the 1:2 stoichiometry. The dissociation of CO turns out to be the rate-limiting step for the oxidation of Mt-trHbN(II)-CO, Mt-trHbO(II)-CO, and Cj-trHbP(II)-CO by HA. Values of the second-order rate constant for HA-mediated oxidation of Mt-trHbN(II)-CO, Mt-trHbO(II)-CO, and Cj-trHbP(II)-CO range between 8.8 × 10 and 8.6 × 10 M s, reflecting different structural features of the heme distal pocket. This study (1) demonstrates that the inhibitory effect of CO is linked to the dissociation of this ligand, giving a functional basis to previous studies, (2) represents the first comparative investigation of the oxidation of ferrous carbonylated bacterial 2/2 globins belonging to the N, O, and P groups by HA, (3) casts light on the correlation between kinetics of HA-mediated oxidation and carbonylation of globins, and (4) focuses on structural determinants modulating the HA-induced oxidation process. [ABSTRACT FROM AUTHOR]

Main conclusion: Annotated hemoglobin genes in Chlamydomonas reinhardtii form functional globins, despite unusual architectures. Spectral characteristics show subtle biochemical differences. Multiple globins might help the alga to cope with its versatile environment. The unicellular green alga C. reinhardtii is a photosynthetic, often soil-dwelling organism, subjected to a changeable environment in nature. The alga contains 12 genes encoding so-called truncated hemoglobins that feature a two-on-two helical fold instead of the three-on-three helix arrangement of the long-studied vertebrate globins or plant symbiotic and non-symbiotic hemoglobins. In plants, non-symbiotic hemoglobins often play a role in acclimation to stress, and we could show recently that one of the C. reinhardtii globin genes is vital for anoxic growth. Here, three further globin encoding transcripts ( Cre16.g661000.t1.1, Cre16.g661300.t2.1 and Cre16.g662750.t1.2) were heterologously expressed along with the recently studied THB1. UV-Vis and X-ray absorption spectroscopy analyses show that the sequences indeed encode functional hemoglobins, despite their uncommon primary sequences, which include long C-termini without any predictable function, or a split heme-binding domain. The proteins show some variations regarding the coordination of the heme iron or the interaction with diatomic ligands, indicating different functionalities. The respective transcripts are not responsive to the nitrogen source, in contrast to results reported for THB1, but they accumulate in darkness. This work advances experimental data on the very large globin family in general, and, more specifically, on hemoglobins in photosynthetic organisms. [ABSTRACT FROM AUTHOR]

Background The nature of ligand motion in proteins is difficult to characterize directly usingexperiment. Specifically, it is unclear to what degree these motions are coupled. Methods All-atom simulations are used to sample ligand motion in truncated Hemoglobin N. A transition network analysis including ligand- and protein-degrees of freedom is used to analyze the microscopic dynamics. Results Clustering of two different subsets of MD trajectories highlights the importance of a diverse and exhaustive description to define the macrostates for a ligand-migration network. Monte Carlo simulations on the transition matrices from one particular clustering are able to faithfully capture the atomistic simulations. Contrary to clustering by ligand positions only, including a protein degree of freedom yields considerably improved coarse grained dynamics. Analysis with and without imposing detailed balance agree closely which suggests that the underlying atomistic simulations are converged with respect to sampling transitions between neighboring sites. Conclusions Protein and ligand dynamics are not independent from each other and ligand migration through globular proteins is not passive diffusion. General significance Transition network analysis is a powerful tool to analyze and characterize the microscopic dynamics in complex systems. This article is part of a Special Issue entitled Recent developments of molecular dynamics. [ABSTRACT FROM AUTHOR]

Although truncated hemoglobin O, (trHbO), is ubiquitous among mycobacteria, its physiological function is not very obvious and may be diverse. In an attempt to understand role of trHbO in cellular metabolism of a non-pathogenic mycobacterium, we analysed expression profile of the glbO gene, encoding trHbO, in M. smegmatis and studied implications of its overexpression on physiology of its host under different environmental conditions. Quantitative RT-PCR indicated that transcript level of the glb O gene remains low at a basal level under aerobic growth cycle of M. smegmatis but its level gets induced significantly during low oxygen, oxidative stress and macrophage infection. Overexpression of the glb O gene enhanced growth of M. smegmatis under hypoxia, promoted pellicle biofilm formation and provided resistance towards oxidative stress. Additionally, glb O gene overexpressing M. smegmatis exhibited enhanced cell survival over isogenic control cells and altered the level of pro- and anti- inflammatory cytokines during intracellular infection. These results suggested important role of trHbO, in supporting the cellular metabolism and survival of M, smegmatis both under low oxygen and oxidative stress. [ABSTRACT FROM AUTHOR]

Hemoglobins are ubiquitous proteins that sense, store and transport oxygen, but the physiological processes in which they are implicated is currently expanding. Recent examples of previously unknown hemoglobin functions, which include scavenging of the signaling molecule nitric oxide ( NO), illustrate how the implication of hemoglobins in different cell signaling processes is only starting to be unraveled. The extent and diversity of the hemoglobin protein family suggest that hemoglobins have diverged and have potentially evolved specialized functions in certain organisms. A unique model organism to study this functional diversity at the cellular level is the green alga Chlamydomonas reinhardtii because, among other reasons, it contains an unusually high number of a particular type of hemoglobins known as truncated hemoglobins ( THB1- THB12). Here, we reveal a cell signaling function for a truncated hemoglobin of Chlamydomonas that affects the nitrogen assimilation pathway by simultaneously modulating NO levels and nitrate reductase ( NR) activity. First, we found that THB1 and THB2 expression is modulated by the nitrogen source and depends on NIT2, a transcription factor required for nitrate assimilation genes expression. Furthermore, THB1 is highly expressed in the presence of NO and is able to convert NO into nitrate in vitro. Finally, THB1 is maintained on its active and reduced form by NR, and in vivo lower expression of THB1 results in increased NR activity. Thus, THB1 plays a dual role in NO detoxification and in the modulation of NR activity. This mechanism can partly explain how NO inhibits NR post-translationally. [ABSTRACT FROM AUTHOR]

The hemoglobins of the cyanobacteria Synechococcus and Synechocystis (GlbNs) are capable of spontaneous and irreversible attachment of the b heme to the protein matrix. The reaction, which saturates the heme 2-vinyl by addition of a histidine residue, is reproduced in vitro by preparing the recombinant apoprotein, adding ferric heme, and reducing the iron to the ferrous state. Spontaneous covalent attachment of the heme is potentially useful for protein engineering purposes. Thus, to explore whether the histidine–heme linkage can serve in such applications, we attempted to introduce it in a test protein. We selected as our target the heme domain of Chlamydomonas eugametos LI637 (CtrHb), a eukaryotic globin that exhibits less than 50% sequence identity with the cyanobacterial GlbNs. We chose two positions, 75 in the FG corner and 111 in the H helix, to situate a histidine near a vinyl group. We characterized the proteins with gel electrophoresis, absorbance spectroscopy, and NMR analysis. Both T111H and L75H CtrHbs reacted upon reduction of the ferric starting material containing cyanide as the distal ligand to the iron. With L75H CtrHb, nearly complete (> 90%) crosslinking was observed to the 4-vinyl as expected from the X-ray structure of wild-type CtrHb. Reaction of T111H CtrHb also occurred at the 4-vinyl, in a 60% yield indicating a preference for the flipped heme orientation in the starting material. The work suggests that the His–heme modification will be applicable to the design of proteins with a non-dissociable heme group. [ABSTRACT FROM AUTHOR]

We examined the role of wheat truncated-hemoglobin ( TatrHb) in nitric oxide (NO) scavenging in transgenic Arabidopsis plants by assessing the response to an NO donor/scavenger and salt stress. The degree of increase in Na and decrease in K levels in the transgenic plants were more than those in the wild-type plants, and the ratio of Na to K increased in the transgenic plants under salt stress. Endogenous NO increased dramatically in the salt-treated wild-type plants but not in the transgenic plants. Additionally, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm) in transgenic plants decreased more significantly than that in the wild-type plants, indicating that the transgenic plants suffered more severe photosynthetic damage because of salt stress than that by the wild type. Similar results were observed in germination experiments by using Murashige and Skoog media containing 100 mM sodium chloride. The Fv/Fm decreased in the leaves of salt-treated transgenic plants, indicating that transgenic seeds were more sensitive to salt stress than that by the wild-type seeds. In addition, the negative effect on seed germination was more severe in transgenic plants than in the wild types under NaCl treatment conditions. The results support the hypothesis that plant trHb shares NO scavenging functions and characteristics with bacterial trHb. [ABSTRACT FROM AUTHOR]

Abstract: Background: Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures. Methods: We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position. Results: The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible. Conclusions: This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability. General significance: These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins. [Copyright &y& Elsevier]

ABSTRACT The X-ray structures of the hemoglobin from Synechococcus sp. PCC 7002 (GlbN) were solved in the ferric bis-histidine (1.44 Å resolution) and cyanide-bound (2.25 Å resolution) states with covalently attached heme. The two structures illustrate the conformational changes and cavity opening caused by exogenous ligand binding. They also reveal an unusually distorted heme, ruffled as in c cytochromes. Comparison to the solution structure demonstrates the influence of crystal packing on several structural elements, whereas comparison to GlbN from Synechocystis sp. PCC 6803 shows subtle differences in heme geometries and environment. The new structures will be instrumental in elucidating GlbN reactivity. Proteins 2014; 82:528-534. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The responses of transcriptome and phenolic compounds were determined with Populus tremula L. × Populus tremuloides Michx. expressing the hemoglobin (Hb) of Vitreoscilla (VHb) and non-transformant (wt) line. After 24-h exposure of leaves to Conistra vaccinii L., the transcript levels of endogenous non-symbiotic class 1 Hb ( PttHb1) and truncated Hb ( PttTrHb) genes were modestly reduced and increased, respectively, in both wt and VHb-expressing line. Besides the herbivory exposed leaves showing the most significant transcriptome changes, alterations were also detected in the transcriptome of nonorthostichous leaves positioned directly above the exposed leaves. Both wt and VHb-expressing line displayed similar herbivory-induced effects on gene expression, although the extent of responses was more pronounced in the wt than in the VHb-expressing line. The contents of phenolic compounds were not altered due to herbivory and they were alike in the wt and VHb-expressing line. In addition, we determined the relative growth rates (RGRs) of Orthosia gothica L., Ectropis crepuscularia Denis & Schiff. and Orgyia antiqua L. larvae, and found no variation in the RGRs between the lines. Thus, VHb-expressing P. tremula × tremuloides lines showed to be comparable with wt in regards to the food quality of leaves. [ABSTRACT FROM AUTHOR]

Abstract: Kinetic studies were performed on ligand rebinding to a cold-adapted globin of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 (Ph-2/2HbO). This 2/2 hemoglobin displays a rapid spectroscopic phase that is independent of CO concentration, followed by the standard bimolecular recombination. While the geminate recombination usually occurs on a ns timescale, Ph-2/2HbO displays a component of about 1μs that accounts for half of the geminate phase at 8°C, indicative of a relatively slow internal ligand binding. The O2 binding kinetics were measured in competition with CO to allow a short-time exposure of the deoxy hemes to O2 before CO replacement. Indeed Ph-2/2HbO is readily oxidised in the presence of O2, probably due to a superoxide character of the Fe O2 bond induced by of a hydrogen-bond donor amino-acid residue. Upon O2 release or iron oxidation a distal residue (probably Tyr) is able to reversibly bind to the heme and as such to compete for binding with an external ligand. The transient hexacoordinated ferrous His–Fe–Tyr conformation after O2 dissociation could initiate the electron transfer from the iron toward its final acceptor, molecular O2 under our conditions. The hexacoordination via the distal Tyr is only partial, indicating a weak interaction between Tyr and the heme under atmospheric pressure. Hydrostatic high pressure enhances the hexacoordination indicating a flexible globin that allows structural changes. The O2 binding affinity for Ph-2/2HbO, poorly affected by the competition with Tyr, is about 1Torr at 8°C, pH7.0, which is compatible for an in vivo O2 binding function; however, this globin is more likely involved in a redox reaction associating diatomic ligands and their derived oxidative species. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: The ferric form of truncated hemoglobin II from Thermobifida fusca (Tf-trHb) and its triple mutant WG8F-YB10F-YCD1F at neutral and alkaline pH, and in the presence of CN− have been characterized by resonance Raman spectroscopy, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations. Tf-trHb contains three polar residues in the distal site, namely TrpG8, TyrCD1 and TyrB10. Whereas TrpG8 can act as a potential hydrogen-bond donor, the tyrosines can act as donors or acceptors. Ligand binding in heme-containing proteins is determined by a number of factors, including the nature and conformation of the distal residues and their capability to stabilize the heme-bound ligand via hydrogen-bonding and electrostatic interactions. Since both the RR Fe–OH− and Fe–CN− frequencies are very sensitive to the distal environment, detailed information on structural variations has been obtained. The hydroxyl ligand binds only the WT protein giving rise to two different conformers. In form 1 the anion is stabilized by H-bonds with TrpG8, TyrCD1 and a water molecule, in turn H-bonded to TyrB10. In form 2, H-bonding with TyrCD1 is mediated by a water molecule. Unlike the OH− ligand, CN− binds both WT and the triple mutant giving rise to two forms with similar spectroscopic characteristics. The overall results clearly indicate that H-bonding interactions both with distal residues and water molecules are important structural determinants in the active site of Tf-trHb. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: The 2/2 hemoglobin of the cyanobacterium Synechococcus sp. PCC 7002, GlbN, coordinates the heme iron with two histidines and exists either with a b heme or with a covalently attached heme. The binding of exogenous ligands displaces the distal histidine and induces a conformational rearrangement involving the reorganization of internal void volumes. The formation of passageways within the resulting conformation is thought to facilitate ligand exchange and play a functional role. Here we monitored the perturbation induced by pressure on the ferric bis-histidine and cyanide-bound states of GlbN using 1H–15N HSQC NMR spectroscopy. We inspected the outcome with a statistical analysis of 170 homologous 2/2 hemoglobin sequences. We found that the compression landscape of GlbN, as represented by the variation of an average chemical shift parameter, was highly sensitive to ligand swapping and heme covalent attachment. Stabilization of rare conformers was observed at high pressures and consistent with cavity redistribution upon ligand binding. In all states, the EF loop was found to be exceptionally labile to pressure, suggesting a functional role as a semi-flexible hinge between the adjacent helices. Finally, coevolved clusters presented a common pattern of compensating pressure responses. The high-pressure dissection combined with protein sequence analysis established locations with volumetric signatures relevant to residual communication of 2/2 hemoglobins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Heme containing proteins are associated with peroxidase activity. The proteins like hemoglobin, myoglobins, cytochrome c and micro-peroxidase other than peroxidases have been shown to exhibit weak peroxidase-like activity. This weak peroxidase-like activity in hemoglobin-like molecules is due to heme moiety. We conducted molecular dynamics (MD) studies to decipher the unfolding path of Ba-Glb (a truncated hemoglobin from Bacillus anthracis) and the role of heme moiety to its unfolding path. The similar unfolding path is also observed in vitro by UV/VIS spectroscopy. The data confirmed that the unfolding of Ba-Glb follows a three state process with a meta-stable (intermediate) state between the native and unfolded conformations. The present study is supported by several unfolding parameters like root-mean-square-deviation (RMSD), dictionary of protein secondary structure (DSSP), and free energy landscape. Understanding the structure of hemoglobin like proteins in unicellular dreaded pathogens like B. anthracis will pave way for newer drug discovery targets and in the disease management of anthrax. [ABSTRACT FROM AUTHOR]

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539-552, ). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10-10 M s and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment. [ABSTRACT FROM AUTHOR]

Abstract: Truncated hemoglobin HbO from Mycobacterium tuberculosis displays very slow exchange of diatomic ligands with its environment. Using femtosecond spectroscopy, we show that upon photoexcitation, ligands rebind with unusual speed and efficiency. Only ∼1% O2 can escape from the heme pocket and less than 1% NO. Most remarkably, CO rebinding occurs for 95%, predominantly in 1.2ns. The general CO rebinding properties are unexpectedly robust against changes in the interactions with close by aromatic residues Trp88 (G8) and Tyr36 (CD1). Molecular dynamics simulations of the CO complex suggest that interactions of the ligand with structural water molecules as well as its rotational freedom play a role in the high reactivity of the ligand and the heme. The slow exchange of ligands between heme and environment may result from a combination of hindered ligand access to the heme pocket by the network of distal aromatic residues, and low escape probability from the pocket. [Copyright &y& Elsevier]

Eukaryotic assimilatory nitrate reductase (NR) is a multi-domain protein that catalyzes the rate-limiting step in nitrate assimilation. This protein is highly conserved and has been extensively characterized in plants and algae. Here, we report hybrid NRs (NR2-2/2HbN) identified in two microalgal species, Heterosigma akashiwo and Chattonella subsalsa, with a 2/2 hemoglobin (2/2Hb) inserted into the hinge 2 region of a prototypical NR. 2/2Hbs are a class of single-domain heme proteins found in bacteria, ciliates, algae and plants. Sequence analysis indicates that the C-terminal FAD/NADH reductase domain of NR2-2/2HbN retains identity with eukaryotic NR, suggesting that the 2/2Hb domain was inserted interior to the existing NR domain architecture. Phylogenetic analysis supports the placement of the 2/2Hb domain of NR2-2/2HbN within group I (N-type) 2/2Hbs with high similarity to mycobacterial 2/2HbNs, known to convert nitric oxide to nitrate. Experimental data confirms that H. akashiwo is capable of metabolizing nitric oxide and shows that Ha NR2- 2/2HbN expression increases in response to nitric oxide addition. Here, we propose a mechanism for the dual function of NR2-2/2HbN in which nitrate reduction and nitric oxide dioxygenase reactions are cooperative, such that conversion of nitric oxide to nitrate is followed by reduction of nitrate for assimilation as cellular nitrogen. [ABSTRACT FROM AUTHOR]

Abstract: Within the 2/2 hemoglobin sub-family, no group II 2/2Hbs from proteobacteria have been so far studied. Here we present the first structural characterization of a group II 2/2Hb from the soil and phytopathogenic bacterium Agrobacterium tumefaciens (At-2/2HbO). The crystal structure of ferric At-2/2HbO (reported at 2.1Å resolution) shows the location of specific/unique heme distal site residues (e.g., His(42)CD1, a residue distinctive of proteobacteria group II 2/2Hbs) that surround a heme-liganded water molecule. A highly intertwined hydrogen-bonded network, involving residues Tyr(26)B10, His(42)CD1, Ser(49)E7, Trp(93)G8, and three distal site water molecules, stabilizes the heme-bound ligand. Such a structural organization suggests a path for diatomic ligand diffusion to/from the heme. Neither a similar distal site structuring effect nor the presence of distal site water molecules has been so far observed in group I and group III 2/2Hbs, thus adding new distinctive information to the complex picture of currently available 2/2Hb structural and functional data. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State. [Copyright &y& Elsevier]

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous HO. [ABSTRACT FROM AUTHOR]

Truncated hemoglobins (trHbs) are distributed from bacteria to unicellular eukaryotes and have roles in oxygen transport and nitric oxide detoxification. It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein. The O association and dissociation rate constants of T. pyriformis trHb were 5.5 μMs and 0.18 s, respectively. The autooxidation rate constant was 3.8 × 10 h. These values are similar to those of HbN from Mycobacterium tuberculosis. The three-dimensional structure of an Fe(II)-O complex of T. pyriformis trHb was determined at 1.73-Å resolution. Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound O molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues. Mutations at Tyr25, Gln46, and Gln50 increased the O dissociation and autooxidation rate constants. An Fe(III)-HO complex of T. pyriformis trHb was formed following reaction of the Fe(II)-O complex of T. pyriformis trHb, in a crystal state, with nitric oxide. This suggests that T. pyriformis trHb functions in nitric oxide detoxification. Graphical Abstract: [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O/NO chemistry, O delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein. © 2011 IUBMB IUBMB Life, 63(3): 214-220, 2011 [ABSTRACT FROM AUTHOR]

Abstract: The thermostable truncated hemoglobin from the actinomyces Thermobifida fusca (Tf-trHb) displays a robust peroxidase activity, with optimum at acidic pH values, in experiments with the redox mediator ABTS. However, typical peroxidase substrates, such as phenolic or aromatic amine compounds, appear to be poor substrates for Tf-trHb. In turn, the protein is able to catalyze a unique dehydrogenation reaction of dibenzylbutanolides, suggested intermediates in the biosynthesis of podophyllotoxin, in the presence of hydrogen peroxide. Dibenzylbutanolides with a free 4″-hydroxyl group were thus converted into the corresponding 2,7″-dehydroderivatives thus setting up the basis for an efficient biotransformation of this important precursor. In particular, Tf-trHb mediated oxidation of trans-2-(4″-hydroxy-3″,5″-dimethoxybenzyl)-3-(3′,4′-methylenedioxy-7′β-hydroxybenzyl)butanolide 1 into the corresponding benzylidene-benzoyl-γ-butyrolactone 2 was obtained at high yield and with excellent selectivity. [Copyright &y& Elsevier]

The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 is a monomeric 123-residue Group I 2/2 hemoglobin. Here, we report 1H, 15N, and 13C assignments for the ferric (low-spin, S = ½) protein with a b heme cofactor and after post-translational modification leading to a c-like heme. [ABSTRACT FROM AUTHOR]

Frankia strain CcI3 produces 2 truncated hemoglobins, HbN and HbO. Using ion-exchange chromatography, we characterized the expression of the relative amounts of HbN and HbO in -N (nitrogen-fixing) cultures and +N (nitrogen-supplemented) cultures. The -N cultures maintained an approximately constant ratio of HbO to HbN throughout the life of the culture, with HbO constituting 80%-85% of the total hemoglobin produced. In contrast, in +N cultures, HbN was observed to increase over time and HbO decreased. Total hemoglobin as a fraction of total protein was approximately constant throughout the growth phase in -N cultures, while it decreased somewhat in +N cultures. Subjecting -N cultures to a NO generator resulted in increased production of HbN, relative to the controls. Nitrite accumulated in +N cultures, but not in -N cultures. This suggests that the greater amount of HbN in +N cultures might be due to NO produced by the reduction of nitrite. The effects of O2 concentration were determined in +N cultures. Cultures grown in 1% O2 produced about 4 times more HbO than cultures grown in 20% O2. Overall, these results provide evidence for a role of HbN in NO oxidation and for a role of HbO in adaptation to low oxygen concentrations. [ABSTRACT FROM AUTHOR]

Since the release in 2005 of the genome sequence and annotation of the first Antarctic marine bacterium, the number of genomes of psychrophilic microorganisms in public databases has steadily increased. Unfortunately, the lack of effective molecular tools for the manipulation of these environmental strains still hampers our understanding of their peculiar strategies to thrive in freezing conditions, limiting the functional genomics approaches to differential analyses only. Over the past two decades, our research group established the first effective gene cloning/expression technology in the Antarctic Gram-negative marine bacterium Pseudoalteromonas haloplanktis TAC125. The setup of a genome mutagenesis technique (based on homologous recombination and counterselection events) further supported the use of this strain, which became an attractive model for studying microbial adaptations to freezing lifestyle. Moreover, to further extend the functional analyses to its essential genes, the set-up of a conditional gene silencing approach is desirable. In this paper, we report the development of an asRNA regulatory system in the Antarctic bacterium, testing the feasibility of Hfq-dependent and PTasRNA strategies previously developed in Escherichia coli. Stable and efficient silencing of two chromosomal genes was obtained by using PTasRNAs, reaching very high levels of downregulation. [ABSTRACT FROM AUTHOR]

Truncated hemoglobins (trHbs) build a sub-class of the globin family, found in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants; among these, selected human pathogens are found. The trHb fold is based on a 2/2 α-helical sandwich, consisting of a simplified and reduced-size version of the classical 3/3 α-helical sandwich of vertebrate and invertebrate globins. Phylogenetic analysis indicates that trHbs further branch into three groups: group I (or trHbN), group II (or trHbO), and group III (or trHbP), each group being characterized by specific structural features. Among these, a protein matrix tunnel, or a cavity system implicated in diatomic ligand diffusion through the protein matrix, is typical of group I and group II, respectively. In general, a highly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues in the different trHb groups. Notably, some organisms display genes from more than one trHb group, suggesting that trHbN, trHbO, and trHbP may support different functions in vivo , such as detoxification of reactive nitrogen and oxygen species, respiration, oxygen storage/sensoring, thus aiding survival of an invading microorganism. Here, structural features and proposed functions of trHbs from human pathogens are reviewed. • The trHb fold is a subset of the classical globin fold, being characterized by a 2/2 α-helical sandwich. • Phylogenetic analysis indicates that trHbs branch into three groups: I (N), II (O), and III (P). • In trHbN and trHbO, diatomic ligand diffusion to the heme-iron occurs through protein matrix tunnels or cavity systems. • Proposed trHb functions are deoxification of reactive nitrogen and oxygen species, respiration, oxygen storage/sensoring. • TrHbs help the survival of selected human pathogens in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Synechocystis sp. PCC 6803 hemoglobin is a cyanobacterial Group I truncated hemoglobin. In the absence of an exogenous ligand, its single heme group is coordinated by His46 (E10, distal) and His70 (F8, proximal). The protein can undergo a post-translational modification by which His117 (H16, in the C-terminal helix) reacts with the heme 2-vinyl group to form a Markownikoff adduct. The new C–N bond prevents heme loss, alters the dynamics of the protein, and influences ligand binding to the heme group. To explore the factors conditioning the formation of the cross-link, variants of the protein that contained an alanine or a leucine at position 46 (E10) were prepared. A double replacement (His46Leu and Tyr22 (B10) to Phe) was also performed to perturb the network of interactions stabilizing bound exogenous ligand. The single and double replacements affected the optical and NMR properties of the globin, each in a different fashion. Heme–protein cross-linking, as promoted by sodium dithionite, was retarded by the replacement of His46, but reactivity was recovered when imidazole or cyanide was used as exogenous ligand. In addition, a significant amount of a second product was systematically obtained when dithionite treatment was performed on the cyanide-bound proteins. This species was identified by NMR spectroscopy to be an adduct to the 4-vinyl group. It was concluded that the specificity and rate of the cross-linking reaction depended critically on the nature of the sixth ligand to the heme iron. [Copyright &y& Elsevier]

Abstract: Analysis of amino acids sequences and protein folds has recently unraveled the structural bases and details of several proteins from the recently discovered “truncated hemoglobin” family. The analysis here presented, in agreement with previous surveys, shows that truncated hemoglobins can be classified in three main groups, based on their structural properties. Crystallographic analyses have shown that all three groups adopt a 2-on-2 α-helical sandwich fold, resulting from apparent editing of the classical 3-on-3 α-helical sandwich of vertebrate and invertebrate conventional globins. Specific structural features distinguish each of the three groups. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and TyrB10 is almost invariant through the three groups. A strongly intertwined network of hydrogen bonds stabilizes the heme bound ligand, despite variability of the heme distal residues observed in the different proteins considered. Details of ligand recognition in the three groups are discussed at the light of residue conservation and of differing ligand diffusion pathways to the heme. Based on structural analyses of the family-specific fold, we endorse a recent proposal of leaving the “truncated hemoglobins” term, that does not represent properly the observed 2-on-2 α-helical sandwich fold, and adopting the simple “2/2Hb” term to concisely address this protein family. [Copyright &y& Elsevier]

The discovery of protein sequences belonging to the widespread 'truncated hemoglobin' family has been followed in the last few years by extensive analyses of their three-dimensional structures. Truncated hemoglobins can be classified in three main groups, in light of their overall structural properties. The three groups adopt a 2-on-2 α-helical sandwich fold, based on four main α-helices of the classical 3-on-3 α-helical sandwich found in vertebrate and invertebrate globins. Each of the three groups displays sequence and structure specific features. Among these, a protein matrix tunnel system is typical of group I, a Trp residue at the G8 topological site is conserved in groups II and III, and residue TyrB10 is almost invariant in the three groups. Despite sequence variability in the heme distal site region, a strongly intertwined, but varied, network of hydrogen bonds stabilizes the heme ligand in the three protein groups. Fine mechanisms of ligand recognition and stabilization may vary based on group-specific distal site residues and on differing ligand diffusion pathways to the heme. Taken together, the structural considerations here presented underline that 'truncated hemoglobins' result from careful editing of the 3-on-3 α-helical globin sandwich fold, rather than from simple 'truncation' events. Thus, '2/2Hb' appears the most proper term to concisely address this protein family. [ABSTRACT FROM AUTHOR]

Abstract: Mycobacterium leprae GlbO has been proposed to represent merging of both O2 uptake/transport and scavenging of nitrogen reactive species. Peroxynitrite reacts with M. leprae GlbO(II)–NO leading to GlbO(III) via the GlbO(III)–NO species. The value of the second order rate constant for GlbO(III)–NO formation is >1×108 M−1 s−1 in the absence and presence of CO2 (1.2×10−3 M). The CO2-independent value of the first order rate constant for GlbO(III)–NO denitrosylation is (2.5±0.4)×101 s−1. Furthermore, peroxynitrite reacts with GlbO(II)–O2 leading to GlbO(III) via the GlbO(IV). Values of the second order rate constant for GlbO(IV)on are (4.8±0.5)×104 and (6.3±0.7)×105 M−1 s−1 in the absence and presence of CO2 (=1.2×10−3 M), respectively. The value of the second order rate constant for the peroxynitrite-mediated GlbO(IV)on (= (1.5±0.2)×104 M−1 s−1) is CO2-independent. These data argue for a role of GlbO in the defense of M. leprae against nitrosative stress. [Copyright &y& Elsevier]

Abstract: As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia coli hmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli. [Copyright &y& Elsevier]

Abstract: Ferrous oxygenated (Fe(II)O2) hemoglobins (Hb’s) and myoglobins (Mb’s) have been shown to react very rapidly with NO, yielding NO3 − and the ferric heme–protein derivative (Fe(III)), by means of the ferric heme-bound peroxynitrite intermediate (Fe(III)OONO), according to the minimum reaction scheme:For most Hb’s and Mb’s, the first step (indicated by k on) is rate limiting, the overall reaction following a bimolecular behavior. By contrast, the rate of isomerization and dissociation of Fe(III)OONO (indicated by h) is rate limiting in NO scavenging by Fe(II)O2 murine neuroglobin, thus the overall reaction follows a monomolecular behavior. Here, we report the characterization of the NO scavenging reaction by Fe(II)O2 truncated Hb GlbO from Mycobacterium leprae. Values of k on (=2.1×106 M−1 s−1) and h (=3.4s−1) for NO scavenging by Fe(II)O2 M. leprae GlbO have been determined at pH 7.3 and 20.0°C, the rate of Fe(III)OONO decay (h) is rate limiting. The Fe(III)OONO intermediate has been characterized by optical absorption spectroscopy in the Soret region. These results have been analyzed in parallel with those of monomeric and tetrameric globins as well as of flavoHb and discussed with regard to the three-dimensional structure of mycobacterial truncated Hbs and their proposed role in protection from nitrosative stress. [Copyright &y& Elsevier]

Abstract: In its resting state, the truncated globin of the cyanobacterium Synechocystis sp. PCC 6803 exhibits hexacoordination of the heme iron, with His46 (E10) and His70 (F8, proximal) serving as axial ligands. Diatomic ligands displace the distal His46 (E10) from the ferric and ferrous iron and promote considerable structural changes in the B helix, E helix, and EF regions. Here, Zn(II)-substituted hemoglobin was used to explore the role of distal ligands in stabilizing the heme pocket structure. NMR data showed that the Zn ion was coordinated by the four pyrrole nitrogens and by His70 (F8) only. The proximal side of the Zn–porphyrin adopted a geometry recognizable as that of the wild-type protein. Decoordination of His46 (E10) to form the pentacoordinate Zn resulted in an incomplete transition to the conformation observed in the ferric, cyanide-bound protein. The NMR data also demonstrated that the H helix underwent complex dynamic processes near His117, a residue readily reacting with the wild-type heme 2-vinyl group in a post-translational modification. [Copyright &y& Elsevier]

Abstract: A hypothetical open reading frame from Bacillus subtilis genome, yjbI [NCBI genome database Accession No. A69844] having homology to many globin and globin-like proteins from different microbial genomes, was selectively amplified from the chromosomal DNA of B. subtilis strain DB104 based on genome sequence database of B. subtilis strain 168. The gene was cloned and over-expressed in Escherichia coli under the transcriptional control of tandem ¿ PL and PR promoters, and the protein was purified to homogeneity. The single-chain monomeric hemoglobin-like protein is stable to the extent of 5.45kcal/mol at 25°C, binds carbon mono-oxide, and shows optical spectra characteristic of hemoproteins. The protein also exhibits peroxidase-like activity. This is the first report of a truncated bacterial globin endowed with peroxidase-like activity. The activity is enhanced in the presence of urea and guanidine hydrochloride, more so in the presence of the latter. Presumably, only a small portion of the protein is involved in peroxidase activity, which is exposed with increasing concentration of the denaturants. [Copyright &y& Elsevier]

Abstract: Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20–40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 α-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 α-helical globin fold, achieved through deletion/truncation of α-helices and specific residue substitutions. Despite their ‘minimal’ polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed. [Copyright &y& Elsevier]

Leprosy is an old, still dreaded infectious disease caused by the obligate intracellular bacterium Mycobacterium leprae. During the infectious process, M. leprae is faced with the host macrophagic environment, where the oxidative stress and NO release, combined with low pH, low pO2, and high pCO2, contribute to limit the growth of the bacilli. Comparative genomics has unraveled massive gene decay in M. leprae, linking the strictly parasitic lifestyle with the reductive genome evolution. Compared with Mycobacterium tuberculosis and Mycobacterium bovis, the leprosy bacillus has lost most of the genes involved in the detoxification of reactive oxygen and nitrogen species. The very low reactivity of the unique truncated hemoglobin retained by M. leprae could account for the susceptibility of this exceptionally slow-growing microbe to NO. [ABSTRACT FROM AUTHOR]

Truncated hemoglobins (trHb''s) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb''s branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb''s, such as 20–40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 α-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3×103 M−1 s−1) is similar to that observed for A. thaliana trHbO-3 (1.4×104 M−1 s−1) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7×106 M−1 s−1). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells. [Copyright &y& Elsevier]

THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate–protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions. [Display omitted] • THB1, a truncated hemoglobin from a green alga, has His-Fe-Lys heme coordination. • The crystal structure of cyanide bound (His-Fe-CN) THB1 was determined. • Heme peripheral interactions perturbed by the distal ligand switch were targeted. • pH titrations and molecular dynamic simulations were performed. • Determinants of Lys affinity for the heme iron were identified. [ABSTRACT FROM AUTHOR]