ABSTRACT. Nitrobindins (Nbs) form a new class of evolutionary conserved heme-proteins characterized by a 10-stranded anti-parallel β-barrel fold. In Nbs, the heme-Fe atom is coordinated to a proximal His residue and is stable in the ferric form. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in both NO/O2 and reactive nitrogen and oxygens species (RNS and ROS, respectively) metabolism.To date, Mycobacterium tuberculosis Nb (Mt-Nb), Arabidopsis thaliana Nb (At-Nb), Homo sapiens Nb (Hs-Nb), and Danio rerio Nb (Dr-Nb) have been characterized by our research group from both the structural and functional viewpoints. Interestingly, while Mt-Nb, At-Nb and Dr-Nb are single-domain proteins, Hs-Nb has been described as a domain of the human protein named THAP4. THAP4 is composed of an N‐terminal modified zinc finger domain able to bind DNA and a C‐terminal Nb domain. To define the role of THAP4 in RNS detoxification, silencing experiments have been performed in breast cancer MCF-7 human cells, in which the expression levels of THAP-4 results higher compared to other human cell lines. Besides, transcriptomic analyses have been performed in both wild-type and silenced cells after treatment with the NO-donor DEA NONOate. In addition, the THAP4 interactome has been retrieved using BioGRID and IntAct molecular interaction databases. Finally, the Gene Ontology Resource and the web server QuickGo have been used to investigate the biological processes involving THAP4. Overall, data obtained suggest an evolutionary conserved structure and anti-oxidant function of Nbs, and highlight a possible role of human THAP4 as a sensing protein that couples the heme-based Nb reactivity with gene transcription.

ABSTRACT. The great oxidation event that led to an abundance of oxygen in the Earth’s atmosphere delivered an attractive alternative as a respiratory electron acceptor, which has clearly had a dramatic impact upon the evolution of electron transport components. The first part of this talk will touch upon the evolution of oxygen-binding respiratory oxidoreductases and highlight the diversity of these protein complexes. The second part of the talk will focus on Cytochrome bd complexes, which are found exclusively in prokaryotes and generate a proton motive force by coupling quinol oxidation to the reduction of dioxygen. Previous work has demonstrated that cytochrome bd complexes are important during infection for a variety of bacterial pathogens, including E. coli and M. tuberculosis, demonstrating their potential as drug targets. Herein, in silico tools were used to screen a library of approved drugs for their ability to inhibit cytochrome bd-I from E. coli. In order to investigate the efficacy and specificity of the top hits, mutant strains of E. coli that express either cytochrome bd-I or cytochrome bo’ as the sole respiratory oxidase were used as a test system, and the expected haem signals of these respiratory oxidases were confirmed for these strains using difference spectroscopy. Membranes were isolated from these strains, and candidate drugs from the in silico analyses were tested for their ability to inhibit oxygen consumption by cytochrome bd-I and cytochrome bo’ using an oxygen electrode. Selected drugs were identified as potent inhibitors of cytochrome bd-I, and further work has been undertaken to aid our understanding of their mechanisms of action and potential for broader applications in antimicrobial chemotherapy.

ABSTRACT. Heme sensor proteins detect changes of either heme (so called heme-responsive sensor proteins) or gas signalling molecules (so called heme-based gas sensor proteins) concentration in many cell types and subsequently modulate their specific functions such as enzymatic activity or DNA binding. Hence, these proteins are intensively discussed as potential therapeutic targets in many pathological processes including bacterial infections or cancer [Shimizu T. et al 2019. Chem. Soc. Rev. 48:5624-5657].

Specifically, heme regulated inhibitor (HRI) has been chosen as a model heme-responsive sensor protein in our study. It is a kinase of eukaryotic initiation factor 2α and therefore affects protein synthesis throughout this mechanism. If heme molecule interacts with HRI, the kinase activity is inhibited. Recently, we have focused on the kinetic analysis of HRI in the presence of various nucleotide triphosphates (NTPs) as their concentration in cells could be modulated as a result of cancer associated processes [Shugar D. 1996. Acta Biochim. Pol. 43:9-24]. In addition to that, HRI has been reported as a potential nitrogen monoxide sensor in cells [Martinkova M. et al 2007. FEBS Lett. 581:4109-4114]. Furthermore, various sulphur species (mostly hydrogen sulfide) has been recently discussed as potential signalling molecules [Kimura H. 2015. Antioxid. Redox Signal. 22:362-375], so we have studied their effect on the kinase activity of HRI.

The effect of HRI incubation with various NTPs as well as selected sulphur species on its kinase activity will be discussed. The results of this study will broaden our knowledge about heme sensor proteins mechanism of function as well as their importance in pathological processes including cancer.

Supported by the grant 158120 from the Grant Agency of Charles University.

ABSTRACT. Globin-coupled histidine kinase from Anaeromyxobacter sp. Fw109-5 (AfGcHK) is an oxygen sensor enzyme in which oxygen binding to Fe(II) heme in the globin sensor domain substantially enhances its autophosphorylation activity. Here, we reconstituted AfGcHK with cobalt protoporphyrin IX (Co-AfGcHK) in place of heme (Fe-AfGcHK) and characterized spectral and catalytic properties of the full-length proteins. Spectroscopic analyses indicated that Co(III) and Co(II)-O2 complexes were in a 6-coordinated low-spin state in Co-AfGcHK, like Fe(III) and Fe(II)-O2 complexes of Fe-AfGcHK. Although both Fe(II) and Co(II) complexes were in a 5-coordinated state, Fe(II) and Co(II) complexes were in high-spin and low-spin states, respectively. The autophosphorylation activity of Co(III) and Co(II)-O2 complexes of Co-AfGcHK were fully active, whereas that of the Co(II) complex was moderately active. This contrasts with Fe-AfGcHK, where Fe(III) and Fe(II)-O2 complexes were fully active and the Fe(II) complex was inactive. Collectively, activity data and coordination structures of Fe-AfGcHK and Co-AfGcHK indicate that all fully active forms were in a 6-coordinated low-spin state, whereas the inactive form was in a 5-coordinated high-spin state. The 5-coordinated low-spin complex was moderately active—a novel finding of this study. These results suggest that the catalytic activity of AfGcHK is regulated by its heme coordination structure, especially the spin state of its heme iron. This study presents the first successful preparation and characterization of a cobalt porphyrin-substituted globin-coupled oxygen sensor enzyme, and may lead to a better understanding of the molecular mechanisms of catalytic regulation in this family.

Ref. Kitanishi, K. et al. (2021) ACS Omega 6, 34912-34919.

ABSTRACT. The coordination of hydrogen sulfide, H2S, to ferric hemeproteins has been reported in more than 40 examples, and forms moderately stable hexacoordinated low spin complexes, [FeIII(SH-)].1 The metal centered reduction has been reported in some cases, with varying timescales. Subsequent aerobic reactivity of the metmyoglobin complex, MbFeIII(SH-) has been reported and yields myoglobin, MbFeII, with the concomitant formation of thiosulfate, sulfite and polysulfides.2 Combining kinetic and spectroscopic methods, we proposed a mechanism for the reduction of MbFeIII by excess sulfide, under argon atmosphere.Asymmetric S-shaped time-traces for the formation of MbFeII, under varying sulfide concentrations or pH were observed, pointing to an autocatalytic behavior. Further analysis of the time-traces at selected times revealed a secondary sigmoidal dependence on the initial concentration of sulfide, suggesting a full time-dose response. The slow initial phase is suggested to depend on the resonant form FeII(SH*) of the starting complex, yielding minor quantities of MbFeII and sulfanyl radical, SH*. The overall rate of the reaction is augmented with increasing pH, pointing to hydrosulfide, SH-, as a critical species in the early steps. We propose the intermediacy of the disulfanuidyl radical anion, HSS*2-, promoted under alkaline conditions by reaction of HS- and HS*, as a source of disulfane (HSS-).3 Significantly, the formation of MbFeII after the addition of HSS- to MbFeIII(SH-) is faster than the isolated addition of HS- or HSS- to MbFeIII(H2O), suggesting a synergistic effect, and pointing to HSS- as a key species in the steep increase of the reduction rate of MbFeIII by sulfide. Kinetic simulations of the sigmoidal traces assisted the evaluation of the proposed mechanism.The process has been termed reductive sulfhydration, after the well described reductive nitrosylation; this denomination should be discussed, as ferrous forms do not form stable complexes with sulfide

ABSTRACT. Carbon monoxide (CO) is an important signaling molecule that has been implicated in physiological processes ranging from inflammation response to cellular proliferation to circadian regulation. Given that CO is chemically inert under most physiological conditions, hemoproteins, which exhibit a strong affinity for CO, represent a class of likely protein targets for CO signaling. Importantly, hemoproteins involved in CO signaling must selectively respond to CO in the presence other heme-binding small molecules, such as oxygen (O2) and nitric oxide (NO). To better understand CO selectivity in biological signaling, we examined the CO-sensing transcription factor, RcoM (regulator of CO metabolism), from the soil bacterium Paraburkholderia xenovorans. The RcoM heme, which is constitutively coordinated by His74, undergoes a unique redox-mediated ligand switching mechanism on the opposite heme face: Cys94, which binds to Fe(III) heme, is replaced by Met104 upon heme reduction. This weakly bound Met is replaced by CO, which exhibits nanomolar affinity for the RcoM Fe(II) heme. Using electronic absorption spectroscopy, we characterized ligand binding and chemical reactivity in variants of the PxRcoM-1 ortholog bearing alterations to distal heme pocket residues Cys94, Met104, and Met105. We studied heme pocket alterations in full-length PxRcoM-1 and in truncates bearing the C-terminal, heme-binding Per-Arnt-Sim (PAS) domain. Given that RcoM exhibits remarkably high affinity for CO, we directly quantified CO binding equilibrium constants (KCO) through a competition assay using a well-characterized, high-affinity variant of human neuroglobin (Ngb-H64Q-CCC). In general, heme-binding truncates exhibited a more open heme pocket than full-length variants, as evidenced by larger ligand binding equilibrium constants and faster nitrite reduction rates. The influence of alterations to heme pocket residues on ligand binding affinities will be discussed in detail.

ABSTRACT. The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization of C. elegans globin-3 (GLB-3) using a multitude of techniques. GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. The presented spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl ligated low-spin form both in the ferrous and ferric state. Unlike other globins, GLB-3 is not capable of reacting with H2O2, H2S, and nitrite. Site-directed point mutation of the distal histidine to an alanine resulted in nitrite reductase activity, implying the strong distal histidine affinity to the heme iron in the wild-type globin. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh (pH = 2) conditions. Redox potentiometric measurements reveal a positive redox potential (+8 ± 19 mV vs. SHE) similar to other heme proteins. Based on our study and a detailed comparison with other globins, we postulate an electron transfer function for GLB-3.

ABSTRACT. Globin Y (GbY), one of the “new members” of the vertebrate globin family, has been documented in the genomes of lobe-finned and cartilaginous fish, amphibians, several reptiles and in the monotreme platypus, but not in most bony fish, birds, or higher mammals. Until now, there is only few data on the physicochemical properties of GbY, and a reliable hypothesis about its physiological function is still missing. The knowledge of main sites of expression may help to understand its function. Here we analyzed the expression pattern of GbY in the kidney of the frog Xenopus laevis and the "living fossile" Lepisosteus oculatus, an ancient fish with a primitive lung belonging to the Neopterygii, via publicly available RNA-Seq data, immunofluorescence and mRNA in situ hybridization. RNA-Seq and qPCR data pointed to a very high GbY mRNA expression in the adult kidney of both species. Subsequent immunofluorescence combined with mRNA in situ hybridizations showed an allocation of GbY in the proximal tubules covered with microvilli. Only in X. laevis, but not the fish, GbY was localized in the inter-renal, possibly steroidogenic cells, which have a very high fat content. In those inter-renal cells, the GbY signals are in close proximity to lipid droplets, always surrounded by serotonin-positive chromaffine cells. We hypothesize that the observed difference in the distribution of GbY expression in adult kidneys of fish and amphibia may hint at two distinct functions of GbY, of which the role in the proximal tubules is conserved. The association of GbY with lipid droplets in inter-renal cells could imply a role in fatty acid metabolism, like it was suggested for myoglobin and cytoglobin.

ABSTRACT. Recently, a new class of all-β-barrel heme-proteins, named nitrobindins (Nbs), has been identified along the evolutionary ladder (De Simone et al., 2016). Nbs are characterized by a stable solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. To date, the physiological role of Nbs has not been understood. However, in vitro evidence suggest that ferric Nbs from Arabidopsis thaliana (At-Nb), Mycobacterium tuberculosis (Mt-Nb) and Homo sapiens (Hs-Nb) are able to catalyze the conversion of peroxynitrite to nitrate (De Simone et al., 2018, 2020). Noteworthy, in both Mt-Nb (PDB ID: 6R3W; De Simone et al., 2020) and At-Nb (PDB ID: 3EMM; Bianchetti et al., 2010) crystal structures, a water molecule is coordinated to the heme-Fe(III) atom forming a 6-coordinate High Spin (6cHS) His-Fe-H2O. Although nuclear magnetic relaxation dispersion (NMRD) profiles showed a fast exchange between coordinated and bulk water, the reactivity of Mt-Nb and Hs-Nb appears to be modulated by residues of the heme distal pocket (De Simone et al., 2020). In order to investigate the solvent-mediated interactions between the heme distal pocket and the heme-Fe atom, we performed classical molecular dynamics simulations. The present computational approach allowed us to observe the system's dynamics, trying to understand how the solvent and the heme pocket could influence the Nb function. In addition, steered molecular dynamics simulations were performed to obtain the free energy profiles for ligand migration (Capece et al., 2013).

ABSTRACT. Nitrobindins (Nbs) are all-β-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O2 metabolism. The high reactivity of Nbs towards reactive nitrogen and oxygen species reflects the solvent exposure of the metal center [1,2,3]. To date, Arabidopsis thaliana Nb (At-Nb), Mycobacterium tuberculosis Nb (Mt-Nb), and Homo sapiens Nb (Hs-Nb) have been characterized from both structural and functional viewpoints [1,2,4,5]. To study Nb function(s), Danio rerio Nb (Dr-Nb) has been expressed, purified, and spectroscopically characterized. Around neutrality, the UV-Vis and Resonance Raman (RR) spectra of ferric Dr-Nb display a mixture of a 5cHS and a 6cHS aquo species. Similarly to Mt-Nb and Hs-Nb, the ferrous form is mainly 5cHS, characterized by the same Fe-proximal His bond strength. Furthermore, both UV-Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is mostly five-coordinated. Kinetics of Dr-Nb(II) nitrosylation are likely impaired by the crowded network of water molecules which shields the heme pocket. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy way out for the ligand into the outer solvent. Ongoing studies involving the RR spectroscopy of the CO complexes, the kinetics of CO binding, and the resolution of Dr-Nb three-dimensional structure will allow us to understand the structure-function relationships of fish Nb.

ABSTRACT. There is a significant clinical need for a synthetic oxygen therapeutic / blood substitute that is both long-lasting and sterile. Hemoglobin-based oxygen carriers (HBOCs) have been a focus of numerous studies and development as a potential artificial oxygen therapeutic / blood substitute. However, previous attempts have met with many failures seen during clinical trials due to observed adverse effects and toxicities. We have utilised genetic engineering to reengineer the hemoglobin molecule, adding numerous features, whilst keeping a focus on protein and heme stability. Using fetal hemoglobin as a template has made the protein much more stable with lower rates of autoxidation and heme loss. A decrease in nitric oxide dioxygenase activity to limit the nitric oxide scavenging capacity of the HBOC was achieved by using the mutations reported previously by the Olson group. Several new technologies have been added to our HBOC: The addition of through-protein electron transfer pathway to enhance the reaction of ferric and ferryl oxidation states with plasma antioxidants such as ascorbate and urate. Furthermore, we have added a mutation to generate a homogeneously PEGylated HBOC that does not significantly disrupt the allosteric oxygen binding of the HBOC. We have completed a scalable manufacturing process and are currently conducting focused pre-clinical studies to specifically identify potential toxic effects of our HBOC on various tissues including myocardial and renal tissue. This approach seeks to de-risk future full pre-clinical and Phase I/II clinical trials. Initial clinical targets for the product will be as an oxygen therapeutic, potentially treating patients with stroke and other thromboses, sickle cell crisis, trauma, those with impaired immune systems and numerous other potential roles.

ABSTRACT. Androglobin, a multi-domain hemoglobin of the metazoans, has been proposed to play a role in the formation of motile cilia during ciliogenesis and spermatogenesis. The 190 kDa human protein has several domains, including a central circularly permuted globin domain, an N-terminal region containing a calpain-like region and a largely disordered C-terminal region containing sequences for a coiled-coil region, and nuclear localization signal and endoplasmic reticulum retention signal. Previous studies of the expression of the full-length androglobin have reported potential auto-proteolytic activity of the calpain domain (1). The active site of calpain proteases typically contain a cysteine-histidine-asparagine catalytic triad. Examination of the sequence homology of the calpain domain of androglobin with human calpain-7 has identified a partially conserved candidate for the active site cysteine (2). However, the calpain-7 active site histidine and aspartate residues did not show equivalent residues in sequence alignments to androglobin sequences. Nonetheless, a number of conserved residues for the histidine and asparagine are present that could present candidates for these active site residues. Here we examine the recombinant expression of the N-terminal domain and its auto-proteolytic activity. We have also examined potential candidates for the active site residues for their effect on auto-proteolytic activity through site-directed mutagenesis.

(1) Bracke, A.; Hoogewijs, D.; Dewilde, S., Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda. Anal. Biochem. 2018, 543, 62-70.

(2) Hoogewijs, D.; Ebner, B.; Germani, F.; Hoffmann, F. G.; Fabrizius, A.; Moens, L.; Burmester, T.; Dewilde, S.; Storz, J. F.; Vinogradov, S. N.; Hankeln, T., Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes. Mol. Biol. Evol. 2012, 29 (4), 1105-14.

ABSTRACT. Dihydrogen disulfane, H2S2, is an endogenous species that has been reported as effector of the biochemical activity of hydrogen sulfide, H2S, in certain tissues.1 The metal centered reduction of the heme protein indoleamine 2,3 dioxygenase by disodium disulfane, Na2S2, leading to a potent activation of the enzyme, precedes and highlights the interest of heme proteins as biochemical targets of disulfane species.2 Herein, we focused on the reactivity of H2S2, or the conjugated base HSS¯, towards metmyoglobin, MbFeIII. The reaction of excess Na2S2 with MbFeIII, was studied under argon atmosphere at 25ºC. A fast biexponential formation of MbFeII was observed by UV-Vis spectroscopy in the interval 6<pH<8, with varying concentrations of disulfane, suggesting the one-electron oxidation of HSS¯, forming the disulfanyl radical HSS* along with MbFeII. The intermediacy of a coordination complex, with UV-Vis features similar to that of the characterized [MbFeIII(SH¯)] complex,3 was detected at the more acidic pH evaluated. The formation of a hexacoordinated low spin complex was confirmed by resonance Raman spectroscopy at 10ºC. As control experiments of the sulfide-mediated reduction MbFeIII reveal a hysteretic behavior, the participation of contaminant H2S/HS¯ is disregarded, suggesting the formation of a [MbFeIII(SSH¯)] complex. The spectroscopic detection of higher polysulfides HSnH (2<n<8) in the final reaction mixture, may be not only a consequence of the dimerization of HSS*, but also the result of the chemistry of disulfane, that characterizes its aqueous solutions.3 References (1) Kimura, H. Neurochem. Internat. 2013, 63 (5), 492–497 (10.1016/j.neuint.2013.09.003). (2) Nelp, N.T. et al. J Am Chem Soc. 2019, 141, 15288-15300 (10.1021/jacs.9b07338). (3) Bogdándi, V. et al. Br J Pharmacol. 2019, 176(4), 646-670 (10.1111/bph.14394).

ABSTRACT. More than two-thirds of the world does not have adequate blood supplies. Moreover, conventional red blood cells have a limited shelf life. Therefore, Hemoglobin based oxygen carriers (HBOCs) were developed as alternatives to blood transfusion and used as oxygen therapeutics in ischemic conditions. These HBOCs should have high heme affinity, low autooxidation rate, high shelf life, and high apoglobin stability. We can use site-directed mutagenesis to change amino acids surrounding the heme pocket in recombinant human hemoglobin in order to enhance heme stability based on the unprecedented heme stability of Synechocystis hemoglobin, which was successfully engineered in myoglobin. We have analyzed heme retention ability, autooxidation rates, and oxygen-binding properties of some of the mutants, of which one was significantly stable with retarded heme loss. Since these rHbs are expressed in E.coli, these might confer immunogenic problems when administered in animal or human subjects; therefore, it is highly desirable to employ sensitive and selective detection of lipopolysaccharides in recombinant hemoglobin solutions, which must be subsequently depleted.

ABSTRACT. Campylobacter jejuni is the major cause of gastroenteritis in the world and responsible for ~1/4 of diarrhoeal diseases in the world. Despite the increasing number of infections caused by C. jejuni, the molecular mechanisms involved in pathogen’s survival within the host are still poorly understood. Haem biosynthesis plays a crucial role in pathogen´s physiology as it ensures the formation of several essential haem-binding proteins/enzymes. Indeed, haem cofactor is responsible for the function of several key cellular processes such as, respiration, signaling and oxidative stress detoxification. Therefore, most prokaryotes, synthesize haem endogenously via specific Haem Synthesis Pathways (HSP). All the so far known HSP begin with the universal tetrapyrrole precursor δ-aminolaevulinic acid (ALA) and uses a cascade of enzymes to finally produce haem. The best-known pathway, currently named the protoporphyrin dependent pathway (PPD), involves at least eight enzymes to go from ALA to haem and is mostly present in Gram-negative bacteria. Our group participated in the discovery of two other distinct pathways, namely the sirohaem dependent pathway and the coproporphyrin dependent pathway (1–4). That are present mainly in sulphate-reducing bacteria and Gram-positive pathogens, respectively. We described our data on the elucidation of which pathway does C. jejuni uses to synthesise haem. We performed the biochemical characterization of the enzymes involved in this pathway. Moreover, we show results on the identification of a novel putative haem chaperon encoded in C. jejuni.

ABSTRACT. Cytochrome bd complexes are terminal respiratory oxidases found exclusively in the aerobic respiratory chains of prokaryotes that generate a proton motive force by coupling quinol oxidation to the reduction of dioxygen. Previous work has demonstrated that cytochrome bd complexes are important during infection for a variety of bacterial pathogens, including E. coli and M. tuberculosis, demonstrating their potential as drug targets. Herein, in silico tools were used to screen a library of approved drugs for their ability to inhibit cytochrome bd-I from E. coli. In order to investigate the efficacy and specificity of the top hits, mutant strains of E. coli that express either cytochrome bd-I or cytochrome bo’ as the sole respiratory oxidase were used as a test system, and the expected spectral signals of these respiratory oxidases were confirmed for these strains using difference spectroscopy. Membranes were isolated from these strains, and candidate drugs from the in silico analyses were tested for their ability to inhibit oxygen consumption by cytochrome bd-I or cytochrome bo’ using an oxygen electrode. Selected drugs were identified as inhibitors of cytochrome bd-I, and further work has been undertaken to aid our understanding of their mechanisms of action and potential for broader applications in antimicrobial chemotherapy.

ABSTRACT. Tuberculosis is amongst the major worldwide health threats. Besides the spread of the disease, multidrug-resistant strains of Mycobacterium tuberculosis (Mt) have emerged. As a consequence, treatment failure is, unfortunately, becoming more usual and there is urgency for disclosing innovative therapeutic strategies, which has lead to renewed screenings for antimycobacterial substances and the identification of novel targets and mechanisms of action.

Truncated hemoglobin, tHbN, of Mt protects its host from the toxic effects of nitric oxide (NO) due to its potent O2-dependent NO dioxygenase (NOD) activity. This protein converts NO produced by macrophages into the harmless nitrate anion. Based on the studies of our research group about the structure of tHbN and the migration of NO and O2, protein tunnel system composed of short and long branches facilitates ligand entry to the distal heme site. On the other hand, the oxyferrous heme interacts with NO to make nitrate and ferric heme, and subsequently a reductase partner would be needed to recover the ferrous state, thus enabling the protein to start the cycle again.

This communication reports the efforts carried out in the search of putative reductase partners, which to the best of our knowledge has not been identified yet. A careful bioinformatics analysis led to a selected reductase candidate, whose relevance for the survival of the bacillus has been experimentally confirmed. Our work has included building up a 3D homology model of the reductase as well as of the complex formed with tHbN, which has been subsequently refined by extended Molecular Dynamics simulations. Furthermore, calculations have been performed to estimate the feasibility of the electron transfer process between reductase and tHbN. Finally, the 3D model has enabled to disclose druggable pockets that might mediate the inhibitory NOD activity.

ABSTRACT. Understanding adaptation to environmental change is of fundamental importance to predicting the evolvability of species in the Anthropocene. Antarctic icefishes (Channichthyidae) lost the ability to produce red blood cells as the Southern Ocean (SO) cooled and dissolved oxygen concentrations rose, providing a test case for analyzing the evolutionary genomic responses to environmental change and the potential for species resilience as the SO now warms. By integrating paleoclimate records with an extensive phylogenomic dataset, we demonstrate relaxation of purifying selection across erythrocyte-associated genetic regions following a rapid decline in global temperatures and the formation of stable ice sheets. Acceleration of variation in erythrocyte-associated regions continues in modern Antarctic notothenioids, including red-blooded species. For example, we detected predicted deleterious variation in the beta-spectrin gene of red-blooded dragonfishes, one of which has spherocytic erythrocytes like those observed in humans with mutations in this gene. Despite loss-of-function mutations in a few key erythrocyte-specific genes, we show that most of the erythroid genetic toolkit has been maintained in icefishes. Interestingly, there is a bias in the accumulation of drift in putative gene-regulatory regions flanking genes expressed late in erythropoiesis. Together, results indicate that erythropoiesis in icefishes is blocked late in erythrocyte differentiation, consistent with the presence of proerythroblasts in icefishes. Our results provide a comprehensive phylogenomic perspective of the genetic changes in icefishes that led to loss of erythrocytes and a framework for understanding the potential for their adaptive resilience as the SO warms. Supported by US NSF PLR-1444167 (H.W.D.) and OPP-1955368 (JD, HWD).

ABSTRACT. The hypoxic and cold environment at high altitudes requires that endotherms sustain high rates of O2 consumption for thermogenesis and locomotion while facing diminished O2 availability. Haemoglobin (Hb) evolution has contributed to high-altitude adaptation in many high-altitude mammals and birds, but the physiological significance of changes in Hb gene sequence and protein function has remained largely unresolved. I will discuss our research on this issue in the deer mouse (Peromyscus maniculatus). Deer mice exhibit the broadest elevational distribution of any North American mammal, and high-altitude populations have evolved increased Hb-O2 affinity. I will show that the physiological significance of this evolved change in Hb function on aerobic capacity was likely contingent upon antecedent changes at other steps in the O2 transport pathway (e.g., O2 diffusing capacity of active tissues). I will also show that variation in Hb genes has other unanticipated effects on respiratory physiology, beyond the canonical function of this protein in circulatory O2 transport. Our work highlights the importance of integrative approaches that maintain a wide field of vision for uncovering the adaptive significance of haemoglobin evolution.

ABSTRACT. Neuroglobin is part of the globin superfamily and is expressed in the central and peripheral nervous system of every vertebrate class. It is presumed to have a conserved and important physiological function. A wide range of potential functions has been described, including a protective role in neurons in hypoxic and oxidative stress related insults. However, the underlying mechanisms of neuroprotection of neuroglobin remain poorly understood. Here, we have investigated the function of mouse and zebrafish neuroglobin as well as mouse myoglobin in a murine neuronal cell culture (HN33) under hypoxia (1% O2) and oxidative stress (0.5 µM H2O2). Transfection of mouse neurons with all three globins resulted in reduced Caspase activity in normoxia, hypoxia and, most significantly, under oxidative stress compared to the mock control. Cell viability, measured via CellTiter-Glo assay showed higher viability of cells transfected with zebrafish neuroglobin and mouse myoglobin under hypoxia and of mouse neuroglobin cells under ROS stress. To investigate the cellular effects of globin overexpression, we analyzed the transcriptomes of the transfected HN33 cells after treatment with normoxia, hypoxia and ROS stress. Transfection of mouse and zebrafish neuroglobin resulted in upregulation of genes regulating neuronal cell death, response to oxygen levels and glycolytic processes in normoxic conditions. Mock and myoglobin transfected cells showed a conserved cellular response to hypoxia with upregulation of glycolytic genes. Little regulation occurred in neuroglobin transfected cells after subjecting them to hypoxia. In addition, no significant regulation was found between cells transfected with mouse neuroglobin in normoxic conditions and after ROS treatment. This indicates that overexpression of neuroglobin but not myoglobin results in a general cellular answer, enhancing cell survival in hypoxic and oxidative stress conditions without high levels of regulation of gene expression.

ABSTRACT. In the literature, there are many studies on vertebrate cytoglobin, but the properties of non-mammalian cytoglobins are largely uncharacterized. As most teleost fish, zebrafish harbor two cytoglobin paralogs, cytoglobin 1 and cytoglobin 2. Dre-cygb1 is expressed ubiquitously in most zebrafish tissues, whereas Dre-cygb2 is highly expressed in neuronal tissue. Here we describe the effects of CRISPR/Cas9 induced cygb1 deficiency on zebrafish physiology and development. We report enhanced occurrence of age-dependent degenerative changes in adult cygb1-/-, mainly weight loss phenotype accompanied by surface respiration in male zebrafish. Transcriptome analysis revealed both tissue specific and global knockout effects. In brain and liver the lipid metabolism, hypoxic response (without stress) and inflammation pathways were dysregulated. In the cygb1-/- liver apolipoprotein D (apoda.2) was strongly upregulated. Apoda.2 is related with aging processes and might function as a “good Samaritan” helping cells to cope with oxidative stress. In the cygb1-/- brain we found enhanced expression of hemoglobin alpha and beta (hbaa1; hbba1) and glutathione peroxidase (gpx1a), that points to a local hypoxic adaptation. Further, we conducted hypoxia experiments in adult WT and in cygb1-/- larvae (33hpf) to evaluate the effect of the knockout during development. Cygb1-/- larvae were more susceptible to hypoxia (~0.7 kPa) since enolase 1 (eno1), heatshock protein 27 (hsp27) and vascular endothelial growth factor (vegfa) expression was enhanced. Additionally, cygb1-/- larvae strongly downregulated protein translation during hypoxia. The penta-coordination of cygb1 and its slow autoxidation rate together with our results might point to a role for cygb1 as an intracellular oxygen carrier protein in zebrafish.

ABSTRACT. In the freezing waters of the Southern Ocean, the dominant suborder of Antarctic teleosts, Notothenioidei, have developed unique adaptations to cope with cold, including, at the extreme, the loss of hemoglobin in icefish. As a consequence, icefish are thought to be the most vulnerable of the Antarctic fish species to ongoing ocean warming. Notothenioidei are one of the most interesting models to study how low temperature and high oxygen concentrations, the main environmental stressors over millions of years, have affected oxygen supply and delivery in Antarctic marine environments. Some icefish also fail to express myoglobin but all appear to retain neuroglobin, cytoglobin-1, cytoglobin-2, and globin-X. Temperature up-regulates globin expression more effectively in white-blooded than in red-blooded fish while hypoxia strongly up-regulates globins in red-blooded fish, particularly in the gills, suggesting that globins function as regulators of temperature and hypoxia tolerance. The structural and functional properties of neuroglobins and cytoglobins of Antarctic notothenioids have been studied and their adaptive features have been inferred from comparisons with human proteins. Similar to the human proteins, Antarctic fish neuroglobin, cytoglobin-1 and cytoglobin-2 can reversibly bind oxygen and carbon monoxide in the Fe2+ form, and show six-coordination by distal His in the absence of exogenous ligands. The 3D structure of Dissostichus mawsoni cytoglobin-1 has been determined through X-ray crystallography at 3 Å resolution (data collected at ESRF, Grenoble, France). At the light of a remarkable 3D-structure conservation, the observed differences in ligand-binding kinetics may reflect specific features in the dynamics of the heme distal region and protein matrix cavities, suggesting adaptation to functional requirements posed by the cold environment.

ABSTRACT. The subterranean blind mole rat Nannospalax galili, which populates underground burrows, is adapted to a temporary lack of oxygen (hypoxia), even surviving ~3% O2 for 14h. The adaptation to hypoxia is accompanied by an enhanced haematocrit and increased concentrations of haemoglobin. Additionally, elevated mRNA- and protein level for other oxygen-binding proteins such as myoglobin, neuroglobin and cytoglobin compared to rat have been detected in various Spalax tissues. One of the mole rats most interesting features is its extended lifespan. Spalax become five to seven times older than its close relatives, mouse and rat, without displaying clear signs of ageing or ageing-related disorders. We hypothesize that the longevity phenotype in Spalax, which is paralleled by a resistance against tumor formation, might be mechanistically linked to the hypoxia adaptation. To infer Spalax-specific gene regulation patterns, we produced RNA-Seq data of liver, kidney and spleen heart from Spalax individuals subjected to 6% O2 or normoxia and compared these data to the hypoxia-sensitive rat. In all three organs, we observed a significantly higher hypoxia-induced transcriptional stress response in the rat. In Spalax organs, on the other hand, we detected constitutively different, often elevated mRNA levels in comparison to rat. These constitutively increased transcript levels may enable Spalax to react faster to acute changes in the oxygen level of its habitat, alleviating the need to regulate response genes after the onset of stress. The body-wide transcriptome differences that were detected between Spalax and rat involve many pathways associated with genome stability maintenance and DNA repair, suggesting an explanation for the extraordinary lifespan of Spalax. In the future, it will be interesting to link the differential gene expression patterns to epigenomic changes which may orchestrate the Spalax-specific transcription.