ABSTRACT. In heme-containing oxygen-sensing proteins, heme acts as the sensing site for binding oxygen molecules and indirectly regulates many physiological functions, including the activities of protein kinases, in response to O2 availability. Conceptually, these proteins are always composed of at least two domains: one is a sensing domain (heme-based oxygen sensing), and the other is a functional domain. However, the structure-function relationship and mechanisms of communication between these domains have not been fully understood.

Therefore, we selected several model systems, including a globin-coupled histidine kinase, AfGcHK, to study the signal transduction in these hemoproteins. The AfGcHK is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Once the O2 (as the first signal) binds to the heme iron complex in the sensor domain of AfGcHK, the functional domain is stimulated, leading to autophosphorylation at a conserved His residue in the functional domain. The phosphate group of phosphorylated AfGcHK is transferred to the cognate response regulator.

Since most heme-based oxygen sensors form very flexible structures, studying them in their full-length, wild-type forms is challenging. However, only such approaches to studying the full-length proteins can reveal the signal transduction mechanism between the sensing and function domains. Thus, the detailed enzyme kinetic study was combined with the hydrogen-deuterium exchange experiments associated with mass spectrometry. It was suggested that the dimerization interface of the sensing domain is essential for signal transmission from the sensing domain to the functional domain. All data together will be discussed to illustrate the signal transduction mechanism in the model system.

Supported by the grant 8F20011 from The Ministry of Education, Youth and Sports.

ABSTRACT. Iron is an essential micronutrient that is required by bacterial pathogens to proliferate and cause disease. During infections, microbial pathogens forage iron from human hemoglobin (Hb), as it contains ∼70% of the human body's total iron content in the form of heme. Bacteria access Hb when it is released from erythrocytes that have either spontaneously ruptured or been lysed by bacterial cytotoxins. Here, I discuss my laboratory’s recent efforts to understand how pathogenic Staphylococcus aureus and Streptococcus pyogenes bacteria harvest iron from Hb. These microbes display receptors that remove heme from Hb despite its pico-molar affinity for this ligand. Using a combination of structural, biophysical and computational methods we show that these pathogens harvest heme using distinct mechanisms. In S. aureus, the surface displayed IsdB/IsdH receptors actively extract heme by distorting Hb’s F-helix, accelerating heme release more than a 1,000-fold by reducing the energy needed to rupture the axial HisF8 Nϵ-Fe3+ bond. Directed inter-domain motions within the receptor play a critical role in the extraction process by transiently positioning the heme extraction unit near the F-helix. In contrast, recent studies of the Hb receptor from S. pyogenes, called Shr, reveal that this microbe employs a distinct molecular solution to capture Hb’s heme. New structural data indicate that it preferentially recognizes the heme-loaded form of Hb by directly binding to heme’s exposed propionate groups. Based on native mass spectrometry, SAXS, and heme transfer measurements we show that Shr initially removes heme from the β subunits in Hb. We propose that the receptor transiently unlatches from the β subunits to facilitate heme release and capture by the receptor’s heme binding domain. Collectively, the results of this work provide new insight into how microbial pathogens exploit metal containing oxygen binding and sensing proteins as nutrient sources.

ABSTRACT. Globins are phylogenetically ancient proteins and thus also belong to the standard gene repertoire of arthropods. Three ancestral globin lineages, termed HbL (hemoglobin-like), GbX (globin X) and GbXL (globin X-like) have been defined in arthropod genomes, but arthropod taxa differ in the presence and copy number of these gene lineages. Brachyceran Diptera, for example, lack the GbX and GbXL lineages, but harbour different numbers of globins of the HbL class. The model species Drosophila melanogaster (Dmel) contains a “major” HbL copy named glob1 plus two other gene duplicates, glob2 and 3, which both are exclusively expressed in testis tissue. Dmel glob1 is found in all developmental stages, with particularly high amounts in fat body and trachea. Our reanalysis of expression levels and gene structure revealed that glob1 is expressed in two different modes: one promotor conveys ubiquitous, but low expression whereas activation of the second one often results in high levels of mRNA. Recent studies on three different types of experimental knockdown systems suggested an unexpected variability of glob 1 deficiency phenotypes, ranging from a reduction in lifespan under hypoxia or even normoxia to severe developmental defects. However, residual expression levels, potential off target effects and the influence from the two different transcription start sites probably complicated the interpretation of these results. We therefore established a complete genetic knockout of Dmel glob1 via CRISPR/Cas9. Preliminary transcriptomic and phenotypic analyses confirm developmental defects, but to a much lesser extent than described in earlier studies. In contrast, we found no evidence for a reduced life span under normoxic conditions. These results demonstrate the need for a true genetic KO system for functional analysis of phenotypical defects. Future studies with a focus on high glob1-expressing organs such as the fat body may shed light on the physiological function of glob1.

ABSTRACT. The genome of the nematode model organism Caenorhabditis elegans encodes 34 globins, most of which have not been functionally characterized yet. Globin 3 is a small, cysteine-rich, C. elegans globin that is predicted to be spliced in two isoforms (210 and 282 AA respectively). It is expressed predominantly in a specific set of neurons, the somatic gonad, and vulval muscles. This expression pattern agrees with the abnormal behaviour and sterility of the homozygous mutant.

In the glb-3 knockout mutant, locomotion is heavily impaired with reduced speed and exaggerated head curvatures as major hallmarks, hinting at dysregulation of motor neurons. Also, pharyngeal pumping is reduced by about 50%, which links to the presence of GLB-3 in pharyngeal neurons in wild-type worms. glb-3 mutants are less responsive to organic attractants but show a normal response to O2 and CO2. Although impaired food sensing has been linked to longevity, glb-3 mutants have a normal lifespan.

Mutation of glb-3 causes complete sterility resulting from severe underdevelopment and malformation of the gonad. In the oviduct, the number of oocytes is often reduced in number and size, while the terminal oocyte often appears bloated.

The overall expression pattern as well as the protein structure of GLB-3 resembles that of GLB-12, a known superoxide generator that works in concert with superoxide dismutase to signal to the germline. For glb-3, we found a mild interaction with the mitochondrial sod-2 and sod-3 in the neuronal phenotypes (pharyngeal pumping rate and chemosensing, respectively).

In summary, GLB-3 is likely a redox active protein that optimizes the activity of a specific set of neurons and regulates oocyte maturation in the gonads of C. elegans.

ABSTRACT. Ferritins play the key role in iron metabolism in practically all life forms. Typically made up of 24 subunits, one ferritin molecule takes a shape of a sphere with a hollow cavity. Ferritins oxidise soluble Fe2+ to poorly soluble Fe3+ and deposit the ferric iron in the form of a mineral inside the cavity (up to several thousand iron atoms). Such ubiquitous iron storage proteins seems to be an evolutionary response of the contrariety of the high importance of iron for life and its scarce availability.

Bacterioferritins are distinct type of ferritins that contain haem prosthetic groups – at the interface of two subunits. Bacterioferritin from Escherichia coli (EcBfr) is a homo-24-mer, each subunits containing a di-iron motif called ferroxidase centre (FC). Thus, there are two distinct iron motifs in EcBfr – each molecule contains 24 FCs and up to 12 haem groups. It appear that the two motifs play roles in two opposing processes: the FC is where O2 binds and oxidises iron in the mineralisation process; whereas the haem, when reduced, can provide an electron via an electron transfer (ET) pathway to the mineral core thus enabling iron reduction and mobilisation for cell’s needs.

We have found another ET pathway from reduced haem - to the FC.[1] We also found that iron oxidation at FC by H2O2 is 1000-fold faster than by O2.[2] Taken together these two findings allow hypothesis that primary function of EcBfr is antioxidant defence against H2O2, not iron sequestering, and that FC acts as an active site of a true enzyme exhibiting redox cycling while relaying electrons from the haem to H2O2 thus reducing it to water and thereby providing an antioxidant defence.

1. Pullin et al., (2021), Angew Chem Int Ed Engl 60, 8376-8379. 2. Pullin et al., (2021), Angew Chem Int Ed Engl 60, 8361-8369.

ABSTRACT. The Escherichia coli respiratory chain contains three terminal oxidases that catalyze the reduction of O₂ to 2H₂O to generate ATP: a heme copper cytochrome bo3 and two bd-type oxidases, bd-I and bd-II containing the low-spin heme b₅₅₈ and the two high-spin hemes b₅₉₅ and d. E. coli bd-II is relatively poorly characterized and its function less clear [1], but it seems to be involved in different physiological processes compared to the other oxidases as its genes are expressed at different environmental and O2 conditions. Remarkably, in the presence of H2O2, bd-II provides a fitness advantage during anaerobic growth to E. coli in the inflamed murine intestine and to Salmonella in the streptomycin-treated gut [2]. Therefore, we investigated whether of E. coli bd-II plays a role in H2O2 metabolism and tolerance, in addition to its function in bioenergetics. Polarographic O₂ measurements have shown that preparations of the untagged bd-II oxidase from E. coli strain decomposes H₂O₂ to O₂ with a high rate by producing half a mole of O₂ per mole of H₂O₂. Such catalase activity is insensitive to N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (a ligand to a quinol binding site) and CO that binds to the reduced heme d. In contrast, the catalase reaction is inhibited by cyanide (IC₅₀=4.5 0.5 μM), azide and it vanishes when cytochrome bd-II is converted into the fully reduced state or inactivated by thermal denaturation, suggesting a role of heme-b₅₉₅ in the hydrogen peroxide scavenging activity. The ability of cytochrome bd-II to detoxify H₂O₂ could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

[1] Grauel et al., (2021) Nat. Commun., 12:6498 [2] Borisov et al., (2021) Antioxid. Redox Signal.34, 16: 1280-1318

ABSTRACT. THB1 is a monomeric truncated hemoglobin from the green alga Chlamydomonas reinhardtii. In the absence of exogenous ligands and at neutral pH, the heme group of THB1 is coordinated by two protein residues, Lys53 and His77. THB1 is thought to function as a nitric oxide dioxygenase, and the distal binding of O2 requires the cleavage of the Fe-Lys53 bond accompanied by protonation and expulsion of the lysine from the heme cavity into the solvent. Nuclear magnetic resonance spectroscopy and crystallographic data have provided dynamic and structural insights of the process, but the details of the mechanism were not fully elucidated. We applied a combination of computer simulations and site-directed mutagenesis experiments to shed light on this issue. Molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations were performed to explore the nature of the transition between the decoordinated and lysine-bound states of the ferrous heme in THB1. Lys49 and Arg52, which form ionic interactions with the heme propionates in the X-ray structure of lysine-bound THB1, were observed to assist in maintaining Lys53 inside the protein cavity and play a key role in the transition. Lys49Ala, Arg52Ala and Lys49Ala/Arg52Ala THB1 variants were prepared, and the consequences of the replacements on the Lys (de)coordination equilibrium were characterized experimentally. Additionally, the "Lys-off" X-ray structure, represented by the cyanide adduct of the Fe(III) protein, allowed to hypothesize 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. The results reinforced the dynamic role of protein-propionate interactions and strongly suggested that cleavage of the Fe-Lys53 bond and ensuing conformational rearrangement is facilitated by protonation of the amino group inside the distal cavity.

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 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. Symbiotic hemoglobins typically display features associated with oxygen transport. These features include: pentacoordinate heme irons, high concentrations, modest oxygen affinity dictated by a rapid on rate and a corresponding fast off rate. Phytoglobins, previously referred to as class 1 non-symbiotic hemoglobins, differ in each of these characteristics typical of oxygen transporting globins. Prior studies have shown that the oxygen transport ability of hemoglobin in plants evolved in at least two convergent pathways, one from the class 2 globins, and a unique pathway found in phytoglobins from two plant species whose proteins share 93 percent identity. Phytoglobin from the plant Parasponia andersonii is a typical oxygen transport globin while the phytoglobin from Trema tomentosa has hexacoordinate heme iron and a low oxygen dissociation rate, both characteristics of a poorer oxygen transporter. Determining which amino acid substitutions between Trema and para phytoglobins lead to changes in oxygen dissociation rate can lead to a better understanding of how globin structure relates to physiological function. This work shows that site directed mutants near the distal histidine and CD corners of Trema phytoglobin affect both bis-histidyl hexacoordination and oxygen dissociation rates. A single mutation near the distal histidine (M72T) shows an oxygen dissociation rate 3-5 times faster than the WT protein and a double mutant of M72T and a mutation near the protein’s CD corner (I59V) showed a tenfold increase in dissociation rate. These changes are indicative of a protein with more oxygen transporting ability, as observed in wild-type Para Hb. These results begin to address the complex relationships observed between globin structure, bis-histidyl hexacoordination, and physiological function.

ABSTRACT. During periods of hypoxic stress, like those caused by soil flooding, harmful amounts of nitrite and nitric oxide accumulate in cells. It has been shown that during these events, phytoglobins as well as hemoglobins found in bryophytes are upregulated. Previous studies have shown that phytoglobins from vascular plants are able to reduce nitrite and hydroxylamine. This opens a possible anaerobic respiration mechanism with ammonium as a final product that may increase plant survival and nitrogen utilization. Previous phylogenetic studies have shown that a common ancestral globin predates modern vascular plant hemoglobins, including those found in the phytoglobin class as well as the class 2 hemoglobins and the later evolved leghemoglobins which are involved in oxygen transport. Hemoglobin from the moss, Physcomitrella patens, is a representative member of this ancestral protein family and little work has been done to probe its function. Here we demonstrate through spectrophotometric studies that hemoglobin from Physcomitrella patens efficiently reduces both nitrite and hydroxylamine at rates comparable to or faster than phytoglobins. These results will advance our understanding of the reductive reactions that hemoglobins are able to catalyze in support of nitrogen metabolism. Further, by characterizing primordial globins in this manner, we can gain further insight into the evolution of physiological functions in the globin superfamily.

ABSTRACT. Myoglobin (Mb) is primarily expressed in mitochondria-rich myocytes where it fulfils a prime role in oxygen storage and intracellular transport, but it also has additional non-classical functions such as the detoxification of nitric oxide or reactive oxygen species. Mb expression has also been reported for brown fat, where Mb probably affects cellular metabolism via its fatty-acid binding properties. In addition, Mb is expressed in secretory cells of mammalian epithelia and in several human tumour entities. Importantly, previous studies have associated expression of Mb with favourable prognosis in both breast and prostate cancer. To study the role of Mb in a cancer context, we evaluated cell type-specificity of Mb expression and its influence on transcriptome-wide gene expression at single-cell resolution, making use of publicly available single-cell RNA-sequencing (scRNA-seq) expression data of breast and prostate cancer patients. Cluster analysis revealed strong association of Mb expression with the (mature) luminal cell-type in both breast and prostate cancer. Across mature luminal cells in HER2-positive breast cancer profiles, we discovered Mb-associated transcriptional heterogeneities. A particular cell population displaying high expression of proliferation marker MKI67 exhibited lower on average expression of Mb, suggesting an association of Mb expression with the differentiation status of luminal cells in cancerous breast tissue. Differential gene expression and gene ontology analyses were employed to further dissect transcriptional heterogeneity linked to Mb expression. The results suggest an association of Mb expression with secretory properties in breast and prostate cancer cells, confirming the previously documented correlation of Mb expression with the luminal cell-type. Thus, we conclude that Mb transcripts may be considered a molecular marker of better differentiated and secretory mature luminal cell-type in both breast and prostate cancer.

ABSTRACT. The oxygenation-linked binding and release of protons to surface-exposed histidine residues of vertebrate hemoglobin (Hb) is an important contributor to O2 uptake and delivery (via the Bohr effect) and blood acid-base balance. We tested the premise that reductions in titratable histidine content enhance O2 delivery in lineages of small endothermic birds and mammals, which have basal mass-specific O2 requirements that are ~75-fold higher than the largest terrestrial species. Specifically, we hypothesized that reductions in specific Hb buffer value (βHb) should produce an exaggerated reduction in red blood cell pH for a given acid (CO2) load, which then through the Bohr effect would augment O2 offloading to metabolically active tissues while safeguarding O2 uptake at the lungs. Consistent with our expectations, calculated βHb values for 369 avian and 449 mammalian species based on the primary structures of their component globin chains revealed strong (18-47%) independent reductions in predicted βHb in five clades (hummingbirds, passerines, shrews, bats and afroinsectivorans) relative to the phylogenetic mean of their respective classes. Notably, convergent replacements of histidine residues at five positions largely underlie reductions in βHb in these high metabolic rate clades. Theoretical modelling employing measured βHb in shrews (which is 46% lower than in adult human Hb) suggests this trait alone increases O2 offloading by ~42% per transit through the systemic capillaries at rest. Reductions in βHb should also facilitate an elevated hematocrit (a common adaptation in small endothermic clades) that further increase O2 carrying capacity/delivery. Finally, since the red blood cells of embryonic birds and most fetal mammals contain the same hemoglobin isoforms as found in adults, evolutionary reductions in βHb are expected to increase maternal-fetal gas exchange (mammals), while aiding systemic O2 offloading in both prenatal birds and mammals.

ABSTRACT. Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mM), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam Spisula solidissima (SsHb), composed of 162 amino acids, was previously characterized as a hexacoordinate heme iron protein by UV-visible and resonance Raman spectroscopy. Furthermore, kinetic and equilibrium measurements showed a moderate oxygen affinity, with P50 ~0.6 torr, and no cooperativity. Phylogenetic analysis demonstrated a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. Here, we present the high-resolution (1.7 Å) crystal stucture of SsHb and we describe its properties within the hexacoordinated Hb family.

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 stably in the ferric form. The high reactivity of Nbs towards reactive nitrogen and oxygen species (RNS and ROS, respectively) reflect the highly solvent exposure of the metal center. Interestingly, while Mt-Nb and At-Nb are single-domain proteins, Hs-Nb has been described as a domain of the human protein named THAP4, whose function is still unknown. THAP4 is composed of an N‐terminal modified zinc finger domain that binds DNA and a C‐terminal Nb domain. Here, we aim at shedding light of THAP4 role in human cells. First, its expression levels and cellular localization have been analyzed in several human cell lines (i.e., human embryonic kidney (HEK293), human breast cancer (MCF7), human neuroblastoma (SH-SY5Y), and human glioblastoma (U251MG). We found that THAP4 is mainly expressed in HEK293, MCF7, and SH-SY5Y cell lines and localized into the nucleus. To define THAP4 role in RNS detoxification, transcriptomic analyses were performed after cells treatment with either the peroxynitrite-donor SIN-1 or the NO-donor DEA NONOate. In addition, THAP4 interactors have been identified by mass-spectrometry and 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. Myoglobin (Mb) is a carrier and shuttle of oxygen in the heart of vertebrates. We intend to investigate how Mb content and its affinity for O2 and H2S affect whole animal metabolic rate and heart mitochondrial respiration by generating a knockout (KO) and knockin (KI) of the myoglobin gene (mb) in zebrafish models using the CRISPR-Cas9 gene editing system. The approach chosen for the KO relies on co-injecting 4 guide RNAs (gRNAs) together with Cas9 mRNA in 1-cell zebrafish embryos. This "shotgun" approach is intended to generate a large inframe deletion in the mb gene. The reason we are straying from the mainstream approach of introducing an early frameshift, and the resulting premature termination codon (PTC), is to avoid the upregulation of homologous genes through genetic compensation. In the KI model, we want to generate a missense mutation of the distal histidine (E7) to a glutamine to alter Mb heme ligand affinity. To generate the KI we are injecting a 300 nucleotide long single stranded DNA template carrying the mutation, Cas9 mRNA and a single gRNA in 1-cell embryos. The gRNA together with Cas9 will introduce a double stranded break and the single stranded donor template will hopefully be utilized for homology directed repair. We plan to utilize Oroboros instruments, a intermittent-flow respirometry system and transcriptomics to respectively investigate mitochondrial respiration, metabolic rate and differentially expressed genes in adult KO, KI, and wildtype zebrafish.

ABSTRACT. Neuroglobin (NGB) is a stress sensor protein, that exerts, when overexpressed, cytoprotective effects against oxidative stress and neurodegeneration. We defined that the activation of Estrogen Receptor β (ERβ)-mediated pathways, induced by 17β-estradiol (E2) and exogenous ligands (resveratrol - Res), is pivotal for NGB-upregulation and its cell-autonomous effect in promoting cell survival. Recent data have also indicated that NGB could exist at the extracellular level, opening to the possibility of a new functional role of exogenous NGB in the nervous system. Following this evidence, we investigated the possibility of extracellular NGB release in the presence of NGB inducers and its possible role outside cells. Data indicate that oxidative stress (H2O2) exposure and the activation of ERβ promote NGB secretion by neuron-derived cells (SH-SY-5Y) through the exosomal and non-exosomal pathways, suggesting a paracrine function of the protein. To strengthen this idea, we evaluated the effect of cell-derived extracellular NGB, by using conditioned media (CM) and exosomes derived from wild type (WT) and NGB overexpressing cells (NGB-HA). Obtained results demonstrated that NGB-enriched CM, collected by NGB-HA cells, prevent the early mitochondrial fragmentation and, in turn, reduce apoptotic cell death in SH-SY-5Y cells after oxidative stress treatment or mitochondrial toxicity induced by 3-nitropropionic acid (3NP). Furthermore, we observed a similar anti-apoptotic effect by using the NGB-enriched exosomes and exosome-deprived CM, or the recombinant globin, suggesting that NGB release as free protein or through extracellular vesicles can be neuroprotective, independently. Altogether, obtained results strengthen the idea that NGB could operate in the extracellular compartment as a transmission factor in non-cell-autonomous mechanisms of neuroprotection, opening to the possibility of exogenous NGB as a new targetable neurotrophic protein in neurodegenerative disease.

ABSTRACT. The light-absorbing retina within the eye has an exceptionally high oxygen demand, which imposes two conflicting needs: high rates of blood perfusion and an unobstructed light path devoid of blood vessels. Improved oxygen release from hemoglobin via the Root effect represents a physiological solution to improve the oxygen supply to the retina without increasing the density of light-scattering blood vessels. In this presentation, I will 1) present a new molecular mechanism underlying Root effect-mediated oxygen supply to the retina, 2) show how the evolutionary origin of this mechanism supported the evolution of sharp vision in the ray-finned fishes, and 3) discuss how biochemical constraints in hemoglobin of terrestrial vertebrates led to fundamentally different respiratory mechanisms for retinal oxygen supply in sharp-sighted tetrapods.

ABSTRACT. The globin proteins are found throughout evolution in all kinds of organisms and their function primarily depends on their heme active site. Based on its redox status, the iron heme can bind different ligands or can favor electron transfer reactions to exert different functions such as decomposition or production of NO, detoxification from reactive oxygen species or intracellular signaling. These biological functions are relevant in many systems and presumably have some implications in development and tissue regeneration. Our goal is to understand the role of globins in biology and we use the zebrafish as model to unravel the function of globins during development and heart regeneration. We analyzed transcript and protein levels and found that myoglobin, cytoglobin1 and cytoglobin2 are expressed in the adult heart and in the embryos in different cell types. We generated the zebrafish knock-out using CRISPr/Cas9 mutagenesis and discovered that myoglobin and cytoglobin1 are important during heart regeneration while cytoglobin2 appears to be involved in heart development. The advent of genome sequencing resulted in the discovery of a host of previously unidentified globin proteins expressed outside of red blood cells and skeletal muscle, changing the paradigm through which we understand this well studied family of proteins. While heme globins such as myoglobin and cytoglobin are highly conserved in vertebrates, their physiological functions have been unknown, largely related to inconsistent and mild to absent observed phenotypes in mice. Here we present valuable zebrafish models, amenable of genetic manipulations, useful to potentially filling the gap in knowledge on the role of globins in biological systems.

ABSTRACT. The annual feather moult in penguins is critically important for survival and reproduction and a recognised energetic bottleneck, because penguins cannot enter water and hunt for several weeks during this time and need to renew their whole plumage from internal stores in what is known as a catastrophic feather moult.

Feathers consist of more than 90% proteins that are exceptionally rich in the sulphur (S)-containing amino acid cysteine, enabling crosslinked disulphide bridges for structural stability. Because of a low average S content of non-feather proteins, an exceptionally high amount of body protein needs to be broken down to provide S for the complete renewal of the feather coat.

Pre-moult ‘fattening’ of penguins includes a built-up of pectoral muscle mass with high levels of O2-storing myoglobin (Mb) that supports the prolonged breath-hold feeding dives of these animals and is subsequently broken down during moult fast. Here we test for a novel role of Mb as S store that is recycled during catastrophic feather moult by analysing Mb primary structures of all extant penguin species obtained from feather DNA extracts and recently released whole genome sequences.

We found a 2- to 3.5-fold higher S content in penguin compared to other avian or non-avian Mbs. Mb S content was negatively correlated with body mass, consistent with a lower surface area to volume ratio in larger birds and hence lower relative feather mass. Ancestral reconstructions of maximum Mb concentration and specific S content revealed concurrent increases already in the last common ancestor of extant penguins, suggesting a unique co-option of O2-storing Mb as a S store as long as 16-20 million years ago.

ABSTRACT. Despite the fact that the Bohr and Haldane effects are of equal size at the molecular level due to their thermodynamic linkage, the influence of the Bohr effect on the delivery of blood borne oxygen to the respiring tissues has been deemed secondary to the influence of the Haldane effect on the uptake of carbon dioxide by the blood. Here we show that this is wrong. Using a simple two-ligand, two-state formulation we modelled the simultaneous oxygen and proton binding to hemoglobin as well as the resulting acid-base changes of the surrounding solution. When the Bohr effect is blocked in this model system, we see a dramatic increase in the oxygen affinity, with a fall in oxygen half saturation pressure (P50) from 27 to 6mmHg. We also show that the P50 and the Bohr factor are not independent but directly related. Thus, changes in hemoglobin structure that lead to changes in the Bohr factor will inevitably also change hemoglobin oxygen affinity. The physiological importance of the Bohr effect on oxygen unloading cannot be assessed by comparing oxygen equilibrium curves measured in the lab at different constant Pco2 or pH because each of these curves are already shaped by the Bohr/Haldane effect.

ABSTRACT. Androglobin, is a multi-domain hemoglobin with a circularly permuted globin domain which is split by an IQ calmodulin binding motif. Expression of this 190 kDa protein in vivo is correlated with overexpression of FOXJ1, a crucial transcription factor of ciliogenesis(1). Since its initial discovery in 2012, efforts to generate and study a stable form of the circularly permuted globin by recombinant expression have proven unsuccessful. Using a remote homologue alignment method, molecular modelling and molecular dynamics, we identified the alignment to other hemoglobins. Validation of our proposed alternative helix alignment lies, at least in part, in the generation of a stable recombinant form of the globin domain protein, which we have characterized. As expressed, the heme iron is hexacoordinate in the ferrous form but partially pentacoordinate in the ferric form, similar to that observed for some other globins such as cytoglobin. A disulfide bond is also detected in a corresponding position to that observed in neuroglobin, stabilizing the heme binding and significantly affecting the reactivity with some ligands. This opens up future research into examining the behavior of the androglobin in vitro and hence its potential mechanism of action in ciliogenesis and spermatogenesis.

(1) Koay, T. W.; Osterhof, C.; Orlando, I. M. C.; Keppner, A.; Andre, D.; Yousefian, S.; Suarez Alonso, M.; Correia, M.; Markworth, R.; Schodel, J.; Hankeln, T.; Hoogewijs, D., Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis. J. Biol. Chem. 2021, 296, 100291.

ABSTRACT. In the present study, we have attempted to reconstruct step-by-step the tetrameric human hemoglobin (Hb) of the form α2β2, by the controlled association of its isolated α- and β-subunits, and trace the origin of cooperativity. Normally, β-units form a tetramer of the form β4, showing no cooperativity. Alkylation of its βCys112 with iodoacetamide or N-acetyl succinimide, produced monomers. Alkylation of βCys93 did not prevent association into oligomers. However, while alkylation of this residue with iodoacetamide did not affect the affinity for oxygen, alkylation with N-ethyl maleimide clearly reduced its affinity for oxygen ten times. Alpha- and β-semiHbs are dimeric forms in which a heme-containing α- or β-subunit is attached to its complementary heme-depleted subunit (i.e., apo subunit), respectively. They do not show cooperativity, but their oxygen affinities are intermediate to the affinity for the first and last oxygen in native tetrameric Hb, indicating that the formation of the α-β interface modulates the affinities for oxygen of the isolated subunits. Strikingly, when the residue βCys112 in these semihemoglobins were alkylated with bulky moieties, such as thiopyridyls, their oxygen affinity showed cooperativity and resembled that of a system with two oxygen-binding sites. Tetramer formation was confirmed by size-exclusion chromatography, indicating that the cross-talk between both the two heme-carrying β-subunits was indeed established, irrespective of the fact that both the α-subunits lacked heme. Moreover, chemical modification of the βCys112, which is located in the α-β interface seem to play a pivotal role in the cooperativity involving both the β-subunits. The binding for the first and second oxygen molecules showed affinities that were lower and higher than that of the monotonical unmodified semiHb, respectively.