Horse Heart Myoglobin Research Articles

Water stabilizes an alternate turn conformation in horse heart myoglobin

Comparison of myoglobin structures reveals that protein isolated from horse heart consistently adopts an alternate turn conformation in comparison to its homologues. Analysis of hundreds of high-resolution structures discounts crystallization conditions or the surrounding amino acid protein environment as explaining this difference, that is also not captured by the AlphaFold prediction. Rather, a water molecule is identified as stabilizing the conformation in the horse heart structure, which immediately reverts to the whale conformation in molecular dynamics simulations excluding that structural water.

Hidden Complexity in the Mechanism of the Autoreduction of Myoglobin Compound II.

The non-native oxidation of horse heart myoglobin with hydrogen peroxide produces compound II which autoreduces by utilizing an internal oxidation site. Here, we utilize full UV–visible time-dependent kinetics with global kinetic singular value decomposition analysis to explore the mechanism and uncover more detail about the high-valent heme spectral features. By varying the hydrogen peroxide and myoglobin concentration, we were able to uncover more detailed spectra of myoglobin compound II and the autoreduction rate under several different pH conditions. The compound II spectra demonstrate pH-dependent features with an inflection point around pH 5.7 ± 0.1. The rate of autoreduction of compound II, k2, increases with lower pH with a half-power proton dependence and no indication of a pKa > 3.9 ± 0.2, indicating that the autoreduction is still dependent on the protonation of the ferryl oxo species. The k2 also demonstrates both hydrogen peroxide and myoglobin dependency. At myoglobin concentrations greater than 6.6 μM, the k2 is myoglobin-independent, but for lower concentrations, a pH-sensitive concentration dependence is seen.

Understanding the Role of Surfactants in the Interaction and Hydrolysis of Myoglobin by Zr‐MOF‐808

AbstractControlled protein hydrolysis is an important procedure in proteomics applications and is used to aid the understanding of protein structure and function. The hydrolysis of hydrophobic proteins is particularly challenging, as due to their poor solubility the use of surfactants, which typically inactivate natural enzymes, is often required. Such limitations of natural enzymes prompted the development of chemical catalysts for the selective hydrolysis of proteins. In this study, the nanozymatic potential of MOF‐808, a Zr6O8 based metal organic framework, has been investigated towards protein hydrolysis in the presence of several surfactants which differ in structure and polarity. The influence of ionic SDS (sodium dodecyl sulfate), neutral TX‐100 (Triton X‐100) and zwitterionic Zw3‐12 (n‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonate) and CHAPS (3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate) surfactants on the hydrolysis of horse heart myoglobin in the presence of MOF‐808 has been examined. The hydrolysis of horse heart myoglobin by MOF‐808 was followed using sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS‐PAGE), which showed that nanozymatic activity of MOF‐808 can be tuned by using appropriate surfactants. To understand the observed reactivity patterns, the interactions between surfactant, MOF and protein were further investigated using a range of spectroscopic methods, which included Dynamic Light Scattering (DLS), 1H NMR, UV‐Vis, Circular Dichroism and UV‐Vis Spectroscopy. While the presence of SDS increased the number of observed peptide fragments due to protein unfolding and increased protein‐MOF interaction, the use of zwitterionic and neutral surfactant reduced the hydrolytic efficiency, most likely by hindering the efficient protein‐MOF interaction.

A zirconium metal-organic framework with SOC topological net for catalytic peptide bond hydrolysis

The discovery of nanozymes for selective fragmentation of proteins would boost the emerging areas of modern proteomics, however, the development of efficient and reusable artificial catalysts for peptide bond hydrolysis is challenging. Here we report the catalytic properties of a zirconium metal-organic framework, MIP-201, in promoting peptide bond hydrolysis in a simple dipeptide, as well as in horse-heart myoglobin (Mb) protein that consists of 153 amino acids. We demonstrate that MIP-201 features excellent catalytic activity and selectivity, good tolerance toward reaction conditions covering a wide range of pH values, and importantly, exceptional recycling ability associated with easy regeneration process. Taking into account the catalytic performance of MIP-201 and its other advantages such as 6-connected Zr6 cluster active sites, the green, scalable and cost-effective synthesis, and good chemical and architectural stability, our findings suggest that MIP-201 may be a promising and practical alternative to commercially available catalysts for peptide bond hydrolysis.

Structural change of myoglobin structure after binding with spermidine

Myoglobin is the iron-rich protein of 153 or 154 amino acid residues, comprising a prosthetic group of heme (iron-containing porphyrin). It is responsible for the reversible binding and transporting of molecular oxygen in muscle tissue. There are proximal and distal histidine groups. The proximal His group joined specifically to the iron center and a distal histidine group on the inverse confront, not bonded to the iron. Horse heart myoglobin contains two tyrosine residues and two tryptophan residues. Multi-spectroscopic, thermodynamic, molecular docking, and molecular dynamics simulations were used in the present study to investigate the variations instability, structure, and function of horse heart myoglobin (Mb) in the presence of polyamine spermidine. Reports from the different experiments have shown that spermidine is bound to myoglobin. Steady-state fluorescence analysis showed a static quenching system as the primal quenching mechanism in the myoglobin − spermidine interaction, which is confirmed by UV − Visible and molecular docking studies. Thermodynamic and molecular docking findings demonstrate spermidine binding to myoglobin is driven mainly by van der Waals force and hydrogen bonding forces. The CD spectrum showed the secondary variation in myoglobin structure with a rise in α-helicity after ligand binding and a small decrease in myoglobin β-sheet after.

Fluoride binding to characteristic heme-pocket centers: Insights into ligand stability

The studies on the L. pectinata hemoglobins (HbI, HbII, and HbIII) are essential because of their biological roles in hydrogen sulfide transport and metabolism. Variation in the pH could also play a role in the transport of hydrogen sulfide by HbI and oxygen by HbII and HbIII, respectively. Here, fluoride binding was used to further understand the structural properties essential for the molecular mechanism of ligand stabilization as a function of pH. The data allowed us to gain insights into how the physiological roles of HbI, HbII, HbIII, adult hemoglobin (A-Hb), and horse heart myoglobin (Mb) have an impact on the heme-bound fluoride stabilization. In addition, analysis of the vibrational assignments of the met-cyano heme complexes shows varied strength interactions of the heme-bound ligand. The heme pocket composition properties differ between HbI (GlnE7 and PheB10) and HbII/HbIII (GlnE7 and TyrB10). Also, the structural GlnE7 stereo orientation changes between HbI and HbII/HbIII. In HbI, its carbonyl group orients towards the heme iron, while in HbII/HbIII, the amino group occupies this position. Therefore, in HbI, the interactions to the heme-bound fluoride ion, cyanide, and oxygen with GlnE7 via H-bonding are not probable. Still, the aromatic cage PheB10, PheCD1, and PheE11 may contribute to the observed stabilization. However, a robust H-bonding networking stabilizes HbII and HbIII, heme-bound fluoride, cyanide, and oxygen ligand with the OH and NH2 groups of TyrB10 and GlnE7, respectively. At the same time, A-Hb and Mb have moderate but similar ligand interactions controlled by their respective distal E7 histidine.

Electrostatic Interaction of Globins with Phospholipid Membranes

For the structures of sperm whale myoglobin (swMb), horse heart myoglobin (hhMb), hemoglobin I (HbI) from botfly Gasterophilus intestinalis (giHbI), and monomeric and dimeric hemoglobins HbI and HbII from Lucina pectinata mollusk (lpHbI and lpHbII), roles of electrostatic interactions in stabilization of native conformations of the heme cavity were analyzed, as well as their possible destabilization by interaction with the negatively charged phospholipid membranes. The native conformation of the heme cavity in these globins, both its proximal and distal parts, was shown to be sustained by a system of hydrogen bonds involving the proximal and distal protein residues, the heme propionates, and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom either outside or within the protoporphyrin plane, which affects the efficiency of ligand binding. In the distal part, the hydrogen bond network formed with the distal protein residue involved (HisE7 in swMb, hhMb, and giHbI and GlnE7 in lpHbII), should stabilize the conformation in which the protein can donate hydrogen to the O2 ligand. The hydrogen bond between the E7 distal residue and O2 ligand prevents rapid dissociation of the latter and plays an important role in the regulation of ligand affinity. The hydrogen bond networks found in the proximal and, especially, in the distal heme environments, must be disrupted when oxyglobin binds to the membrane surface. This disruption caused by interaction of negatively charged phospholipid heads with lysine or arginine residues exposed towards the membrane, altered local pH near the membrane, and the effect of negative electrostatic field of the membrane, can decrease the affinity of the protein for the O2 ligand, thus facilitating its dissociation at the physiological pO2 values in the cell.

Eliminating sweet spot in MALDI-MS with hydrophobic ordered structure as target for quantifying biomolecules

In matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the analyte is usually distributed unevenly throughout the sample spot. The area with aggregated analyte molecules contributing abundant signal, is termed as “sweet spot”, which results in poor detection reproducibility and makes it impossible to quantify analytes without internal standards. We proposed a strategy to eliminate sweet spot in MALDI-MS by using a hydrophobic ordered structure as target. The target is fabricated by creating a hydrophobic silicon nanopillar array and subsequently decorating it uniformly with poly(methyl methacrylate) nanodots for capturing analytes. The sweet spot is eliminated by distributing analyte molecules uniformly on this target, and then result in a uniform MS image, which demonstrates an ideal reproducibility. Finally, with the target assisted MALDI-MS as biosensor was suitable to analyze practical sample such as bacitracin A in milk. Horse heart myoglobin and, angiotensin III molecules can be quantified without internal standard using α-cyano-4-hydroxycinnamic acid as matrix. This biosensor presented good linearity, high salts tolerance and high signal-to-noise ratio (up to 271.8), even the 1 mol/L salt concentration. This strategy could provide an alternative for improving the performance of MALDI-MS.

Discrete Hf18 Metal-oxo Cluster as a Heterogeneous Nanozyme for Site-Specific Proteolysis.

The selective hydrolysis of proteins by non-enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal-oxo cluster [Hf18 O10 (OH)26 (SO4 )13 ⋅(H2 O)33 ] (Hf18 ), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18 . X-ray scattering and ESI-MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

Discrete Hf18 Metal‐oxo Cluster as a Heterogeneous Nanozyme for Site‐Specific Proteolysis

AbstractThe selective hydrolysis of proteins by non‐enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal‐oxo cluster [Hf18O10(OH)26(SO4)13⋅(H2O)33] (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18. X‐ray scattering and ESI‐MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

ЭЛЕКТРОСТАТИЧЕСКОЕ ВЗАИМОДЕЙСТВИЕ ГЛОБИНОВ С ФОСФОЛИПИДНЫМИ МЕМБРАНАМИ

The present study aimed to analyze the role of electrostatic interactions contributing to the stability of the native conformations of the heme group in the structure of sperm whale myoglobin (SWMb), horse heart myoglobin (HHMb), hemoglobin I (HbI) from the botfly Gasterophilus intestinalis (giHbI) and monomeric and dimeric hemoglobins HbI and HbII from the mollusk Lucina pectinata (lpHbI and lpHbII) as well as investigate a possible reason of destabilization due to interaction with negatively charged phospholipid membranes. It was shown that the native conformation of the heme cavity in these globins, both in its proximal and distal sides, is sustained by a system of hydrogen bonds involving the proximal and distal protein residues, both heme propionic acid groups and the nearby polar amino acids on the protein surface (His, Arg, Lys). The hydrogen bond network in the proximal part of the heme pocket controls the position of the Fe atom outside or in the protoporphyrin plane, affecting the efficiency of ligand binding, while in the distal part of the heme cavity the hydrogen bond network formed with the participation of the distal protein residue (HisE7 in swMb, hhMb and giHbI, and GlnE7 in lpHbII) should, apparently, stabilize conformation where the protein is able to donate hydrogen to O2 ligand...

Catalase-Like Antioxidant Activity is Unaltered in Hypochlorous Acid Oxidized Horse Heart Myoglobin.

Activated neutrophils release myeloperoxidase that produces the potent oxidant hypochlorous acid (HOCl). Exposure of the oxygen transport protein horse heart myoglobin (hhMb) to HOCl inhibits Iron III (Fe(III))-heme reduction by cytochrome b5 to oxygen-binding Iron II (Fe(II))Mb. Pathological concentrations of HOCl yielded myoglobin oxidation products of increased electrophoretic mobility and markedly different UV/Vis absorbance. Mass analysis indicated HOCl caused successive mass increases of 16 a.m.u., consistent serial addition of molecular oxygen to the protein. By contrast, parallel analysis of protein chlorination by quantitative mass spectrometry revealed a comparatively minor increase in the 3-chlorotyrosine/tyrosine ratio. Pre-treatment of hhMb with HOCl affected the peroxidase reaction between the hemoprotein and H2O2 as judged by a HOCl dose-dependent decrease in spin-trapped tyrosyl radical detected by electron paramagnetic resonance (EPR) spectroscopy and the rate constant of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) oxidation. By contrast, Mb catalase-like antioxidant activity remained unchanged under the same conditions. Notably, HOCl-modification of Mb decreased the rate of ferric-to-ferrous Mb reduction by a cytochrome b5 reductase system. Taken together, these data indicate oxidizing HOCl promotes Mb oxidation but not chlorination and that oxidized Mb shows altered Mb peroxidase-like activity and diminished rates of one-electron reduction by cytochrome b5 reductase, possibly affecting oxygen storage and transport however, Mb-catalase-like antioxidant activity remains unchanged.

Addition of sodium ascorbate to extend the shelf-life of tuna meat fish: A risk or a benefit for consumers?

We investigate the effects of antimicrobial (sodium citrate tribasic, E331) and antioxidant (ascorbic acid, E300 and sodium ascorbate, E301) additives on the meat drip from defrosted yellowfin tuna fish loins obtained from the local market and horse heart myoglobin. The effects have been followed by electronic absorption, its second derivative spectra, and resonance Raman spectroscopies. Upon addition of the additives, a final form is reached after about 24 h. It is characterized by a 4 nm red-shifted Soret band compared to that typical of the oxy species (418 nm) but with similar Q bands. Resonance Raman experiments carried out in 16O2 and 18O2 allowed us to identify the presence of the native oxy form coexisting with a second oxygen bound species, characterized by a ν(FeO2) stretching frequency upshifted 7 cm−1 compared to the native oxy form and with a greater (33 cm−1) isotopic shift in 18O2. These data suggest the presence of a highly bent ligand conformation. The new species induced by the addition of the additives imparts a red colour to the tuna fish meat, a characteristic that is of some concern. In fact, the presence of the new red form can mask the aging of the product that, consequently, might contain histamine. Furthermore, the electronic absorption spectrum is very similar to that of the tuna fish myoglobin carbon monoxide complex, which has important regulatory consequences. Carbon monoxide treatment of tuna is banned in the EU for masking the effects of aging on the appearance of meats.

Recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites.

BackgroundN-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from >20% of allergic patients but have low or no allergenic activity.ObjectivesTo engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE.MethodsOne or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-Five™ insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes.ResultsHHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4 > nCyn d 1 > nPla a 2 > nJug r 2 > nCup a 1 > nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2.ConclusionDefined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity.

Thermal Stability of Globins: Implications of Flexibility and Heme Coordination Studied by Molecular Dynamics Simulations.

Proteins are sensitive to temperature, and abrupt changes in the normal temperature conditions can have a profound impact on both structure and function, leading to protein unfolding. However, the adaptation of certain organisms to extreme conditions raises questions about the structural features that permit the structure and function of proteins to be preserved under these adverse conditions. To gain insight into the molecular basis of protein thermostability in the globin family, we have examined three representative examples: human neuroglobin, horse heart myoglobin, and Drosophila hemoglobin, which differ in their melting temperatures and coordination states of the heme iron in the absence of external ligands. In order to elucidate the possible mechanisms that govern the thermostability of these proteins, microsecond-scale classical molecular dynamics simulations were performed at different temperatures. Structural fluctuations and essential dynamics were analyzed, indicating that the flexibility of the CD region, which includes the two short C and D helixes and the connecting CD loop, is directly related to the thermostability. We observed that a larger inherent flexibility of the protein produces higher thermostability, probably concentrating the thermal fluctuations observed at high temperature in flexible regions, preventing unfolding. Globally, the results of this work improve our understanding of thermostability in the globin family.

Human nitrobindin: the first example of an all-β-barrel ferric heme-protein that catalyzes peroxynitrite detoxification.

Nitrobindins (Nbs), constituting a heme‐protein family spanning from bacteria to Homo sapiens, display an all‐β‐barrel structural organization. Human Nb has been described as a domain of the nuclear protein named THAP4, whose physiological function is still unknown. We report the first evidence of the heme‐Fe(III)‐based detoxification of peroxynitrite by the all‐β‐barrel C‐terminal Nb‐like domain of THAP4. Ferric human Nb (Nb(III)) catalyzes the conversion of peroxynitrite to NO3− and impairs the nitration of free l‐tyrosine. The rate of human Nb(III)‐mediated scavenging of peroxynitrite is similar to those of all‐α‐helical horse heart and sperm whale myoglobin and human hemoglobin, generally taken as the prototypes of all‐α‐helical heme‐proteins. The heme‐Fe(III) reactivity of all‐β‐barrel human Nb(III) and all‐α‐helical prototypical heme‐proteins possibly reflects the out‐to‐in‐plane transition of the heme‐Fe(III)‐atom preceding peroxynitrite binding. Human Nb(III) not only catalyzes the detoxification of peroxynitrite but also binds NO, possibly representing a target of reactive nitrogen species.

Rotational Diffusion Depends on Box Size in Molecular Dynamics Simulations.

We show that the rotational dynamics of proteins and nucleic acids determined from molecular dynamics simulations under periodic boundary conditions suffer from significant finite-size effects. We remove the box-size dependence of the rotational diffusion coefficients by adding a hydrodynamic correction kB T/6 ηV with kB Boltzmann's constant, T the absolute temperature, η the solvent shear viscosity, and V the box volume. We show that this correction accounts for the finite-size dependence of the rotational diffusion coefficients of horse-heart myoglobin and a B-DNA dodecamer in aqueous solution. The resulting hydrodynamic radii are in excellent agreement with experiment.

Fully Anisotropic Rotational Diffusion Tensor from Molecular Dynamics Simulations.

We present a method to calculate the fully anisotropic rotational diffusion tensor from molecular dynamics simulations. Our approach is based on fitting the time-dependent covariance matrix of the quaternions that describe the rigid-body rotational dynamics. Explicit analytical expressions have been derived for the covariances by Favro, which are valid irrespective of the degree of anisotropy. We use these expressions to determine an optimal rotational diffusion tensor from trajectory data. The molecular structures are aligned against a reference by optimal rigid-body superposition. The quaternion covariances can then be obtained directly from the rotation matrices used in the alignment. The rotational diffusion tensor is determined by a fit to the time-dependent quaternion covariances, or directly by Laplace transformation and matrix diagonalization. To quantify uncertainties in the fit, we derive analytical expressions and compare them with the results of Brownian dynamics simulations of anisotropic rotational diffusion. We apply the method to microsecond long trajectories of the Dickerson-Drew B-DNA dodecamer and of horse heart myoglobin. The anisotropic rotational diffusion tensors calculated from simulations agree well with predictions from hydrodynamics.

Recombinant Glycoproteins Resembling Carbohydrate-Specific IgE Epitopes from Plants, Venoms and Mites

Background: N-linked glycans present in venoms, pollen and mites are recognized by IgE antibodies from more than 20% of allergic patients but have low or no allergenic activity. Objectives: To engineer recombinant glycoproteins resembling carbohydrate-specific IgE epitopes from venoms, pollen and mites which can discriminate carbohydrate-specific IgE from allergenic, peptide-specific IgE. Methods: One or two N-glycosylation sites were engineered into the N-terminus of the non-allergenic protein horse heart myoglobin (HHM) using synthetic gene technology. HHM 1 and HHM 2 containing one or two N-glycosylation sites were expressed in baculovirus-infected High-FiveTM insect cells and a non-glycosylated version (HHM 0) was obtained by mutating the glycosylation motif. Recombinant HHM proteins were analyzed regarding fold and aggregation by circular dichroism and gel filtration, respectively. IgE reactivity was assessed by ELISA, immunoblotting and quantitative ImmunoCAP measurements. IgE inhibition assays were performed to study cross-reactivity with venom, plant and mite-derived carbohydrate IgE epitopes. Results: HHM-glycovariants were expressed and purified from insect cells as monomeric and folded proteins. The HHM-glycovariants exhibited strictly carbohydrate-specific IgE reactivity, designed to quantify carbohydrate-specific IgE and resembled IgE epitopes of pollen, venom and mite-derived carbohydrates. IgE-reactivity and inhibition experiments established a hierarchy of plant glcyoallergens (nPhl p 4>nCyn d 1>nPla a 2>nJug r 2>nCup a 1>nCry j 1) indicating a hitherto unknown heterogeneity of carbohydrate IgE epitopes in plants which were completely represented by HHM 2. Conclusion: Defined recombinant HHM-glycoproteins resembling carbohydrate-specific IgE epitopes from plants, venoms and mites were engineered which made it possible to discriminate carbohydrate- from peptide-specific IgE reactivity. Funding Statement: This study was supported by the grants P26728, F4605, F4604 and DK W1248 from the Austrian Science Fund (FWF). Rudolf Valenta is recipient of a Megagrant of the Government of the Russian Federation, grant number 14.W03.31.0024. Declaration of Interests: Rudolf Valenta has received research grants from Biomay AG, Vienna, Austria and Viravaxx, Vienna, Austria. He serves as a consultant for Biomay AG and Viravaxx. Dr. Lupinek reports personal fees from Thermo Fisher Scientific, outside the submitted work. All other authors have nothing to disclose. Ethics Approval Statement: The anonymized analysis of the sera was approved by the ethical committee of the Medical University of Vienna (EK1641/2014).

Fe(III) – Sulfide interaction in globins: Characterization and quest for a putative Fe(IV)-sulfide species

The present study reports findings regarding the contrast between H2S interaction with bovine hemoglobin (Hb) and horse heart myoglobin (Mb), in terms of binding and dissociation kinetics, affinities, and mechanism. At pH9.5, oxidation of ferric-sulfide adducts in presence of no free sulfide, using hexachloroiridate as oxidant is examined using stopped-flow UV–vis, EPR, vibrational spectroscopy and mass spectrometry. Oxidation of the ferric-sulfide adduct in such conditions occurs with a putative unstable Fe(IV)-sulfide adduct as intermediate that finally leads to a paramagnetic ferric species with distinct EPR features. As detected by MS spectrometry, this final species appears to be a truncated form of globin at a distinct Tyr. In case of Hb, only β-chain is truncated at Tyr144.