Quantifying pH-induced changes in plasma strong ion difference during experimental acidosis: clinical implications for base excess interpretation.

It is commonly assumed that changes in plasma strong ion difference (SID) result in equal changes in whole blood base excess (BE). However, at varying pH, albumin ionic-binding and transerythrocyte shifts alter the SID of plasma without affecting that of whole blood (SIDwb), i.e., the BE. We hypothesize that, during acidosis, 1) an expected plasma SID (SIDexp) reflecting electrolytes redistribution can be predicted from albumin and hemoglobin's charges, and 2) only deviations in SID from SIDexp reflect changes in SIDwb, and therefore, BE. We equilibrated whole blood of 18 healthy subjects (albumin = 4.8 ± 0.2 g/dL, hemoglobin = 14.2 ± 0.9 g/dL), 18 septic patients with hypoalbuminemia and anemia (albumin = 3.1 ± 0.5 g/dL, hemoglobin = 10.4 ± 0.8 g/dL), and 10 healthy subjects after in vitro-induced isolated anemia (albumin = 5.0 ± 0.2 g/dL, hemoglobin = 7.0 ± 0.9 g/dL) with varying CO2 concentrations (2-20%). Plasma SID increased by 12.7 ± 2.1, 9.3 ± 1.7, and 7.8 ± 1.6 mEq/L, respectively (P < 0.01) and its agreement (bias[limits of agreement]) with SIDexp was strong: 0.5[-1.9; 2.8], 0.9[-0.9; 2.6], and 0.3[-1.4; 2.1] mEq/L, respectively. Separately, we added 7.5 or 15 mEq/L of lactic or hydrochloric acid to whole blood of 10 healthy subjects obtaining BE of -6.6 ± 1.7, -13.4 ± 2.2, -6.8 ± 1.8, and -13.6 ± 2.1 mEq/L, respectively. The agreement between ΔBE and ΔSID was weak (2.6[-1.1; 6.3] mEq/L), worsening with varying CO2 (2-20%): 6.3[-2.7; 15.2] mEq/L. Conversely, ΔSIDwb (the deviation of SID from SIDexp) agreed strongly with ΔBE at both constant and varying CO2: -0.1[-2.0; 1.7], and -0.5[-2.4; 1.5] mEq/L, respectively. We conclude that BE reflects only changes in plasma SID that are not expected from electrolytes redistribution, the latter being predictable from albumin and hemoglobin's charges.NEW & NOTEWORTHY This paper challenges the assumed equivalence between changes in plasma strong ion difference (SID) and whole blood base excess (BE) during in vitro acidosis. We highlight that redistribution of strong ions, in the form of albumin ionic-binding and transerythrocyte shifts, alters SID without affecting BE. We demonstrate that these expected SID alterations are predictable from albumin and hemoglobin's charges, or from the noncarbonic whole blood buffer value, allowing a better interpretation of SID and BE during in vitro acidosis.

Nitrobindin versus myoglobin: A comparative structural and functional study.

Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-ß-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-ß-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O2/NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-ß-barrel heme-proteins.

Nitric oxide binding to ferrous nitrobindins: A computer simulation investigation.

Nitrobindins (Nbs) represent an evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom, coordinated by a proximal His residue. Interestingly, even if the distal side is exposed to the solvent, the value of the second order rate constants for ligand binding to the ferrous derivative is almost one order of magnitude lower than those reported for myoglobins (Mbs). Noteworthy, nitric oxide binding to the sixth coordination position of the heme-Fe(II)-atom causes the cleavage or the severe weakening of the proximal His-Fe(II) bond. Here, we provide a computer simulation investigation to shed light on the molecular basis of ligand binding kinetics, by dissecting the ligand binding process into the ligand migration and the bond formation steps. Classical molecular dynamics simulations were performed employing a steered molecular dynamics approach and the Jarzinski equality to obtain ligand migration free energy profiles. The formation of the heme-Fe(II)-NO bond took into consideration the iron atom displacement from the heme plane. The ligand migration is almost unhindered, and the low rate constant for NO binding is due to the large displacement of the Fe(II) atom with respect to the heme plane responsible for the barrier for the Fe(II)-NO bond formation. In addition, we investigated the weakening and breaking of the proximal His-Fe(II) bond, observed experimentally upon NO binding, by means of a combination of classical molecular dynamics simulations and quantum-classical (QM-MM) optimizations. In both human and M. tuberculosis Nbs, a stable alternative conformation of the proximal His residue interacting with a network of water molecules was observed.

Ferrous nitrosylated cytochrome c: The unusual strength of the proximal His18-Fe bond.

NO binding to horse heart cytochrome c (hhcyt c) has been investigated as a function of pH by both optical absorption and EPR spectroscopies. Lowering pH from 3.5 to 1.5 induces: (i) a blue-shift of the maximum of the optical absorption spectrum in the Soret region from 415 to about 404 nm, and (ii) the appearance of a strong three hyperfine splitting in the gz region of the EPR spectrum. Both spectroscopic features indicate the cleavage of the proximal His18-Fe(II)-NO bond giving rise to the five-coordinated Fe(II)-NO species. By quantification of the relative weight for the six- and the five-coordinated component in the EPR spectra, the pKa value was determined. The apparent pKa of the proximal His Nε atom (1.8 ±â€¯0.1) is unusually low for a ferrous nitrosylated form since in all investigated ferrous NO-bound heme-proteins the pKa value for the cleavage of the proximal His-Fe(II) bond ranges between 3.7 and 5.8. The pKa value of ferrous nitrosylated hhcyt c indicates that the strength of the proximal His18-Fe(II) bond (= 27.9 kJ/mol) is about 10-22 kJ/mol higher than that observed in all investigated heme-proteins. The strong coordination of the heme-Fe atom by His18 is extremely important to maintain the redox efficiency of cyt c and to keep apoptosis under control. This is a crucial point in tissues, such as retina, where apoptosis might trigger macular degenerative processes.

Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3-. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2- to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M-1 s-1, 9.3 M-1 s-1, 1.4 × 101 M-1 s-1, and 5.8 M-1 s-1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2--based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-ß-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-ß-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.

Heme Scavenging and Delivery: The Role of Human Serum Albumin.

Heme is the reactive center of several metal-based proteins that are involved in multiple biological processes. However, free heme, defined as the labile heme pool, has toxic properties that are derived from its hydrophobic nature and the Fe-atom. Therefore, the heme concentration must be tightly controlled to maintain cellular homeostasis and to avoid pathological conditions. Therefore, different systems have been developed to scavenge either Hb (i.e., haptoglobin (Hp)) or the free heme (i.e., high-density lipoproteins (HDL), low-density lipoproteins (LDL), hemopexin (Hx), and human serum albumin (HSA)). In the first seconds after heme appearance in the plasma, more than 80% of the heme binds to HDL and LDL, and only the remaining 20% binds to Hx and HSA. Then, HSA slowly removes most of the heme from HDL and LDL, and finally, heme transits to Hx, which releases it into hepatic parenchymal cells. The Hx:heme or HSA:heme complexes are internalized via endocytosis mediated by the CD91 and CD71 receptors, respectively. As heme constitutes a major iron source for pathogens, bacteria have evolved hemophores that can extract and uptake heme from host proteins, including HSA:heme. Here, the molecular mechanisms underlying heme scavenging and delivery from HSA are reviewed. Moreover, the relevance of HSA in disease states associated with increased heme plasma concentrations are discussed.

Strategies of Pathogens to Escape from NO-Based Host Defense.

Nitric oxide (NO) is an essential signaling molecule present in most living organisms including bacteria, fungi, plants, and animals. NO participates in a wide range of biological processes including vasomotor tone, neurotransmission, and immune response. However, NO is highly reactive and can give rise to reactive nitrogen and oxygen species that, in turn, can modify a broad range of biomolecules. Much evidence supports the critical role of NO in the virulence and replication of viruses, bacteria, protozoan, metazoan, and fungi, thus representing a general mechanism of host defense. However, pathogens have developed different mechanisms to elude the host NO and to protect themselves against oxidative and nitrosative stress. Here, the strategies evolved by viruses, bacteria, protozoan, metazoan, and fungi to escape from the NO-based host defense are overviewed.

The Balancing of Peroxynitrite Detoxification between Ferric Heme-Proteins and CO2: The Case of Zebrafish Nitrobindin.

Nitrobindins (Nbs) are all-ß-barrel heme proteins and are present in prokaryotes and eukaryotes. Although their function(s) is still obscure, Nbs trap NO and inactivate peroxynitrite. Here, the kinetics of peroxynitrite scavenging by ferric Danio rerio Nb (Dr-Nb(III)) in the absence and presence of CO2 is reported. The Dr-Nb(III)-catalyzed scavenging of peroxynitrite is facilitated by a low pH, indicating that the heme protein interacts preferentially with peroxynitrous acid, leading to the formation of nitrate (~91%) and nitrite (~9%). The physiological levels of CO2 dramatically facilitate the spontaneous decay of peroxynitrite, overwhelming the scavenging activity of Dr-Nb(III). The effect of Dr-Nb(III) on the peroxynitrite-induced nitration of L-tyrosine was also investigated. Dr-Nb(III) inhibits the peroxynitrite-mediated nitration of free L-tyrosine, while, in the presence of CO2, Dr-Nb(III) does not impair nitro-L-tyrosine formation. The comparative analysis of the present results with data reported in the literature indicates that, to act as efficient peroxynitrite scavengers in vivo, i.e., in the presence of physiological levels of CO2, the ferric heme protein concentration must be higher than 10-4 M. Thus, only the circulating ferric hemoglobin levels appear to be high enough to efficiently compete with CO2/HCO3- in peroxynitrite inactivation. The present results are of the utmost importance for tissues, like the eye retina in fish, where blood circulation is critical for adaptation to diving conditions.

Nitrosylation of ferric zebrafish nitrobindin: A spectroscopic, kinetic, and thermodynamic study.

Nitrobindins (Nbs) are all-ß-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and isomerizing peroxynitrite to NO3- and NO2-. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV-Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH ≤ 7.0, Dr-Nb(III) binds reversibly NO, whereas at pH ≥ 7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (∼ 1.5 ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., klim) increase linearly with the OH- concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH--based catalysis. From the dependence of klim on [OH-], the value of the second-order rate constant kOH- was obtained (5.2 × 103 M-1 s-1). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO2, and the other being tightly bound to the heme-Fe(II) atom.

Dissociation of the proximal His-Fe bond upon NO binding to ferrous zebrafish nitrobindin.

Nitrobindins (Nbs) are all-ß-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, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pH 5.5 and 9.1 at 22.0 °C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6 × 106 M-1 s-1 and 2.3 × 106 M-1 s-1 and between 5.3 × 10-2 s-1 and 8.2 × 10-2 s-1, respectively. Interestingly, both UV-Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.

Hydroxylamine-induced oxidation of ferrous nitrobindins.

Hemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 ß-strands, respectively, represent the molecular models of all-ß-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M-1 s-1, 6.5 × 104 M-1 s-1, and 2.2 × 104 M-1 s-1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-ß-barrel heme-proteins.

Serum albumin and nucleic acids biodistribution: From molecular aspects to biotechnological applications.

Serum albumin (SA) is the most abundant protein in plasma and represents the main carrier of endogenous and exogenous compounds. Several evidence supports the notion that SA binds single and double-stranded deoxynucleotides and ribonucleotides at two sites, with values of the dissociation equilibrium constant (i.e., Kd ) ranging from micromolar to nanomolar values. This can be relevant from a physiological and pathological point of view, as in human plasma circulates cell-free nucleic acids (cfNAs), released by different tissues via apoptosis, necrosis, and secretions, circulates as single and double-stranded NAs. Albeit SA shows low hydrolytic reactivity toward DNA and RNA, the high plasma concentration of this protein and the occurrence of several SA receptors may be pivotal for sequestering and hydrolyzing cfNAs. Therefore, pathological conditions like cancer, characterized by altered levels of human SA or by altered SA post-translational modifications, may influence cfNAs distribution and metabolism. Besides, the stability, solubility, biocompatibility, and low immunogenicity make SA a golden share for biotechnological applications related to the delivery of therapeutic NAs (TNAs). Indeed, pre-clinical studies report the therapeutic potential of SA:TNAs complexes in precision cancer therapy. Here, the molecular and biotechnological implications of SA:NAs interaction are discussed, highlighting new perspectives on SA plasmatic functions.

Haptoglobin and the related haptoglobin protein: the N-terminus makes the difference.

Haptoglobin related protein (Hpr) is a component of the trypanosome lytic factor (TLF), a complex acting in the innate immune response against African trypanosomes. Like haptoglobin (Hp), Hpr binds hemoglobin (Hb) in the blood, but unlike Hp, Hpr does not bind the CD163 receptor. Moreover, unlike Hp, Hpr retains the N-terminal signal peptide that is required for the association with Apolipoprotein L-1 (ApoL-1), a component of the TLF complex. Here, the molecular model of human Hpr has been built based on the high sequence identity with human Hp (91%). The structural bases of Hpr:Hpr dimerization and Hpr recognition by Hb and Trypanosoma brucei brucei Hp receptor (TbHpHbR) have been analyzed in parallel with those of Hp:Hp, Hp:Hb, and TbHpHbR:Hp:Hb complexes. We show that the Cys33-Cys33 intermolecular disulfide bridge that stabilizes the Hp1:Hp1 complex is replaced by the Phe33, Pro34, and Phe48 hydrophobic core in the Hpr:Hpr dimer. Moreover, we show that the N-terminal peptide of Hpr participates in the stabilization of the Hpr:Hpr dimer. Thus, the N-terminal peptide seems to have been retained in Hpr to mediate its critical role in the human innate immunity towards Trypanosoma brucei brucei infection.Communicated by Ramaswamy H. Sarma.

Thalassemias: from gene to therapy.

Thalassemias (α, ß, γ, δ, δß, and εγδß) are the most common genetic disorders worldwide and constitute a heterogeneous group of hereditary diseases characterized by the deficient synthesis of one or more hemoglobin (Hb) chain(s). This leads to the accumulation of unstable non-thalassemic Hb chains, which precipitate and cause intramedullary destruction of erythroid precursors and premature lysis of red blood cells (RBC) in the peripheral blood. Non-thalassemic Hbs display high oxygen affinity and no cooperativity. Thalassemias result from many different genetic and molecular defects leading to either severe or clinically silent hematologic phenotypes. Thalassemias α and ß are particularly diffused in the regions spanning from the Mediterranean basin through the Middle East, Indian subcontinent, Burma, Southeast Asia, Melanesia, and the Pacific Islands, whereas δß-thalassemia is prevalent in some Mediterranean regions including Italy, Greece, and Turkey. Although in the world thalassemia and malaria areas overlap apparently, the RBC protection against malaria parasites is openly debated. Here, we provide an overview of the historical, geographic, genetic, structural, and molecular pathophysiological aspects of thalassemias. Moreover, attention has been paid to molecular and epigenetic pathways regulating globin gene expression and globin switching. Challenges of conventional standard treatments, including RBC transfusions and iron chelation therapy, splenectomy and hematopoietic stem cell transplantation from normal donors are reported. Finally, the progress made by rapidly evolving fields of gene therapy and gene editing strategies, already in pre-clinical and clinical evaluation, and future challenges as novel curative treatments for thalassemia are discussed.

Structural and (Pseudo-)Enzymatic Properties of Neuroglobin: Its Possible Role in Neuroprotection.

Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and ß subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100-200 µM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O2 buffer and delivery. In contrast, the very low levels of Ngb (~1 µM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O2 metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

Serum Albumin: A Multifaced Enzyme.

Human serum albumin (HSA) is the most abundant protein in plasma, contributing actively to oncotic pressure maintenance and fluid distribution between body compartments. HSA acts as the main carrier of fatty acids, recognizes metal ions, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays esterase, enolase, glucuronidase, and peroxidase (pseudo)-enzymatic activities. HSA-based catalysis is physiologically relevant, affecting the metabolism of endogenous and exogenous compounds including proteins, lipids, cholesterol, reactive oxygen species (ROS), and drugs. Catalytic properties of HSA are modulated by allosteric effectors, competitive inhibitors, chemical modifications, pathological conditions, and aging. HSA displays anti-oxidant properties and is critical for plasma detoxification from toxic agents and for pro-drugs activation. The enzymatic properties of HSA can be also exploited by chemical industries as a scaffold to produce libraries of catalysts with improved proficiency and stereoselectivity for water decontamination from poisonous agents and environmental contaminants, in the so called "green chemistry" field. Here, an overview of the intrinsic and metal dependent (pseudo-)enzymatic properties of HSA is reported to highlight the roles played by this multifaced protein.

Oxygen-mediated oxidation of ferrous nitrosylated nitrobindins.

The O2-mediated oxidation of all-ß-barrel ferrous nitrosylated nitrobindin from Arabidopsis thaliana (At-Nb(II)-NO), Mycobacterium tuberculosis (Mt-Nb(II)-NO), and Homo sapiens (Hs-Nb(II)-NO) to ferric derivative (At-Nb(III), Mt-Nb(III), and Hs-Nb(III), respectively) has been investigated at pH 7.0 and 20.0 °C. Unlike ferrous nitrosylated horse myoglobin, human serum heme-albumin and human hemoglobin, the process in Nb(II)-NO is mono-exponential and linearly dependent on the O2 concentration, displaying a bimolecular behavior, characterized by kon = (6.3 ±â€¯0.8) × 103 M-1 s-1, (1.4 ±â€¯0.2) × 103 M-1 s-1, and (3.9 ±â€¯0.5) × 103 M-1 s-1 for At-Nb(II)-NO, Mt-Nb(II)-NO, and Hs-Nb(II)-NO, respectively. No intermediate is detected, indicating that the O2 reaction with Nb(II)-NO is the rate-limiting step and that the subsequent conversion of the heme-Fe(III)-N(O)OO- species (i.e., N-bound peroxynitrite to heme-Fe(III)) to heme-Fe(III) and NO3- is much faster. A similar mechanism can be invoked for ferrous nitrosylated human neuroglobin and rabbit hemopexin, in which the heme-Fe(III)-N(O)OO- species is formed as well, although the rate-limiting step seems represented by the reshaping of the six-coordinated heme-Fe(III) complex. Although At-Nb(II)-NO and Mt-Nb(II)-NO are partially (while Hs-Nb(II)-NO is almost completely) penta-coordinated, density functional theory (DFT) calculations rule out that the cleavage of the proximal heme-Fe-His bond in Nb(II)-NO is responsible for the more stable heme-Fe(III)-N(O)OO- species. Moreover, the oxidation of the penta-coordinated heme-Fe(II)-NO adduct does not depend on O2 binding at the proximal side of the metal center. These features may instead reflect the peculiarity of Nb folding and of the heme environment, with a reduced steric constraint for the formation of the heme-Fe(III)-N(O)OO- complex.

Subunits of the PBAP Chromatin Remodeler Are Capable of Mediating Enhancer-Driven Transcription in Drosophila.

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural-functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

Binding of direct oral anticoagulants to the FA1 site of human serum albumin.

The anticoagulant therapy is widely used to prevent and treat thromboembolic events. Until the last decade, vitamin K antagonists were the only available oral anticoagulants; recently, direct oral anticoagulants (DOACs) have been developed. Since 55% to 95% of DOACs are bound to plasma proteins, the in silico docking and ligand-binding properties of drugs apixaban, betrixaban, dabigatran, edoxaban, and rivaroxaban and of the prodrug dabigatran etexilate to human serum albumin (HSA), the most abundant plasma protein, have been investigated. DOACs bind to the fatty acid (FA) site 1 (FA1) of ligand-free HSA, whereas they bind to the FA8 and FA9 sites of heme-Fe(III)- and myristic acid-bound HSA. DOACs binding to the FA1 site of ligand-free HSA has been validated by competitive inhibition of heme-Fe(III) recognition. Values of the dissociation equilibrium constant for DOACs binding to the FA1 site (ie, calc KDOAC ) derived from in silico docking simulations (ranging between 1.2 × 10-8 M and 1.4 × 10-6 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (ie, exp KDOAC ; ranging between 2.5 × 10-7 M and 2.2 × 10-6 M). In addition, this study highlights the inequivalence of rivaroxaban binding to mammalian serum albumin. Given the HSA concentration in vivo (~7.5 × 10-4 M), values of KDOAC here determined indicate that the formation of the HSA:DOACs complexes in the absence and presence of FAs and heme-Fe(III) may occur in vivo. Therefore, HSA appears to be an important determinant for DOACs transport.