Taming hemoglobin chemistry-a new hemoglobin-based oxygen carrier engineered with both decreased rates of nitric oxide scavenging and lipid oxidation.

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation.

Hell's Gate Globin-I from Displays a Unique Temperature-Independent pH Sensing Mechanism Utililized a Lipid-Induced Conformational Change.

Hell's Gate globin-I (HGb-I) is a thermally stable globin from the aerobic methanotroph . Here we report that HGb-I interacts with lipids stoichiometrically to induce structural changes in the heme pocket, changing the heme iron distal ligation coordination from hexacoordinate to pentacoordinate. Such changes in heme geometry have only been previously reported for cytochrome c and cytoglobin, linked to apoptosis regulation and enhanced lipid peroxidation activity, respectively.

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction.

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain.

Insights into the function of cytoglobin.

Since its discovery in 2001, the function of cytoglobin has remained elusive. Through extensive in vitro and in vivo research, a range of potential physiological and pathological mechanisms has emerged for this multifunctional member of the hemoglobin family. Currently, over 200 research publications have examined different aspects of cytoglobin structure, redox chemistry and potential roles in cell signalling pathways.

Globin Associated Oxidative Stress.

Globins have been studied for their "pseudo-peroxidase" activity for over 70 years, being an ideal model of other kinetically more rapid metalloenzymes [...

Modulating Nitric Oxide Dioxygenase and Nitrite Reductase of Cytoglobin through Point Mutations.

Cytoglobin is a hexacoordinate hemoglobin with physiological roles that are not clearly understood. Previously proposed physiological functions include nitric oxide regulation, oxygen sensing, or/and protection against oxidative stress under hypoxic/ischemic conditions. Like many globins, cytoglobin rapidly consumes nitric oxide under normoxic conditions.

The peroxidatic activities of Myoglobin and Hemoglobin, their pathological consequences and possible medical interventions.

Under those pathological conditions in which Myoglobin and Hemoglobin escape their cellular environments and are thus separated from cellular reductive/protective systems, the inherent peroxidase activities of these proteins can be expressed. This activity leads to the formation of the highly oxidizing oxo-ferryl species. Evidence that this happens in vivo is provided by the formation of a covalent bond between the heme group and the protein and this acts as an unambiguous biomarker for the presence of the oxo ferryl form.

Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1.

Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods.

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit.

In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules.

Corrigendum: A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

[This corrects the article DOI: 10.3389/fpls.2020.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O and/or nitric oxide (NO). Here, we have identified a gene of the model legume with unique properties. The gene, designated , generates four alternative splice forms encoding Glbs with one or two heme domains and 215-351 amino acid residues.

Engineering hemoglobin to enable homogenous PEGylation without modifying protein functionality.

In order to infuse hemoglobin into the vasculature as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the molecule to enhance vascular retention. This aim can be achieved by PEGylation. However, using non-specific conjugation methods creates heterogenous mixtures and alters protein function.

Sugar beet hemoglobins: reactions with nitric oxide and nitrite reveal differential roles for nitrogen metabolism.

In contrast with human hemoglobin (Hb) in red blood cells, plant Hbs do not transport oxygen, instead research points towards nitrogen metabolism. Using comprehensive and integrated biophysical methods we characterized three sugar beet Hbs: BvHb1.1, BvHb1.

Engineering tyrosine residues into hemoglobin enhances heme reduction, decreases oxidative stress and increases vascular retention of a hemoglobin based blood substitute.

Hemoglobin (Hb)-based oxygen carriers (HBOC) are modified extracellular proteins, designed to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects, in part linked to the intrinsic oxidative toxicity of Hb. Previously a redox-active tyrosine residue was engineered into the Hb β subunit (βF41Y) to facilitate electron transfer between endogenous antioxidants such as ascorbate and the oxidative ferryl heme species, converting the highly oxidizing ferryl species into the less reactive ferric (met) form.

High- and low-affinity PEGylated hemoglobin-based oxygen carriers: Differential oxidative stress in a Guinea pig transfusion model.

Hemoglobin-based oxygen carriers (HBOCs) are an investigational replacement for blood transfusions and are known to cause oxidative damage to tissues. To investigate the correlation between their oxygen binding properties and these detrimental effects, we investigated two PEGylated HBOCs endowed with different oxygen binding properties - but otherwise chemically identical - in a Guinea pig transfusion model. Plasma samples were analyzed for biochemical markers of inflammation, tissue damage and organ dysfunction; proteins and lipids of heart and kidney extracts were analyzed for markers of oxidative damage.

Comparison of the oxidative reactivity of recombinant fetal and adult human hemoglobin: implications for the design of hemoglobin-based oxygen carriers.

Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been engineered to replace or augment the oxygen carrying capacity of erythrocytes. However, clinical results have generally been disappointing, in part due to the intrinsic oxidative toxicity of Hb. The most common HBOC starting material is adult human or bovine Hb.

Effect of Enzymatic pre-treatment of microalgae extracts on their anti-tumor activity.

Background: There is an increasing need to find natural bioactive compounds for pharmaceutical applications, because they have less harmful side effects compared to their chemical alternatives. Microalgae (MA) have been identified as a promising source for these bioactive compounds, and this work aimed to evaluate the anti-proliferative effects of semi-purified protein extracted from MA against several tumor cell lines.

Methods: Tested samples comprised MA cell extracts treated with cellulase and lysozyme, prior to extraction.

Strong modulation of nitrite reductase activity of cytoglobin by disulfide bond oxidation: Implications for nitric oxide homeostasis.

Globin-mediated nitric oxide (NO) dioxygenase and nitrite reductase activities have been proposed to serve protective functions within the cell by scavenging or generating NO respectively. Cytoglobin has rapid NO dioxygenase activity, similar to other globins, however, the apparent rates of nitrite reductase activity have been reported as slow or negligible. Here we report that the activity of cytoglobin nitrite reductase activity is strongly dependent on the oxidation state of the two surface-exposed cysteine residues.

Correction: Ultrafast photochemistry of the bc complex.

Correction for 'Ultrafast photochemistry of the bc complex' by Marten H. Vos et al., Phys.

Ultrafast photochemistry of the bc complex.

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c, two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5-7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study.

Redox and Peroxidase Activities of the Hemoglobin Superfamily: Relevance to Health and Disease.

Significance: Erythrocyte hemoglobin (Hb) and myocyte myoglobin, although primarily oxygen-carrying proteins, have the capacity to do redox chemistry. Such redox activity in the wider family of globins now appears to have important associations with the mechanisms of cell stress response. In turn, an understanding of such mechanisms in vivo may have a potential in the understanding of cancer therapy resistance and neurodegenerative disorders such as Alzheimer's.

Enzymatic pre-treatment of microalgae cells for enhanced extraction of proteins.

Crude proteins and pigments were extracted from different microalgae strains, both marine and freshwater. The effectiveness of enzymatic pre-treatment prior to protein extraction was evaluated and compared to conventional techniques, including ultrasonication and high-pressure water extraction. Enzymatic pre-treatment was chosen as it could be carried out at mild shear conditions and does not subject the proteins to high temperatures, as with the ultrasonication approach.

Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species.

Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin.

The reaction of hydrogen peroxide with ferric human cytoglobin and a number of distal histidine variants were studied. The peroxidase activity of the monomeric wildtype protein with an internal disulfide bond, likely to be the form of the protein in vivo, exhibits a high peroxidase-like activity above that of other globins such as myoglobin. Furthermore, the peroxidatic activity of wildtype cytoglobin shows increased resistance to radical-based degradation compared to myoglobin.

Coupling of disulfide bond and distal histidine dissociation in human ferrous cytoglobin regulates ligand binding.

Earlier kinetics studies on cytoglobin did not assign functional properties to specific structural forms. Here, we used defined monomeric and dimeric forms and cysteine mutants to show that an intramolecular disulfide bond (C38-C83) alters the dissociation rate constant of the intrinsic histidine (H81) (∼1000 fold), thus controlling binding of extrinsic ligands. Through time-resolved spectra we have unequivocally assigned CO binding to hexa- and penta-coordinate forms and have made direct measurement of histidine rebinding following photolysis.