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Iron surface determinant B (IsdB) is a hemoglobin (Hb) receptor essential for hemic iron acquisition by Staphylococcus aureus. Heme transfer to IsdB is possible from oxidized Hb (metHb), but inefficient from Hb either bound to oxygen (oxyHb) or bound to carbon monoxide (HbCO), and encompasses a sequence of structural events that are currently poorly understood. By single-particle cryo-electron microscopy, we determined the structure of two IsdB:Hb complexes, representing key species along the heme extraction pathway. The IsdB:HbCO structure, at 2.9-Å resolution, provides a snapshot of the preextraction complex. In this early stage of IsdB:Hb interaction, the hemophore binds to the ß-subunits of the Hb tetramer, exploiting a folding-upon-binding mechanism that is likely triggered by a cis/trans isomerization of Pro173. Binding of IsdB to a-subunits occurs upon dissociation of the Hb tetramer into a/ß dimers. The structure of the IsdB:metHb complex reveals the final step of the extraction process, where heme transfer to IsdB is completed. The stability of the complex, both before and after heme transfer from Hb to IsdB, is influenced by isomerization of Pro173. These results greatly enhance current understanding of structural and dynamic aspects of the heme extraction mechanism by IsdB and provide insight into the interactions that stabilize the complex before the heme transfer event. This information will support future efforts to identify inhibitors of heme acquisition by S. aureus by interfering with IsdB:Hb complex formation. [ABSTRACT FROM AUTHOR]

Hemoglobin (Hb) plays its vital role through structural and functional properties evolutionarily optimized to work within red blood cells, i.e., the tetrameric assembly, well-defined oxygen affinity, positive cooperativity, and heterotropic allosteric regulation by protons, chloride and 2,3-diphosphoglycerate. Outside red blood cells, the Hb tetramer dissociates into dimers, which exhibit high oxygen affinity and neither cooperativity nor allosteric regulation. They are prone to extravasate, thus scavenging endothelial NO and causing hypertension, and cause nephrotoxicity. In addition, they are more prone to autoxidation, generating radicals. The need to overcome the adverse effects associated with cell-free Hb has always been a major hurdle in the development of substitutes of allogeneic blood transfusions for all clinical situations where blood is unavailable or cannot be used due to, for example, religious objections. This class of therapeutics, indicated as hemoglobin-based oxygen carriers (HBOCs), is formed by genetically and/or chemically modified Hbs. Many efforts were devoted to the exploitation of the wealth of biochemical and biophysical information available on Hb structure, function, and dynamics to design safe HBOCs, overcoming the negative effects of free plasma Hb. Unfortunately, so far, no HBOC has been approved by FDA and EMA, except for compassionate use. However, the unmet clinical needs that triggered intensive investigations more than fifty years ago are still awaiting an answer. Recently, HBOCs "repositioning" has led to their successful application in organ perfusion fluids. [ABSTRACT FROM AUTHOR]

Abstract: Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH6.2 and pH8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: Polyethylene glycol (PEG)-conjugated hemoglobins, a novel class of blood substitutes, were investigated by a combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with the microspectrophotometric characterization of single bands and the functional analysis of electrophoretically separated fractions. For these intrinsically heterogeneous products, the molecular mass, the size distribution, and the degree of PEGylation are strictly correlated to their side effects and, therefore, are crucial pieces of information to evaluate their safety and efficacy. The PEGylation pattern was shown to strongly depend on the quaternary conformation of hemoglobin during the reaction, and the degree of conjugation was shown to correlate with the oxygen binding properties of the individual electrophoretically separated fractions. Moreover, small but not negligible fractions of underivatized tetramers, known to be responsible for serious side effects, were detected even in preparations with a high average degree of PEGylation. Overall, this approach might be exploited to characterize other products of protein PEGylation, an increasingly relevant technology for the optimization of the pharmacokinetic properties of protein-based drugs. [ABSTRACT FROM AUTHOR]

Abstract: Dynamic light scattering (DLS) is a technique capable of determining the hydrodynamic radius of proteins. From this parameter, a molecular weight can be assessed provided that an appropriate calibration curve is available. To this goal, a globin-based calibration curve was used to determine the polymerization state of a recombinant hemoglobin-based oxygen carrier and to assess the equivalent molecular weight of hemoglobins conjugated with polyethylene glycol molecules. The good agreement between DLS values and those obtained from gel filtration chromatography is a consequence of the high similarity in structure, shape, and density within the globin superfamily. Moreover, globins and heme proteins in general share similar spectroscopic properties, thereby reducing possible systematic errors associated with the absorption of the probe radiation by the chromophore. [Copyright &y& Elsevier]

Abstract: Historically, exogenous administration of hemoglobin solutions to implement the oxygen transport capacity for clinical applications suffered from dramatic drawbacks, resulting in the failure of many attempts. In the last decades, the biochemical and physiological basis responsible for the therapeutic failures has been extensively investigated. It is now widely accepted that they mostly arise because, out of the confined and controlled environment of the red blood cell, hemoglobin exhibits tetramer instability, increased auto-oxidation rate, higher oxygen affinity, altered cooperativity and nitric oxide reactivity. Moreover, it became evident that the design of a hemoglobin-based oxygen carrier that exactly reproduces the “physiological” oxygen-binding curve is not only an overly ambitious task, but may also represent a wrong approach for many potential clinical applications. Under these premises, and given the complex chemical nature of blood, it is obvious that any strategy undertaken to modify the stability and function of the hemoglobin tetramer for clinical use should be driven by a detailed knowledge of its structure, dynamics and mechanism of allosteric regulation. We briefly review the most recent theories and experiments that increased our understanding of the mechanism of homo- and heterotropic effects in human hemoglobin, trying to interpret, on a biophysical basis, how diverse approaches like polymerization, cross-linking, site-directed mutagenesis, surface decoration and encapsulation may affect ligand affinity and allosteric regulation. [Copyright &y& Elsevier]

Abstract: Cooperativity depends on the existence of equilibria among functionally distinct conformational states that are affected by homo and heterotropic effectors. In order to isolate the quaternary conformations of hemocyanin from E. californicum, the 24-meric giant protein was encapsulated in wet, nanoporous silica gels, either in the absence or presence of oxygen. The deoxy- and oxy-hemocyanin gels exhibit a p50 for oxygen of 11 and 2.5 torr, respectively, values in close agreement with those for hemocyanin in solution. The observed Hill coefficients are lower than unity, indicating a conformational heterogeneity within each locked conformational state, a finding in agreement with the assumption that at least four conformational states are required to explain the oxygen binding properties of E. californicum hemocyanin in solution. [Copyright &y& Elsevier]

Encapsulation of hemoglobin in a biocompatible matrix is a potential strategy for obtaining blood substitutes. Such a system would retain most of the immunogenic and functional properties of the physiologically relevant oxygen carrier but would prevent protein extravasation and dimer/dimer dissociation. We applied this approach by entrapping hemoglobin in wet nanoporous silica gel, in the presence and absence of allosteric effectors. Silica gels, although not suitable for intravenous perfusion, are inert and optically transparent, thus allowing a full characterization of the functional and structural properties of encapsulated hemoglobin by spectroscopic techniques. Results indicate that hemoglobin molecules, entrapped using different protocols, exhibit an oxygen affinity that can be modulated between 12 and 140 torr. This tunability could be exploited to meet distinct clinical needs. [ABSTRACT FROM AUTHOR]

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. We inserted different single tyrosine mutations into the α and β subunits of Hb to determine if this effect of βF41Y was unique. Every mutation that was inserted within electron transfer range of the protein surface and the heme increased the rate of ferryl reduction. However, surprisingly, three of the mutations (βT84Y, αL91Y and βF85Y) also increased the rate of ascorbate reduction of ferric(met) Hb to ferrous(oxy) Hb. The rate enhancement was most evident at ascorbate concentrations equivalent to that found in plasma (< 100 μM), suggesting that it might be of benefit in decreasing oxidative stress in vivo. The most promising mutant (βT84Y) was stable with no increase in autoxidation or heme loss. A decrease in membrane damage following Hb addition to HEK cells correlated with the ability of βT84Y to maintain the protein in its oxygenated form. When PEGylated and injected into mice, βT84Y was shown to have an increased vascular half time compared to wild type PEGylated Hb. βT84Y represents a new class of mutations with the ability to enhance reduction of both ferryl and ferric Hb, and thus has potential to decrease adverse side effects as one component of a final HBOC product. fx1 • Cell free Hemoglobin Based Oxygen Carriers can deliver oxygen to tissue. • Oxidation of ferrous hemoglobin to ferric and ferryl leads to oxidative toxicity. • Insertion of tyrosine residues enhances reduction of ferryl to ferric hemoglobin. • Some mutants (e.g. βT84Y) also enhance ferric to ferrous reduction by ascorbate. • βT84Y decreases cellular oxidative stress and enhances vascular retention in vivo. [ABSTRACT FROM AUTHOR]