Search

The blood of the sub-Antarctic fish Eleginops maclovinus (Em) contains three haemoglobins. The major haemoglobin (Hb1Em) displays the Root effect, a drastic decrease in the oxygen affinity and a loss of cooperativity at acidic pH. The carbomonoxy form of HbEm1 has been crystallized in two different crystal forms, orthorhombic (Ortho) and hexagonal (Hexa), and high-resolution diffraction data have been collected for both forms (1.45 and 1.49 A ˚ resolution, respectively). The high-frequency resonance Raman spectra collected from the two crystal forms using excitation at 514 nm were almost indistinguishable. Hb1Em is the first sub-Antarctic fish Hb to be crystallized and its structural characterization will shed light on the molecular mechanisms of cold adaptation and the role of the Root effect in fish haemoglobins.

Dehaloperoxidase (DHP) from the annelid Amphitrite ornata is a catalytically active hemoglobin-peroxidase that possesses a unique internal binding cavity in the distal pocket above the heme. The previously published crystal structure of DHP shows 4-iodophenol bound internally. This led to the proposal that the internal binding site is the active site for phenol oxidation. However, the native substrate for DHP is 2,4,6-tribromophenol, and all attempts to bind 2,4,6-tribromophenol in the internal site under physiological conditions have failed. Herein, we show that the binding of 4-halophenols in the internal pocket inhibits enzymatic function. Furthermore, we demonstrate that DHP has a unique two-site competitive binding mechanism in which the internal and external binding sites communicate through two conformations of the distal histidine of the enzyme, resulting in nonclassical competitive inhibition. The same distal histidine conformations involved in DHP function regulate oxygen binding and release during transport and storage by hemoglobins and myoglobins. This work provides further support for the hypothesis that DHP possesses an external binding site for substrate oxidation, as is typical for the peroxidase family of enzymes.

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted, from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high, affinity for hydrogen sulfide (K= 5.0 × 106 and 28 × 106 M-1 for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though, lower with, respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 × 108 M-1). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH.....-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH -whereas the YCD1 and. YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized, by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron, low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm-1 is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen, sulfide is thought to have a possible physiological significance as H2S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis. © 2010 American Chemical Society. Fil: Nicoletti, Francesco P.. Università degli Studi di Firenze; Italia Fil: Comandini, Alessandra. Università degli studi di Roma "La Sapienza"; Italia Fil: Bonamore, Alessandra. Università degli studi di Roma "La Sapienza"; Italia Fil: Boechi, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Boubeta, Fernando Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; Argentina Fil: Feis, Alessandro. Università degli Studi di Firenze; Italia Fil: Smulevich, Iulietta. Università degli Studi di Firenze; Italia Fil: Boffi, Alberto. Università degli studi di Roma "La Sapienza"; Italia

The present work highlights the important role played by the distal histidine in controlling the binding of heme ligands in dehaloperoxidase (DHP) as compared to myoglobin and peroxidases. In DHP the distal histidine is highly mobile and undergoes a conformational change that places it within hydrogen-bonding distance of anionic ligands and water, where strong hydrogen bonding can occur. The detailed resonance Raman (RR) analysis at room temperature shows the presence of an equilibrium between a 5-coordinate and a 6-coordinate (aquo) high-spin form. The equilibrium shifts toward the aquo form at 12 K. These two forms are consistent with the existing X-ray structures where a closed conformation, with His55 positioned in the distal pocket and H-bonded with the distal water molecule (6-coordinate), and an open solvent-exposed conformation, with the His55 displaced from the distal pocket (5-coordinate form), are in equilibrium. Moreover, the comparison between the Raman data at 298 and 12 K and the results obtained by EPR of DHP in the presence of 4-iodophenol highlights the formation of a pure 5-coordinate high-spin form (open conformation). The data reported herein support the role of His55 in facilitating the interaction of substrate and inhibitor in the regulation of enzyme function, as previously suggested. The two conformations of His55 in equilibrium at room temperature provide a level of control that permits the distal histidine to act as both the acid-base catalyst in the peroxidase mechanism and the stabilizing amino acid for exogenous heme-coordinated ligands.

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.

The unique architecture of the active site of Thermobifida fusca truncated hemoglobin (Tf-trHb) and other globins belonging to the same family has stimulated extensive studies aimed at understanding the interplay between iron-bound ligands and distal amino acids. The behavior of the heme-bound hydroxyl, in particular, has generated much interest in view of the relationships between the spin-state equilibrium of the ferric iron atom and hydrogen-bonding capabilities (as either acceptor or donor) of the OH− group itself. The present investigation offers a detailed molecular dynamics and spectroscopic picture of the hydroxyl complexes of the WT protein and a combinatorial set of mutants, in which the distal polar residues, TrpG8, TyrCD1, and TyrB10, have been singly, doubly, or triply replaced by a Phe residue. Each mutant is characterized by a complex interplay of interactions in which the hydroxyl ligand may act both as a H-bond donor or acceptor. The resonance Raman stretching frequencies of the Fe−OH moiety, together with electron paramagnetic resonance spectra and MD simulations on each mutant, have enabled the identification of specific contributions to the unique ligand-inclusive H-bond network typical of this globin family. Fil: Nicoletti, Francesco P.. Universita Degli Studi Di Firenze; Italia Fil: Bustamante, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Droghetti, Enrica. Universita Degli Studi Di Firenze; Italia Fil: Howes, Barry D.. Universita Degli Studi Di Firenze; Italia Fil: Fittipaldi, Maria. Universita Degli Studi Di Firenze; Italia Fil: Bonamore, Alessandra. Universita Di Roma; Italia Fil: Baiocco, Paola. Italian Institute of Technology; Italia Fil: Feis, Alessandro. Universita Degli Studi Di Firenze; Italia Fil: Boffi, Alberto. Universita Di Roma; Italia Fil: Estrin, Dario Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Smulevich, Giulietta. Universita Degli Studi Di Firenze; Italia

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.

Dehaloperoxidase-hemoglobin (DHP) is a unique multifunctional enzyme with a globin fold. The enzyme serves as the respiratory hemoglobin for the marine worm Amphitrite ornata and has been shown to catalyze the conversion of highly toxic trihalophenols to dihaloquinones as a detoxification function for the organism. Given the simplicity of the structure of A. ornata, it is entirely possible that DHP may play an even more general role in detoxification of the organism from sulfide commonly found in the coastal estuaries where A. ornata thrives. Comparison of DHP with other sulfide-binding hemoglobins shows that DHP possesses several distal cavity structural properties, such as an aromatic cage and a hydrogen-bond-donor amino acid (His55), that facilitate sulfide binding. Furthermore, a complete reduction of the ferric heme occurs after sulfide exposure under aerobic or anaerobic conditions to yield either the oxy or the deoxy ferrous states of DHP, respectively. Oxidation of sulfide by the heme leads to sulfur products that are less toxic to A. ornata. This proposed new function for DHP relies on the highly flexible distal His55 for deprotonation of the bound hydrogen sulfide, similar to H(2)O(2) activation of the peroxidase function, and provides further support for the importance of the flexibility of the distal His55 in this novel globin.

Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO(3)(-) catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 degrees C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO(2); the values of the second order catalytic rate constant (k(on)) are 4.1 x 10(5) and 4.5 x 10(5) m(-1) s(-1), respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO(2). The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO(2), ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 x 10(-4) and 9.7 x 10(-4) m. Under conditions where [ibuprofen] isL, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

Thermobifida fusca hemoglobin (TFH) is a prototypical bacterial (class 2) hemoglobin. It has been identified in a thermophilic actinobacterium and overexpressed. The heme cavity properties are mainly related to the polarity and H-bonding capability of the “distal” amino acids: tyrosine (B10 in the myoglobin helix notation), tyrosine (CD1), tryptophan (G8).Single, double and triple phenyalalanin (Phe) mutants of these key residues have recently become available. We have applied several techniques of time-resolved and steady-state optical spectroscopy to the study of THF carbon monoxide complexes, in an effort to correlate structural and dynamic properties of the protein active site.i) Femtosecond transient absorption has shown that CO can rebind from TFH distal cavity in few ns. TrpG8 substitution with the apolar Phe accelerates the recombination, which takes place in hundreds of ps.ii) Laser flash photolysis has allowed to extend the study of the dynamics to longer times. The yield of the geminate rebinding and the kinetics of recombination from the solvent are both influenced by the single TrpG8 → Phe mutation. Further changes are observed when the three polar amino acids are replaced by Phe. Rate constant distributions have been obtained by the maximum entropy method.iii) Photoacoustic measurements have yielded both volume and enthalpy changes following CO photodissociation in the ns range. The thermodynamic parameters can be related to those measured by laser flash photolysis.iv) Resonance Raman spectroscopy of seven THF mutated variants has given evidence for a H-bonding network in the distal cavity, which involves the bound CO.All the results point to an overwhelming role for TrpG8. This is in agreement with a recent study on a related bacterial hemoglobin from B. subtilis.