Carla Spagnuolo

Pentacoordinate Hemin Derivatives in Sodium Dodecyl Sulfate Micelles: Model Systems for the Assignment of the Fifth Ligand in Ferric Heme Proteins

Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins.

Oxidation reaction of human oxyhemoglobin with nitrite: a reexamination.

The oxidation reaction of human oxyhemoglobin with nitrite is complex and is characterized by a lag period followed by an autocatalytic phase. On the basis of contradictory experimental results, in order to describe the time-course of the reaction, two different mechanisms have been proposed, involving either hydrogen peroxide or the superoxide anion as reaction intermediates. This paper reports a careful reinvestigation of this reaction carried out as a function of reagent concentration, buffer composition, presence of enzymatic scavengers of oxygen radicals or of other compounds which may affect the intermediate steps of the reaction. The results obtained show that: hydrogen peroxide can be definitely identified as the reaction intermediate, in agreement with the mechanism proposed by Kosaka et al. (Biochim. Biophys. Acta 702 (1982) 237-241); the reaction time-course depends in a different way on the concentrations of hemoglobin and nitrite, a finding that cannot be explained on the basis of this mechanism. A more complex reaction scheme is proposed, that provides a satisfactory description in quantitative terms for all the available experimental data.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: The oxidation reaction

The oxidation by ferricyanide of the dimeric (HbI) and tetrameric (HbII) hemoglobins from the bivalve mollusc Scapharca inaequivalvis has been studied in static and kinetic experiments. Both hemoglobins give rise to hemichromes as stable oxidation products. Oxidation of deoxyHbI yields a hemichrome by a simple bimolecular process. No intermediate Met form can be detected during the reaction even in rapid mixing experiments. The HbI hemichrome undergoes a reversible pH-dependent dissociation into monomers. A simple model has been proposed to account for the linkage between proton binding and subunit dissociation. In the case of tetrameric HbII, oxidation yields an intermediate Met form. Thus, the kinetics of the oxidation reaction are always biphasic; the fast reaction is a bimolecular process and yields the Met derivative. The slow reaction is a monomolecular process and corresponds to the conversion of the Met form into the hemichrome; its rate is independent of the state of ligation of the ferrous protein and decreases with increase of pH. The HbII hemichrome is tetrameric when newly formed; it tends to dissociate into lower molecular weight species with the same optical properties. The rate of dissociation is relatively fast at neutral pH (t 1/2 approximately equal to 12 min) and markedly less at alkaline pH values. The HbI and HbII hemichromes are reduced by dithionite yielding the spectra of the native deoxygenated proteins; in the case of HbII, the tetrameric structure of the native protein is re-acquired.

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis. Reaction of the oxidized derivatives with azide and fluoride

The reaction of the oxidized derivatives of the dimeric (HbI) and tetrameric (HbII) Scapharca inaequivalvis hemoglobins with azide and fluoride has been studied. The two oxidized hemoglobins have specific characteristics. Oxidized HbI consists of a dimeric high-spin aquomet form which is in a pH-dependent association-dissociation equilibrium with a monomeric low-spin hemichrome. In contrast, in HbII the high-spin aquomet derivative is only a transient species that converts itself into a tetrameric hemichrome which in turn dissociates into lower molecular weight forms. The reaction of oxidized HbI with azide and fluoride can be described in terms of a simple reaction scheme which assumes that external ligands bind only to the aquomet derivative. In the case of HbII, the reaction route is the same; however, the situation is complicated by the fact that in the dissociated hemichromes the internal protein ligand can no longer be displaced. Therefore, irreversible processes take place whose relevance depends primarily on the affinity of the external ligand for the ferric heme iron.

Oxidation reaction of Scapharaca inaquivalvis hemoglobins with nitrate

Abstract The oxidation reaction with nitrite of the dimeric and tetrameric hemoglobins from the mullosc Scapharca inaequivalvis has been studied kinetically and at equilibrium. In line with previous findings obtained with ferricyanide as oxidant, in both proteins the stable oxidation product is a hemichrome, although the nitrite-methemoglobin complex is formed in significant amount when excess nitrite is employed. The reaction kinetics are characterized by a lag period followed by an autocatalytic phase, as in the case of human hemoglobin. However, with respect to human hemoglobin, in the two molluscan proteins the lag phase is prolonged significantly due to the instability of their met-form, an obligatory intermediate for the onset of autocatalysis. All the data obtained in spectrophotometric, EPR and sedimentation velocity experiments under a variety of experimental conditions conform to the reaction mechanism proposed for human hemoglobin (Spagnuolo et al., Biochim. Biophys. Acta 911 (1987) 59–63) provided hemichrome formation and nitrite binding are taken into account.

High and Low Spin Forms of Oxidized Dimeric Scapharca inaequivalvis Hemoglobin

The intracellular dimeric Hb (Hbl) from the Arcid clam Scapharca inaequivalvis is made up of two identical subunits that exhibit cooperative interaction in O2 binding (1). In a previous study on the oxidized protein, sedimentation velocity experiments showed that this derivative undergoes a reversible pH-dependent dissociation into monomers; monomer formation is more marked at acid pH values. Parallel optical spectroscopy measurements indicated that dissociation is accompanied by a high to low spin transition. The low spin species is characterized by the presence of absorption bands, typical of hemichromes in which the sixth heme ligand is contributed by the protein. The high spin species displays an unusually high absorption at 600 nm (2).

Studies of the Heme Environment in Molluscan Hemoglobins by EPR

The red cells of the mollusc Scapharca inaequivalvis, as those of other Arcid molluscs, contain a dimeric (HbI) and a tetrameric (HbII) hemoglobin component constructed from three myoglobin-like chains assembled into a homodimer and an α 2 β 2 type of structure, respectively. From a structural viewpoint, the heme carrying helices, E and F, are essentially invariant in all three polypeptide chains, but differ in sequence from the corresponding regions of vertebrate hemoglobins and myoglobins. The E and F helices of the Arcid hemoglobins are particularly rich in hydrophobic residues, a feature that has been ascribed to their involvement in the dimeric intersubunit contact (1, 2). This characteristic topology is accompanied by a number of spectroscopic features, i.e. the absorption shoulder and the unusually high ellipticity at 590 nm in both deoxy-HbI and HbII, which indicate that in the deoxygenated derivative the structure at the heme site is constrained (3). Moreover, the anisotropy of the EPR spectrum of deoxy-CoHbI is indicative of a distorted coordination of the hindered proximal histidine (4). As a model for diamagnetic 02 and CO complexes the ferrous NO derivatives of the S.inaequivalvis hemoglobins have been prepared. Since NO contains one unpaired electron the ferrous NO complexes are easily amenable to EPR studies.