Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni–Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within α-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the α- or β-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [α2(Fe–NO)β2(Ni)] but such a direct transformation is less marked in [α2(Ni)β2(Fe–NO)]. Hence the bond between Nε and Fe is fundamental to the structure–function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO

Substrate interactions with human ferrochelatase

Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by ≈100° and is buried an additional 4.5 Å deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase

Binding of Isoquinoline Alkaloids Berberine, Palmatine and Coralyne to Hemoglobin: Sructural and Thermodynamic Characterization Studies

Berberine, palmatine and coralyne, the isoquinoline alkaloids distributed in many botanical families, are extensively investigated due to their potential therapeutic actions and clinical utilities. In this work, their binding characteristics to hemoglobin (Hb) were studied by UV-vis absorption spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal calorimetric titration and differential scanning calorimetric techniques. The results indicated that all the three alkaloids caused strong fluorescence quenching of Hb by the static quenching mechanism, but with differing quenching efficiencies. There was a single binding site on Hb for these alkaloids. According to the theory of Fo¨rster resonance energy transfer, the binding distances between b-Trp37 of Hb and berberine, palmatine and coralyne were evaluated to be 2.78 nm, 2.64 nm and 3.29 nm, respectively. The result of synchronous fluorescence, circular dichroism and 3D fluorescence revealed that the polarity around Trp residues experienced a significant increase in the presence of alkaloids. The binding was favoured by enthalpy and entropy changes. Results of circular dichroism, 3D and synchronous fluorescence studies confirmed that the binding of the alkaloids significantly changed the secondary structure of Hb. The studies revealed that berberine and palmatine bound to a site near to the a1b2 interface on Hb different than coralyne but the affinity of coralyne was one order higher than that of berberine and palmatine