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Raman assisted X-Ray crystallographic study of nitric oxidebinding to deoxygenated hemoglobins
No full textNitric oxide (NO) is a signaling molecule that regulates essential
physiological processes, including neurotransmission, vasodilatation,
and blood clotting [1].
The heme groups of deoxy hemoglobin (Hb) bind NO very strongly,
almost irreversibly, with a Kdiss = 0.9 x 10-12 M. The kinetic constant of
NO dissociation from Hb(NO)4 increases as the reaction progresses,
indicating that partially NO-saturated T-state Hb has a lower NO
affinity than fully bound, R-state, Hb [2].
Previous spectroscopic and crystallographic studies have shown
that NO binding to the heme groups of T-state human hemoglobin
(HbA) produces the breakage of Fe-proximal histidine bonds at the α-
subunits but not at the β-subunits [3]. NO can also react with the thiol
group of the Cys93β of HbA [4].
Until now there are very few crystallographic structures of nitrosylhemoglobin,
due to the high and various reactivity of these species.
Here we report a Raman-assisted crystallographic study of
the NO binding to the hemoglobin isolated from the Antarctic fish
Trematomus bernacchii (HbTb). HbTb is endowed with the Root
effect, i.e. a drastic drop of cooperativity at acidic pH [5,6]. The crystal
structures of the nitrosylated form of T-state HbTb, crystallized at
pH 6.2 and 8.4 (HbTb6NO and HbTb8NO), have been solved. These
structures and the Raman spectra have been compared to those of
nitrosylated HbA, reported in literature [3,7]. The main results of the
analysis reveal a different behavior of α and β chains. In particular, in
both HbTb6NO and HbTb8NO, the α-heme is nitrosylated and shows
a six-coordination, whereas the iron ion at β-heme is clearly oxidized
in high spin form
Issue Introduction
No full textEditoriale
Biopolymers Special Issue dedicated to Prof. Lelio Mazzarella
Editors Giulietta Smulevich & Filomena Sica
Volume 91, Issue 12, December 2009, Page97
Searching for additional functions of fish hemoglobins: evidence of multiple quaternary structures and exogeneous coordination states
No full textAll fish hemoglobins (Hb) show a high auto-oxidation rate, and some fish Hbs are endowed with Root effect (drastic drop of cooperativity at acidic pH). Differently from temperate fish Hbs, at physiological pH Antarctic fish Hbs (AFHbs) in the ferric state show both an aquomet form and two distinct hemichromes within a R / T intermediate quaternary structure (1). Interestingly, AFHbs exhibit a peroxidase activity higher than that observed for mammalian and temperate fish Hbs, thus suggesting that a partial hemichrome state in tetrameric Hbs does not protect them from peroxidation as previously proposed (2). At acidic pH, a combined EPR / x-ray crystallography approach has revealed, only for Root-effect AFHbs, significant amount of pentacoordinated (5C) high-spin Fe(III) species.(3) Furthermore, along the oxidation pathway, a combined x-ray crystallography / Resonance Raman spectroscopy of AFHbs has revealed a hybrid valence state [α(O2)/β(Fe3+, pentacoordinate)].(4) This valence hybrid states prompted us to test a FeSOD activity, that is as low as human Hb. A combined x-ray crystallography / FT-IR study has revealed at least two coordination states of the carbomonoxy form of AFHbs, one corresponding to a His assisted CO binding (band III at 1951 cm-1), and another to a not-His assisted CO binding (band IV at 1968 cm-1). The band IV, typical of both temperate fish (carp and trout) Hbs and AFHbs, assigned to the second CO coordination state, justifies the high auto-oxidation rate of fish Hbs. Furthermore, this novel CO coordination in AFHb occurs within a R-T intermediate quaternary structure. These findings provide an alternative structural explanation of the Root effect, in terms of a three state model .(5) This work was financially supported by PNRA (Italian National Programme for Antarctic Research) 1. Vergara, A., Franzese, M., Merlino, A., Vitagliano, L., di Prisco, G., Verde, C., Lee, H. C., Peisach, J., and Mazzarella, L. (2007) Biophys. J. 93, 2822-2829 2. Feng, L., Zhou, S., Gu, L., Gell, D., Mackay, J., Weiss, M., Gow, A., and Shi, Y. (2005) Nature 435, 697-701 3. Vergara, A., Franzese, M., Merlino, A., Bonomi, G., Verde, C., di Prisco, G., Lee, H., Peisach, J., and Mazzarella, L. . submitted. 4. Vitagliano, L., Vergara, A., Bonomi, G., Merlino, A., Smulevich, G., Howes, B., di Prisco, G., Verde, C., and Mazzarella, L., submitted. 5. Edelstein, S. J. (1996) J. Mol. Biol. 256, 737-74
Crystal structure of the alcohol dehydrogenase from the hyperthermophilic Archaeon Sulfolobus solfataricus at 1.85 A ° resolution
No full textRole of tertiary structures on the Root effect in fish hemoglobins
No full textMany 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 pH 6.2 and pH 8.4.
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
Crystal structure of the alcohol dehydrogenase from the hyperthermophilic Archaeon Sulfolobus solfataricus at 1.85 A ° resolution
No full textFirst atomic structural characterizationof the interaction between a mixedduplex-‐quadruplex aptamer and its target protein
No full textEffects of a thymine residue deletion in TT loops on thrombin-TBA recognition interactions: a crystallographic study
No full text
A regular thymine tetrad and a peculiar supramolecular assembly in the first crystal structure of an all-LNA G-quadruplex
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