Abstract

Crystallographic and spectroscopic evidences on Antarctic fish hemoglobins (AFHbs) have revealed that their ferric tetramers at physiological pH are in a mixed |α(aquo-met)/β(bis-histidyl) coordination state and show a quaternary structure intermediate between the classical R and T states (H state). Ferric bis-histidyl adducts (hemichromes) have been also observed in some mammalian Hbs. In order to clarify whether hemichrome in AFHbs can be converted into a ferrous bis-histidyl adduct (hemochrome), at least in the crystal phase, chemical reduction of ferric hemoglobins fromTrematomus bernacchii(HbTb) single crystals has been followed via Raman microscopy. The results of this analysis reveal that in HbTb, upon reduction, the bis-histidyl coordination is disrupted in favor of a penta-coordinated ferrous deoxy state, with no evidence of hemochrome. These data are in agreement with UV/Vis absorption spectra in solution. Furthermore, our data are also indirectly supported by the observation that upon reduction with dithionite, the ferric HbTb crystals crack and lose their diffraction power: in the crystalline state, the quaternary structure transition from the H to the T state is not compatible with the crystal packing. Altogether these data indicate that if bis-histidyl adducts have a functional significance in AFHbs, this function refers to a stable ferric state, or to a transient, though never detected, ferrous species.

Highlights

  • Tetrameric hemoglobins (Hbs) deserve a special position in the field of protein allostery [1,2,3,4,5]

  • In order to establish whether Antarctic fish Hb (AFHb) hemichromes encapsulated in the crystal phase can be converted into hemochromes, we chemically reduced the ferric hemoglobins from Trematomus bernacchii (HbTb) crystals and followed the process by Raman microscopy

  • Crystals of ferric HbTb were grown at pH 7.6 according to a previous procedure [19, 20]

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Summary

Introduction

Tetrameric hemoglobins (Hbs) deserve a special position in the field of protein allostery [1,2,3,4,5]. Tetrameric Hbs from Antarctic fish are well suited to this purpose, as they form a highly correlated system with peculiar structural and functional similarities and differences. These Hbs have T and R states very similar to those characterized in mammalian Hb [15,16,17]. Intermediate species with the α chain in the oxy, carbomonoxy or met state and the β chain in the hexa-coordinate low-spin, 6cLS, bis-histidyl form (hemichromes) [18,19,20] or in the penta-coordinate high-spin, 5cHS, ferric form have been detected [20,21] These mixed forms present a quaternary organization somewhat intermediate between the classical R and T states (H state) [18,19,20]. These ferric states have been identified in polar and polar-related fish Hbs by using a combination of Raman spectroscopy [22,23,24], EPR [20,21] and X-ray crystallography [18,19,20,24]

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