Structural Characterization of Ferric Hemoglobins from Three Antarctic Fish Species of the Suborder Notothenioidei

Abstract

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.

Occurrence and formation of endogenous histidine hexa‐coordination in cold‐adapted hemoglobins

“…Electron paramagnetic resonance (EPR) data correlate well with the X-ray data collected for several Hbs and model systems (8,38). Indeed, EPR provides unique data on the oxidation, coordination, and spin state of the heme group.…”

“…The cytochrome b 5 hemichromes and model compounds with unhindered imidazoles display the expected unconstrained coordination geometry with values of y d and y p close to 908 (values in the range 85.68-89.18), and maximal p overlap between the axial imidazole planes and the Fe dp orbitals, which is achieved for x close to 08 (values span the range 0.78-21.28) (2,8). The unconstrained geometry of cytochrome b 5 -like hemichromes is also inferred from the values of the Ne 2prox -Fe-Ne 2dist angle, which measures the deviation from linearity of the apical bonds at the iron atom.…”

“…Successively, the formation and stability of hemichrome species in solution, as well as their structural features in the solid state were extensively investigated for several Antarctic fish Hbs. In particular, a detailed analysis of the hemichrome species was obtained by complementing X-ray crystallography with optical and resonance Raman (RR) spectroscopy in solution (29,(31)(32)(33) and in the solid state (2,34), and with electron paramagnetic resonance (EPR) (8)(9)(10). The crystal structure of the hemichrome of HbTb, the Hb from the Antarctic fish Trematomus bernacchii, has been elucidated (8).…”

“…In particular, a detailed analysis of the hemichrome species was obtained by complementing X-ray crystallography with optical and resonance Raman (RR) spectroscopy in solution (29,(31)(32)(33) and in the solid state (2,34), and with electron paramagnetic resonance (EPR) (8)(9)(10). The crystal structure of the hemichrome of HbTb, the Hb from the Antarctic fish Trematomus bernacchii, has been elucidated (8). Crystallographic detection of the hemichrome has also been reported in horse Hb (24) and in the a chain of HbA complexed with the a-Hb-stabilizing-protein (AHSP) (35).…”

“…1) of various Hbs are collected in Table 1 and compared with those obtained from three different structures of the closely related bis-histidyl cytochrome b 5 . In detail, Table 1 reports the tilt angles of the distal (y d ) and proximal (y p ) imidazole with the heme plane, the dihedral angle formed between the distal and the proximal imidazole groups (x), the bond length between the iron atom and the Ne 2 of the proximal and distal His (Fe-Ne 2im ), the Ne 2prox -Fe-Ne 2dist bond angle and the angle u formed by the sp 2 orbital direction of the Ne 2 atom (of both His) and the Ne 2 -Fe bond (8). The heme distortion from planarity was monitored via the root mean square deviations (RMSD) from the least squares plane through the 24 atoms of the porphyrin skeleton (RMSD24A) and through the four pyrrole nitrogen atoms of the heme (RMSD4N).…”